egfr and her2 targeting for radionuclide-based imaging and ...172029/fulltext01.pdf · egfr and...

ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2008

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 350

EGFR and HER2 Targeting forRadionuclide-Based Imaging andTherapy

Preclinical Studies

ERIKA NORDBERG

ISSN 1651-6206ISBN 978-91-554-7197-2urn:nbn:se:uu:diva-8721

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List of papers

I Nordberg E, Steffen AC, Persson M, Sundberg ÅL, Carlsson J, Glimelius B. Cellular uptake of radioiodine delivered by trastuzumab can be modified by the addition of epidermal growth factor. Eur J Nucl Med Mol Imaging 2005;32(7):771-7.

II Friedman M, Nordberg E, Höidén-Guthenberg I, Brismar H,

Adams GP, Nilsson FY, Carlsson J, Ståhl S. Phage display se-lection of Affibody molecules with specific binding to the ex-tracellular domain of the epidermal growth factor receptor. Protein Engineering, Design & Selection (PEDS), 2007;20(4):189-99.

III Nordberg E, Friedman M, Göstring L, Adams GP, Brismar

H, Nilsson FY, Ståhl S, Glimelius B, Carlsson J. Cellular studies of binding, internalization and retention of a radio-labeled EGFR-binding affibody molecule. Nucl Med Biol 2007;34(6):609-18.

IV Nordberg E, Orlova A, Friedman M, Tolmachev V, Ståhl S,

Nilsson FY, Glimelius B, Carlsson J. In vivo and in vitro up-take of 111In, delivered with the affibody molecule (ZEGFR:955)2, in EGFR expressing tumour cells. Oncology Reports, 2008;19:853-7.

V Nordberg E, Ekerljung L, Häggblad S, Carlsson J, Lennarts-

son J, Glimelius B. Effects of an EGFR-binding affibody molecule on intracellular signalling pathways. [Manuscript, 2008].

Reprints were made with kind permission from Springer-Verlag (I), Oxford Univer-sity Press (II), Elsevier (III) and Spandidos Publications Ltd (IV). Front cover: Figure modified from the crystallographic structure of two dimerized EGFR, stabilized with two EGF molecules (Dawson et al. 2007). Printed with kind permission from Elsevier.

Contents

Introduction.....................................................................................................9 What is Cancer? .........................................................................................9 Cancer statistics..........................................................................................9 Cancer detection and treatment today ......................................................10 Tumour targeting......................................................................................10 The epidermal growth factor receptor (EGFR) family.............................11

EGFR/HER1........................................................................................12 HER2 ...................................................................................................12 HER3 and HER4 .................................................................................13 Receptor dimerization and signalling ..................................................13 Endocytosis..........................................................................................15

Targeting the EGFR family......................................................................16 Trastuzumab (Herceptin®) ..................................................................16 Cetuximab (Erbitux®) .........................................................................16 Panitumumab (Vectibix®)...................................................................17 Gefitinib (Iressa®)...............................................................................17 Erlotinib (Tarceva®) ...........................................................................17 Lapatinib (Tykerb®)............................................................................18 Affibody molecules .............................................................................18

Diagnosis and therapy using radionuclides ..............................................20

The present study ..........................................................................................21 Modification of the 125I uptake (paper I) ..................................................21 Selection of EGFR-targeting affibody molecules (II) ..............................24

Selection and affinity studies...............................................................24 Binding studies ....................................................................................25

Characterization of (ZEGFR:955)2 (III & V) .................................................26 Internalization ......................................................................................26 Retention..............................................................................................27 Binding studies ....................................................................................28 Signalling studies.................................................................................29

In vivo uptake of 111In delivered with (ZEGFR:955)2, (IV) ...........................32 Biodistribution in tumour-bearing mice ..............................................32 Gamma camera imaging ......................................................................33

Summary.......................................................................................................35

Future studies ................................................................................................37

Acknowledgments.........................................................................................38

References.....................................................................................................41

Abbreviations

AR Amphiregulin ATE Activated tin ester BTC Betacellulin CAT Chloramine-T CHX-A"-DTPA [®-2-Amino-3-(4-isothiocyanatophenyl)propyl]-

trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid CPM Counts per minute DABI (4-isothiocyanato-benzylammonio)-undecahydro-

closo-dodecaborate (-1) DNA Deoxyribonucleic acid DTPA Diethylenetriaminepentaacetic acid ECD Extracellular domain EGF Epidermal growth factor EGFR Epidermal growth factor receptor Erk Extracellular signal regulated kinase EPR Epiregulin Fab Antigen-binding fragment FACS Fluorescence activated cell sorting FDA Food and drug administration Gy Gray HB Heparin-binding HER Human EGFR IC Intracellular tyrosine kinase domain ID Injected dose IgG Immunoglobulin, class G i.p. Intraperitoneally i.v. Intravenously LET Linear energy transfer Mab Monoclonal antibody MAPK Mitogen-activated protein kinase NA Not available NRG Neuregulin NSCLC Non-small-cell lung cancer PBS Phosphate buffered saline p.i. Post-injection PET Positron emission tomography

PI(3)K Phosphatidylinositol-3-OH kinase PIP2 Phosphatidylinositol (3,4)-biphosphate PIP3 Phosphatidylinositol (3,4,5)-trisphosphate PLC Phospholipase-C PTEN Phosphatase and Tensin homolog s.c. Subcutaneously SD Standard deviation SH2 Src homology 2 SPA Staphylococcal protein A SPECT Single photon emission computed tomography SPMB N-succinimidyl-para-(tri-methylstannyl)benzoate TGF Transforming growth factor TKI Tyrosine kinase inhibitor TM Transmembrane domain VEGF Vascular endothelial growth factor

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Introduction

What is Cancer? Normal cells have a balance between proliferation and programmed cell-death, but if this balance is disturbed and cells start to divide without control they become tumorous.

Tumours can be either benign or malignant. Benign tumours can be large but cannot infiltrate into other tissues or organs. Malignant tumours, how-ever, can both displace and destroy other tissues and establish new daughter tumours (metastases).

It is difficult to ascertain why a person gets cancer. Several factors can cause normal cells to become malignant. Various carcinogens, such as to-bacco smoke, radiation, chemicals and infectious agents, can cause genomic changes that lead to cancer. Such changes are randomly acquired but some genomic changes that lead to cancer can also be inherited.

There are many different types of cancer and they can be classified ac-cording to the tissue that the malignant cells originate from; the main catego-ries include:

� Adenocarcinoma – tumours derived from secretory epithelial cells

(e.g. prostate, breast and colorectal cancers). � Squamous cell carcinoma – tumours derived from epithelial cells

(in skin or in tissues covering some inner organs). � Sarcoma – tumours derived from connective or supportive tissue

(bone, cartilage, fat, muscle and blood vessels). � Leukaemia – derived from blood-forming tissue (bone-marrow). � Lymphoma and Myeloma – cancers that originate in the immune

system. � Glioma – tumours derived from brain and spinal cord.

Cancer statistics Cancer is one of the most common causes of death and can affect people at any age; however, risk increases with age. From a total of 58 million deaths

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worldwide in 2005, cancer was responsible for about 7.6 million (or 13%) of all deaths [1]. That year in Sweden, 91,775 people died and cancer ac-counted for about 23% of all deaths for women and 26% for men. The num-ber of cancer diagnosed in Sweden has during the last decades increased and is presently more than 50,000 per year [2].

Cancer detection and treatment today Symptoms of cancer can be the discovery of a lump, problems with wound-healing, persistent cough, misshapen birthmarks, changed fecal habits and urinary difficulties. Various methods such as palpation, biopsy, endoscopy, tumour markers and different types of imaging techniques are used to locate tumours and establish diagnosis. Screening for cancer is also performed among people who do not show symptoms, for example mammography for breast cancer and cell sample analysis for cervical cancer.

There are several factors to consider when treatment for cancer is se-lected, such as tumour type, how extensive the tumour is (stage), the inten-tion with the therapy (curative or palliative) and the patient’s physical condi-tion. There are many different types of therapies; those used on a regular basis can be divided into three groups : 1. Surgery, i.e. removal of tumours and surrounding tissue. To cure a pa-

tient, all tumour cells capable of tumour regeneration must be removed. 2. Radiotherapy, where different types of radiation are delivered from dif-

ferent sources, i.e. exogenously (external therapy) or endogenously (brachytherapy and radionuclide targeting therapy).

3. Medical therapy, using drugs that are toxic for replicating cells, anti-hormone treatment for tumours requiring hormones for their growth or by using other toxic agents.

Combinations of different therapies are also given in order to improve the results. The effects of chemotherapy and radiotherapy were systematically evaluated by the Swedish council on technology assessment in health care (SBU) group, some years ago [3, 4].

Tumour targeting The optimal way of detecting and treating a cancerous growth is to target the cancer cells exclusively without affecting adjacent tissue. The target should be present in large quantities, preferably be present only on tumour cells and be easily accessible [5].

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Most cancer treatments currently used affect all dividing cells and the dose of the drug or radioactivity then becomes the limiting factor. If instead the tumour cells were targeted more specifically, using molecules carrying the toxic substance, higher doses could be directed at the tumour cells.

The size of the targeting agent is also an important factor, small mole-cules are often trapped in the kidneys following tubular resorption, whereas molecules larger than 70 kDa are not filtrable in the kidneys [6]. It can there-fore be difficult to use small molecules in therapy applications where the dose of the drug or radioactivity can be the limiting factor for the kidneys. Blood clearance also differs for molecules with different molecular weights. Larger molecules stay longer in blood, which increases the uptake in tu-mours but can also, in unfortunate cases, give high doses to normal tissues. Large molecules are most often unsuitable for imaging applications where the radionuclides should stay as briefly as possible in the blood (within one or a few hours), in order to give sharp contrast in tumour images. Studies have also shown that it is likely that small molecules penetrate tumours bet-ter than larger molecules [7, 8].

The epidermal growth factor receptor (EGFR) family The epidermal growth factor receptor (EGFR) family is often overexpressed in different types of cancer. The family comprises four receptors: the human epidermal growth factor 1 (HER1/EGFR), HER2, HER3 and HER4 (see Figure 1) [9]. Each receptor in the family is composed of an extracellular domain consisting of four subdomains (I-IV), a transmembrane domain (TM) and an intracellular tyrosine kinase domain (IC), except HER3 which lacks a functional tyrosine kinase domain [10]. The EGFR family is known to contribute to the development of many organ and tissue systems [11, 12] and regulates many important cellular processes such as proliferation, migra-tion, apoptosis and differentiation. Abnormally expression of the receptors is frequently associated with various malignancies [12, 13].

The receptors have several ligands which are produced either by the same cell that expresses the receptor (autocrine secretion) or from surrounding cells (paracrine secretion) [12, 14].

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Figure 1. The EGFR receptor family. The design of the drawings is inspired by the described crystal structure of EGFR, EGF and cetuximab as published by Li et al. [15].

EGFR/HER1 The EGFR was the first discovered receptor in the EGFR family and the ligand EGF was originally described by Cohen et al. [16]. EGFR binds sev-eral ligands, such as EGF, transforming growth factor-� (TGF-�), am-phiregulin (AR), betacellulin (BTC), heparin-binding EGF (HB-EGF) and epiregulin (EPR) [12, 14].

Large deletions in the EGFR have also been found, the most frequent mu-tant being EGFRvIII. This mutant lacks the dimerization arm and some part of the ligand-binding domain. It is constitutively active at the plasma mem-brane and evades downregulation [17, 18].

EGFR is overexpressed in head and neck, bladder, breast, glioma, pros-tate, ovarian, kidneys and non-small-cell lung cancers (NSCLC) [9, 19].

HER2 The second member in the EGFR family was identified in separate occasions in mice and humans. Transfection assays revealed the oncogene neu in brain tumours in offspring of mice treated with ethylnitrosourea [20]. The human homologue of neu was cloned by homology screening with v-erbB [21, 22] and is called HER2.

HER2 does not bind any known natural ligand. A strong interaction be-tween extracellular subdomains I and III leads to a constantly exposed dimerization arm which is believed to be the reason why HER2 is incapable of binding any ligand [10, 23, 24]. This stabilized conformation makes the

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HER2 a preferred heterodimerization partner [23, 25] and is also believed to spontaneously form HER2 homodimers in HER2 overexpressing cells [26].

HER2 is overexpressed in approximately one-third of all breast cancers [27, 28]. Other cancers in which the HER2 receptor has been found to be overexpressed are ovarian, lung, pancreas, colon, oesophagus, urinary blad-der and cervix cancer [9, 12, 29]

HER3 and HER4 HER3 is the least related family member. It exhibits two substantial differ-ences not found in the other EGFR family members. First there are four amino acid changes in the tyrosine kinase domain which causes the loss of kinase activity [30, 31]. The other difference is the presence of multiple re-peats of a specific carboxyl terminal tyrosine autophosphorylation site that have specific signalling abilities [32].

The HER3 binds two ligands, neuregulins 1 and 2 (NRGs) [12, 33]. It is localized mainly within intracellular compartments but is also expressed in the cellular membrane [34]. The HER3 is expressed in breast, colon, gastric, prostate cancer and is overexpressed in oral squamous cell cancer [9].

HER4 was cloned and sequenced 1992 by Plowman et al. [35]. The HER4 receptor shares recognition and signalling features with EGFR and, similarly to EGFR, binds a large group of ligands, BTC, HB-EGF and EPR, but also the non EGFR-binding ligands NRGs 1-4 [10, 36, 37]. HER4 ex-pression is reduced in breast and prostate cancer, but is expressed in child-hood medullo-blastoma [38, 39]

Receptor dimerization and signalling The receptors in the EGFR family can dimerize either with an identical re-ceptor in the same family (homodimerization) or another receptor in the same family (heterodimerization) [40, 41].

Crystallographic studies have shown that rearrangements and conforma-tional changes play an important role in receptor dimerization and for signal-ling. The EGFR, HER3 and HER4 seem, when not ligand-stimulated, to mostly be in a closed conformation where subdomains II and IV form an intramolecular interaction. However, when a ligand binds to subdomain I and III on the EGFR, this tether is broken and an open, untethered conforma-tion appears. This rearrangement causes the dimerization site on subdomain II to become exposed to the surroundings and the receptor is then able to dimerize with another receptor (see Figure 2) [10, 42-46]. The HER2 recep-tor has a different conformation where a strong interaction between the ex-tracellular subdomains I and III leads to a constantly exposed dimerization arm. This conformation is believed to be the reason why HER2 is the pre-ferred dimerization partner for other activated receptors in the family [10,

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23, 24]. However, some studies also indicate that there are predimerized receptors without any stabilizing ligand [42, 47]

Figure 2. A sketch describing how EGF potentially binds and changes the configu-ration of the subdomains to prepare EGFR for dimerization.

Upon receptor dimerization, specific tyrosine residues within the cytoplas-mic tail become phosphorylated [12]. One theory is that two tyrosine kinase domain monomers form an asymmetric dimer in which a catalytically inac-tive monomer stabilizes the other monomer in a catalytically active confor-mation [48].

The phosphorylated tyrosine residues serve as docking sites for signalling molecules containing a Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains and are responsible for activating numerous downstream pathways (see Figure 3) [9, 10, 12]. The individual receptors in the EGFR family bind to a distinct subset of signalling molecules, also depending on the composition of the dimer [49]. Two major components of this signalling network include:

1. The RAS protein which activates a number of pathways; one of these is

via Raf kinase which stimulates a linear kinase cascade, culminating in activation of the Erk/mitogen-activated protein kinase (MAPK). This pathway is involved in cell proliferation [50, 51].

2. The phosphoinositide 3-kinases (PI3K)/Akt signalling regulates many cellular processes including proliferation and survival. PI3K phosphory-lates phosphatidylinositol (4,5)-bisphosphate (PIP2) and converts it to phosphatidylinositol (3,4,5)-triphosphate (PIP3). Downstream molecules such as Akt are thereafter recruited by PIP3 and several responses are triggered [52, 53]. Phosphatase and Tensin homologue (PTEN) regulates the PI3K/Akt pathway by dephosphorylating PIP3 to phosphatidylinositol (4,5)-biphosphate (PIP2); mutations in this gene are common in cancers [54].

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Figure 3. Receptor dimerization leading to phosphorylation of downstream signal-ling pathways.

Endocytosis EGFR and HER2 are often found in membrane areas called caveole [55]. Activated EGFRs are believed to migrate from caveole and become sorted into clathrin-coated vesicles [10, 56]. The ligand receptor complex is there-after internalized and delivered to a tubular-vesicular network [10] where it can be either recycled or degraded. To be degraded, the receptors are be-lieved to recruit ubiquitin ligases and monoubiquitin-binding proteins. Cbl is a ubiquitin ligase that is recruited to the EGFR with a kinase-activity-dependent approach [10, 57, 58].

The different receptors have different patterns concerning degradation and recycling. For example HER2 containing heterodimers are thought to un-dergo slow endocytosis and frequently recycle to the cell surface, resulting in a more potent signalling [26, 59]. The rate of EGFR endocytosis is be-lieved to increase 5–10-fold following ligand activation, whereas the other receptors in the family appear to have a constant internalization rate [60] and appear not to be degraded following ligand binding [61]. Studies also pro-pose that the receptors have an alternative routing to the nucleus [62, 63].

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Targeting the EGFR family Two groups of EGFR targeting agents are currently approved for clinical use today: the EGFR targeting monoclonal antibodies and the receptor tyrosine kinase inhibitors (TKI). The monoclonal antibodies have several mecha-nisms of action, including interference with the receptor signalling by block-ing the ligand-bindingsite, internalization of receptor-antibody complexes and potential stimulation of an immunologic response. The TKIs block the intracellular tyrosine kinase mediated signalling pathways by binding to or near the ATP-binding pocket on the intracellular kinase domain [12, 13, 64, 65]. There are presently six EGFR family targeting agents that are approved by the U.S. Food and Drug Administration (FDA).

Trastuzumab (Herceptin®) This humanized monoclonal antibody binds to subdomain IV of HER2 (see Figure 4) [24]. It was approved in September 1998 for treatment of HER2-overexpressing breast cancer. The mechanism of trastuzumab action is thought to involve many processes, including downregulation of HER2, blocking of cell-cycle progression and reduction of the number of cells in the S-phase fraction, antibody dependent cellular cytotoxicity, angiogenesis inhibition and sensitizing of tumours against chemo and radiotherapy [66-68] Approximately one-third of patients with HER2-overexpressing metastatic breast cancer respond to trastuzumab [69, 70]. Main side effect: cardiac dysfunction.

Cetuximab (Erbitux®) Cetuximab is a recombinant human/murine monoclonal antibody that binds to subdomain III on the EGFR (see Figure 4) [15]. It was approved in Feb-ruary 2004 for treatment of EGFR-positive metastatic colorectal cancer and in December 2005 for treatment of advanced squamous cell carcinoma of the head and neck, in combination with irradiation.

One of cetuximab’s primary mechanisms of action is that it competes with EGF for the binding site on EGFR, resulting in downregulated recep-tors. Cetuximab has also been shown to retard cell division, increase the proportion of cells in G1 phase [71, 72] and reduce the proportion in S phase. G1 is a more radiosensitive phase which is believed to be one mecha-nism that could contribute to the radio-sensitization effect [73]. The growth inhibitory effect of cetuximab is however often more pronounced in tumour xenografts than in cell cultures. It is believed that anti-angiogenetic factors, such as inhibited vascular endothelial growth factor (VEGF) production, are involved [74, 75].

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Some immunohistochemistry studies have reported however that the in-tensity of EGFR staining did not correlate with the clinical response of cetuximab [76] and some patients who were negative for EGFR expression surprisingly responded to cetuximab therapy [77].

The most common side effect of cetuximab is allergic reactions and skin toxicity. Interestingly, a clear association between the rash from the skin reaction and the drug’s efficacy has been found [78, 79].

Panitumumab (Vectibix®) Panitumumab is a fully humanized monoclonal antibody, approved in Sep-tember 2006 for treatment of patients with EGFR-expressing metastatic co-lorectal cancer with progression, on or following fluoropyrimidine-, ox-aliplatin- and irinotecan-containing treatment. Panitumumab competes with cetuximab for the same binding-site on subdomain III, as seen in Figure 4, and has a similar mechanism of action. Panitumumab does not have any clinical antitumour efficacy if kRAS is mutated in codons 12, 13 and possi-ble 61 based upon a retrospective study [80]. This appears to be same for cetuximab, but presently less well documented. The antibody is internalized as cetuximab, although it appears not to be degraded (possibly recycled). Skin rash similar to that provoked by cetuximab has also been found with panitumumab; the association between rash and efficacy was also observed in the panitumumab studies. The side effects are similar to those with cetuximab, but fewer and less severe allergic reactions [81].

Gefitinib (Iressa®) Gefitinib is a small, reversible TKI that blocks the ATP-binding domain of EGFR (see Figure 4) [13]. It was approved in 2003 for treatment of ad-vanced NSCLC, though it was later withdrawn and from June 2005 the FDA states that gefitinib should only be used in cancer patients who have already taken the medicine and whose doctor believes it is helping them.

However, several studies have shown that NSCLC patients carrying vari-ous somatic mutations of the EGFR gene respond favourably to gefitinib treatment [82-84]. Side effects: skin rash, diarrhoea, nausea and emesis, acute lung injury has also been reported [64].

Erlotinib (Tarceva®) This EGFR targeting TKI (see Figure 4), was approved in 2004 for treat-ment of patients with NSCLC and in 2005 for use in combination with gem-citabine for pancreatic cancer. Erlotinib also has a slight inhibitory effect on the HER2 and inhibits EGFRvIII tyrosine kinase activity. Side effects in-clude skin rash and diarrhoea [64, 85].

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Lapatinib (Tykerb®) Lapatinib is a TKI that can bind reversibly to both EGFR and HER2 (see Figure 4) [86]. It was approved in March 2007 for the treatment of patients with advanced or metastatic breast cancer with HER2-overexpressing tu-mours and who have received prior therapies.

When binding to EGFR, lapatinib does not bind to the active receptor conformation such as erlotinib or gefitinib. Instead it binds to the inactive form which contributes to the slower dissociation rate, resulting in a pro-longed effect compared with other TKI [87]. Side effects include: skin toxic-ity and diarrhoea [88].

Figure 4. Binding-site localisation for approved EGFR family targeting agents.

Affibody molecules Affibody molecules (Affibody®) constitute a fairly new type of molecules that is of interest for targeting the EGFR family. Affibody molecules are derived from an IgG-binding domain of staphylococcal protein A (SPA). SPA consist of an N-terminal signal sequence followed by five homologous Ig-binding domains (E, D, A, B and C). The native B domain of SPA was engineered to increase its chemical stability and was thereafter denoted Z domain. The Z domain is a low molecular weight protein (~6,5 kDa) consist-ing of 58 amino acids retaining the parental bundle structure of three anti-parallel �-helices [89]. Thirteen surface-exposed amino acid residues, lo-cated in helices one and two, can be randomized to create protein libraries from which affibody molecules with specificity to a broad range of proteins can be obtained (see Figure 5) [90, 91].

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Figure 5. A three-helix bundle affibody molecule, derived from the Ig-binding do-main B of staphylococcal protein A. Illustration modified from Affibody AB.

Affibody molecules are especially interesting for imaging applications be-cause of their small size. Small molecules are often better at penetrating tumour tissue and have a faster blood clearance than larger molecules such as antibodies [8, 92]

A ZHER2:4 affibody molecule was isolated by using phage display selection against the extracellular domain of HER2 [93]. When radiolabelled with 125I this HER2 binder showed specific binding to HER2 expressing cells. In or-der to improve its affinity, a bivalent version of the ZHER2:4 affibody (~15 kDa) was created in a head-to-tail conformation. (ZHER2:4)2 showed an im-proved affinity (nanomolar) for the receptor as well as prolonged cellular retention of radioactivity when delivered with radiolabelled affibody [94]. In vivo studies revealed that both the mono- and bivalent versions bound to HER2-expressing xenografts in mice [92, 95]. By affinity maturation, a sec-ond generation affibody, ZHER2:342, with improved affinity was developed. This affibody molecule has recently been thoroughly characterized in animal models using various radionuclides as labels [96, 97]. It has also been tested in a few patients using both 111In for SPECT and 68Ga for PET [98, 99]. Ef-forts have also been done to reduce the renal uptake to allow for systemic therapy. One approach was to fuse an albumin-binding domain (ABD) to the affibody molecule which allowed tumour targeting of 177Lu to microxeno-grafted tumours at curative doses [100].

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Diagnosis and therapy using radionuclides Radionuclides can be used for both imaging and therapy applications. For imaging, two types of radionuclides are used, the gamma (�) and positron (�+) emitters. The gamma emitters can be used either in a regular 2D-imaging gamma-camera or in a gamma camera technique called single pho-ton emission computed tomography (SPECT), where the information from the gamma-rays is transformed into 3D-images. The �+-emitting nuclides are instead used in positron emission tomography (PET). The PET technique is more sensitive and gives better spatial resolution than SPECT, the PET tech-nique is however more expensive, which limits its use in the clinic [101].

In therapeutic applications, three main types of radiation are used: the al-pha- (�) and beta (�-) -particles and auger electrons. The �-particle has, in most cases, a shorter range than a �--particle and therefore deposits its en-ergy in a smaller volume. The longer range of most �--particles gives instead benefit from crossfire irradiation [102, 103]. The auger electrons have the shortest range and are released in cascades making them locally very cyto-toxic and is therefore considered mostly for treatment of single cells when the radionuclides can be located in the cellular nucleus [102, 104].

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The present study

Modification of the 125I uptake (paper I) Targeting the HER2 or EGFR is a well known anti-cancer strategy and it has been indicated that cancers that co-overexpress both receptors have a poorer prognosis than those that overexpress either the one or the other receptor [105]. Thus, it is important to understand the ‘crosstalk’ between the recep-tors to improve patient prognosis and treatment.

It was found in paper I, that epidermal growth factor, EGF, via its interac-tion with EGFR, could modify the HER2-mediated cellular uptake of 125I when delivered as [125I]trastuzumab to A431 cells. When EGF was added together with [125I]trastuzumab, the cellular uptake of 125I was lower during the first hours of incubation compared with cells not treated with EGF. However, after about 10 hours of incubation the levels of 125I were higher in cells treated with EGF (see Figure 6).

Figure 6. Cellular uptake of 125I after 0-48 hours incubation with [125I]trastuzumab (50 ng/ml), treated with 10 ng EGF/ml (open squares) or 0 ng EGF/ml (filled trian-gles). Counts per minute (CPM) per 105 cells are presented (mean values and stan-dard deviations of three values).

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There are at least two potential mechanisms that might explain the decreased 125I uptake after short incubation times. One mechanism could be that the EGF:EGFR association with HER2 blocks the binding site for [125I]trastuzumab, thereby reducing the 125I uptake. No such block has, to the author’s knowledge been reported in the literature.

The other mechanism is probably related to EGFR:HER2 heterodimeriza-tion. EGF (~6 kDa) is a smaller molecule than trastuzumab (~150 kDa), and if the EGF molecule binds to the EGFR faster than [125I]trastuzumab binds to HER2, it is possible that the EGF:EGFR complex can dimerize with and internalize unoccupied HER2 receptors. This would reduce levels of avail-able HER2 receptors and therefore reduce the amount of internalized [125I]trastuzumab (see Figure 7).

Figure 7. EGF binds to EGFR and heterodimerizes and internalizes unoccupied HER2 receptors (downregulation of HER2) before [125I]trastuzumab can bind to these receptors.

When EGF was incubated for longer times (>10 h), a shift could be detected where the 125I uptake increased instead of being decreased, compared with untreated cells. One theory is that cell growth influenced the uptake. A cell number reduction could be seen in A431 cells treated with EGF concentra-tions in the range of 10-1000 ng/ml (see Figure 8a). This EGF-dependent A431 cell reduction has previously been reported in A431 cells [106, 107]. A connection has also been found with EGF sensitivity and cells that co-express high levels of both EGFR and HER2 [108], A431 cells express ~1.5×106 EGFR and ~1.5×105 HER2 receptors. When the cell number was plotted against the trastuzumab mediated 125I uptake it was found that the

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decrease in cell number correlated with the increase in uptake (see Figure 8b).

Figure 8. (a) Cell numbers after 24 hours of exposure to EGF and/or trastuzumab (mean values and standard deviations of six measurements). (b) Uptake quotients plotted as a function of the cell number quotients. Quotients >1 indicate an increased 125I uptake; <1 denotes reduced 125I uptake when EGF was present (y-axis). Cell number quotiens >1 indicate an increased number of cells, while <1 denotes a re-duced cell number when cells were exposed to EGF (x-axis).

If the synthesis of new HER2 receptors is nearly constant, while the cell divisions are fewer due to the EGF exposure, the number of HER2 receptors per cell will, on average, increase (see Figure 9).

Figure 9. A431 cells treated with and without EGF. If fewer cell divisions take place the amount of HER2 receptors per cell will on average increase. If then [125I]trastuzumab is added, the 125I uptake per cell will increase.

The experiment was repeated using different concentrations of [125I]trastuzumab. The data indicated that higher concentrations of [125I]trastuzumab gave a less pronounced initial decrease in 125I uptake, while the second phenomenon, giving increased uptake, became more dominant earlier.

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Selection of EGFR-targeting affibody molecules (II)

Selection and affinity studies Selection and characterization of affibody molecules targeting the EGFR was performed in paper II. From the 58-residue, protein A-derived library, nine affibody molecules were produced and further characterized. Of those nine candidates, three affibody molecules ZEGFR:942, ZEGFR:948 and ZEGFR:955 were selected, based on the binding data from the real-time BIA analysis using a Biacore biosensor instrument. To improve the affinity of the three selected affibody molecules, head to tail dimers were generated. The dimeric affibody molecules were found to bind with an affinity in the range of ~25–50 nM in the biosensor analysis and ~1–3 nM in the cellular study (see Table 1).

Table 1. Dimeric affibody molecules tested both for binding to the extracellular domain (ECD) of EGFR immobilized to a Biacore sensorchip, and in saturation curve analyses in A431 cells Dimer affibody

KDa (nM)

Biosensor Ka

b (M-1s-1) Biosensor

Kdc (s-1)

Biosensor KD

a (nM) Cellular study

(ZEGFR:942)2 ~25 ~6.0 × 105 ~1.6 × 10-2 ~1 (ZEGFR:948)2 ~45 ~1.9 × 105 ~8.1 × 10-3 ~3 (ZEGFR:955)2 ~50 ~4.8 × 104 ~2.4 × 10-3 ~1 aDissociation equilibrium constant bAssociation rate constant cDissociation rate constant

The biosensor study resulted in a better affinity for the (ZEGFR:942)2 than for the other two affibody molecules, whereas in the cellular studies the (ZEGFR:955)2 appeared to be an equally good binder. The better performance of (ZEGFR:955)2 in the cell assays might have been due to the ~10-fold slower off-rate that was observed in the Biacore analysis.

In the cellular saturation study, the number of binding-sites for the three affibody molecules was obtained in A431 cells and compared with the num-ber of binding-sites for EGF in the same cell-line (data not shown). Interest-ingly it was found that the affibody molecules bound to only about one-third of the binding-sites, compared with EGF. Similar results were previously found when the binding-sites for [125I]cetuximab were compared with EGF in A549 lung adenocarcinoma cells [109]. This difference could be ex-plained by the fact that as both (ZEGFR:955)2 and cetuximab have a bivalent nature, it is possible that one molecule could bind two receptors.

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Binding studies Binding specificity to native EGFR was studied with iodinated affibody molecules. The binding of [125I](ZEGFR:942)2, [125I](ZEGFR:948)2 and [125I](ZEGFR:955)2 could be effectively blocked by adding an excess of the same non-radiolabelled affibody molecule, indicating a specific binding to native EGFR (see Figure 10, A2, B2 and C2). Furthermore, the three EGFR-binding affibody molecules were found to bind EGFR in a competitive man-ner, as they inhibited each other’s binding (see Figure 10, A3–4, B3–4 and C3–4). This cellular binding assay suggested that they have the same (or at least compete for the same), epitope. Moreover, since a significant attempt was made to apply the same amount of affibody molecule, the results would suggest that (ZEGFR:942)2 had the lowest, (ZEGFR:948)2 intermediate and (ZEGFR:955)2 the highest binding to cells (see Figure 10, A1, B1 and C1). Flow cytometric analysis confirmed this ranking in binding performance (data not shown).

Figure 10. Binding and specificity of the three EGFR-binding affibody molecules towards A431 cells. (A1) = [125I](ZEGFR:942)2, (B1) = [125I](ZEGFR:948)2 and (C1) = [125I](ZEGFR:955)2. The specificity was tested by simultaneously adding an excess (~500:1) of unradiolabelled affibody molecule together with the same 125I-labelled affibody molecule: (A2) = (ZEGFR:942)2 + [125I](ZEGFR:942)2, (B2) = (ZEGFR:948)2 + [125I](ZEGFR:948)2 and (C2) = (ZEGFR:955)2 + [125I](ZEGFR:955)2. In order to establish whether the affibody molecules had the same binding-site, an excess (~500:1) of unradiolabelled affibody molecule was incubated with each 125I-labelled affibody molecule: (A3) = (ZEGFR:948)2 + [125I](ZEGFR:942)2, (A4) = (ZEGFR:955)2 + [125I](ZEGFR:942)2, (B3) = (ZEGFR:942)2 + [125I](ZEGFR:948)2, (B4) = (ZEGFR:955)2 + [125I](ZEGFR:948)2, (C3) = (ZEGFR:942)2 + [125I](ZEGFR:955)2 and (C4) = (ZEGFR:948)2 + [125I](ZEGFR:955)2. The samples were analysed in a gamma counter and counts per minute (CPM) for 105 cells are shown (mean values and standard deviations from three values).

The cellular studies indicated that (ZEGFR:955)2 was a slightly stronger binder than (ZEGFR:942)2 and (ZEGFR:948)2 and was selected for further characterization.

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Characterization of (ZEGFR:955)2 (III & V)

Internalization The internalization pattern was studied in A431 cells, using confocal micros-copy (see Figure 11). After brief incubation (5 minutes), the green fluores-cence was mainly membrane associated for (ZEGFR:955)2 and cetuximab, while after longer incubation time (2 hours), the fluorescence was more in the cy-toplasm, indicating internalization. In the case of EGF, internalization seemed to be initiated after only 5 minutes. A difference in granularity could also be seen, EGF had a more granular fluorescence pattern in the cytoplasm after 2 hours of incubation, compared with (ZEGFR:955)2 and cetuximab.

Figure 11. Cell binding and internalization of oregon green 488 labelled (ZEGFR:955)2, EGF and cetuximab in A431 cells, using confocal microscopy.

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One theory is that the difference in the granular fluorescence patterns is a result of differences in EGFR trafficking. It is known that EGFRs have a rapidly EGF induced internalization and are mostly degraded in the ly-sosomes [60]. Cetuximab, in contrast, has a relatively slow internalization rate and may be recycled to the cell surface. The EGF-EGFR internalization is also dependent on receptor tyrosine kinase activity, which cetuximab seems not to be. However, cetuximab can cause a reduction in surface EGFR and appears not always to be passively accompanying the receptor. It has also been studied if the recycling pattern is due to pH-sensitive ligand disso-ciation in the acidic environment of early endosomes. This seems not to be the case for cetuximab, whose binding appears to be more resistant to low pH than that’s of EGF [109]. It is possible that neither (ZEGFR:955)2 nor cetuximab takes the same intracellular route as EGF. Further investigation is needed to understand the cellular routing of these different EGFR targeting agents.

Retention The cellular retention of 125I after delivery as [125I](ZEGFR:955)2, [125I]EGF and [125I]cetuximab was studied at various times (see Figure 12). The remaining cell-associated radioactivity after 48 hours of incubation was ~20% for [125I](ZEGFR:955)2 and ~25% for [125I]cetuximab, while almost all cell-associated radioactivity had disappeared after 24 hours of incubation with [125I]EGF.

Figure 12. Cellular retention of 125I delivered [125I](ZEGFR:955)2, [125I]EGF or [125I]cetuximab (men values and standard deviations).

Instead of being excreted out from the cell, like 125I delivered by EGF, it appeared that a fraction of the 125I delivered by the affibody molecules was still associated to the cells for at least 48 hours. These differences in reten-tion of the radioactivity are difficult to explain but are probably a result of different intracellular compartmentalization of the targeting substances.

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Binding studies The binding position of [125I](ZEGFR:955)2 on EGFR was investigated by study-ing whether an excess of EGF or cetuximab could disturb the binding of the affibody molecule. Binding of [125I](ZEGFR:955)2 was effectively blocked by an excess of EGF or cetuximab. Binding of [125I]EGF was also successfully blocked by an excess of cetuximab. However, an excess (ZEGFR:955)2 could not completely block the binding of [125I]EGF, as ~10% could still bind. The binding of [125I]cetuximab could be only partly blocked when an excess of EGF was added, ~15% of the binding remaining. The affibody molecule was even less effective in blocking [125I]cetuximab, as ~40% of the binding re-mained (see Figure 13).

Figure 13. To test if the three different agents competed for the same binding-site, A431 cells were incubated with, simultaneously, an excess (~500:1) of unradio-labelled molecule together with another 125I-labelled molecule: (A) [125I](ZEGFR:955)2 (white), [125I](ZEGFR:955)2 + EGF (black) and [125I](ZEGFR:955)2 + cetuximab (light grey). (B) [125I]EGF (white), [125I]EGF + (ZEGFR:955)2 (dark grey) and [125I]EGF + cetuximab (light grey). (C) [125I]cetuximab (white), [125I]cetuximab + (ZEGFR:955)2 (dark grey) and [125I]cetuximab + EGF (black).

One hypothesis is that affinity is an important factor. Cetuximab has a higher affinity than EGF and (ZEGFR:955)2 which could explain the better blocking ability of cetuximab. Another explanation is based on considerations about the degree of overlap in the binding surfaces of the three substances. It is possible that, due to its extensive binding surface and the more surface local-ized binding [15], cetuximab can at least to some degree, bind to EGFR in spite of the possible presence of (ZEGFR:955)2 or EGF in the same domain.

The results of the blocking experiments give a hint about where on EGFR the new affibody molecule binds. The EGF ligand is known to bind to both domains, I and III [43, 110] and the binding localization for cetuximab is domain III [15]. In view of above information it seems likely that the bind-

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ing site of the new affibody molecule (ZEGFR:955)2 is on domain III. Figure 14 gives a schematic illustration of the assumed binding patterns.

Figure 14. Schematic illustration of the assumed binding patterns of (ZEGFR:955)2, EGF and cetuximab to EGFR. The extracellular domains of EGFR are marked I-IV and the transmembrane region is marked TM. (A) The “closed” configuration of EGFR without ligand. (B) The “open” configuration of EGFR when EGF (marked E) is bound to domains I and III. (C) An excess of non-radioactive cetuximab inhib-its binding of both [125I](ZEGFR:955)2 (marked [125I]Z) and [125I]EGF (marked [125I]E) due to binding of EGFR to a large surface of domain III. (D) An excess of non-radioactive (ZEGFR:955)2 (marked Z) cannot completely inhibit binding of [125I]cetuximab, probably due to blocking of only a part of the large cetuximab bind-ing surface at domain III. (E) Nor could an excess of non-radioactive EGF com-pletely block the binding of [125I]cetuximab, also probably due to the large cetuxi-mab binding surface at domain III.

Signalling studies Cells were treated with EGF, (ZEGFR:955)2 or cetuximab for different periods of time (5–360 min). As expected, EGFR showed strong autophosphoryla-tion after only 5 minutes in response to EGF in both studied cell-lines, A431 and U343. However, when cells were incubated with the affibody molecule (ZEGFR:955)2, no activation could be detected whereas cetuximab treatment seemed to induce weak EGFR autophosphorylation. The level of pEGFR expression can however be questioned in the cetuximab case, as the total EGFR levels were consistently lower in the samples not treated with cetuxi-mab. The variation in the total EGFR levels is difficult to explain; perhaps

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EGFR bound to cetuximab is of some advantage in the cetrifugation steps. Thus, even though the three substances share the same binding-site, they affected the receptor autophosphorylation in different ways (see Figure 15).

Figure 15. Phosphorylated and total EGFR in (a) A431 and (b) U343 cells. Imuno-precipitation with an anti-EGFR antibody was followed by immunoblotting with antibodies specific for phosphotyrosine or total EGFR. Untreated cells were used as negative controls (-) and cells incubated with EGFR for 15 minutes as positive con-trols (+).

Next we investigated what effects EGF, (ZEGFR:955)2 and cetuximab had on activation of the intracellular signalling proteins Erk and Akt. Phosphoryla-tion of Erk was induced by all substances in both U343 and A431 cells, as seen in Figure 16. The increase in Erk phosphorylation was however more transient in A431 than U343 cells.

Figure 16. Expression of phosphorylated Erk1/2 and total Erk was analysed in cells treated with EGF, (ZEGFR:955)2, or cetuximab in (a) A431 and (b) U343 cells. Un-treated cells were used as negative controls (-) and cells incubated with EGF for 15 minutes as positive controls (+).

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The phosphorylation status of the anti-apoptotic kinase Akt was also ana-lysed. In U343 cells it was found that Akt was constitutively phosphorylated and no definite changes were observed after treatment with either EGF, cetuximab or (ZEGFR:955)2 and about the same levels were also seen in the positive and negative controls. By contrast, Akt was not phosphorylated in the absence of stimulus in A431 cells. Phosphorylation was induced by treatment with EGF, (ZEGFR:955)2 and cetuximab (see Figure 17). Note that both Erk and Akt were phosphorylated by (ZEGFR:955)2, even though no auto-phosphorylation of the EGFR could be detected.

Figure 17. Expression of phosphorylated Akt and total Akt was analysed in cells treated with EGF, (ZEGFR:955)2 or cetuximab in (a) A431 and (b) U343 cells. Un-treated cells were used as negative controls (-) and cells incubated with EGF for 15 minutes as positive controls (+).

Activation of the downstream signalling molecules Erk1/2 and Akt was sur-prising, considering that (ZEGFR:955)2 was unable to induce EGFR activation. This paradox may be resolved by assuming that very low levels of phos-phorylated EGFR are sufficient to activate Erk and Akt. Another explanation could be that (ZEGFR:955)2 binds to EGFR in such a way that EGFR integrates with the other receptors in the same family or ‘crosstalk’ with other signal-ling pathways [9]. A third, but less likely possibility is that the affibody molecule induces EGFR autophosphorylation that cannot be detected by the PY99 antibody.

Our results suggest that the signalling pattern induced by (ZEGFR:955)2 is fairly similar to that induced by cetuximab. This makes the affibody mole-cule an interesting candidate for EGFR targeting therapy. However, more research is needed to elucidate the mechanism behind the affibody molecule induced Erk and Akt activation without detectable EGFR autophosphoryla-tion.

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In vivo uptake of 111In delivered with (ZEGFR:955)2, (IV) Since the (ZEGFR:955)2 affibody molecule demonstrates good in vitro proper-ties it was now interesting to investigate the features in vivo. Small mole-cules often have rapid blood clearance, which makes affibody molecules interesting for detection of EGFR-expressing primary tumours and corre-sponding metastases, at least outside the liver.

Biodistribution in tumour-bearing mice To analyse the uptake in tumours and in vital organs, a biodistribution study was made (see Figure 18). The binding of [111In](ZERFR:955)2 in tumours was EGFR-specific, which was tested with the irrelevant affibody molecule [111In](Zabeta3-C28S)2. The tumour uptake of 111In mediated by [111In](ZEGFR:955)2 was ~3.8%ID/g 4 hours post-injection. The radiotracer showed rapid blood clearance, with a concentration less than 0.4 ± 0.1%ID/g in the circulation after 4 hours at which time the tumour to blood ratio was 9:1.

Figure 18. Biodistribution of 111In, delivered as [111In](ZEGFR:955)2, expressed as per-cent injected dose per gram tissue in tumour (A431 cells) bearing nude mice, 4 and 8 hours after injection. The control mice injected with the irrelevant affibody molecule [111In](Zabeta3-C28S)2 were analysed 4 hours after injection (mean values and standard deviations).

The 111In uptake in the kidneys and liver was high when delivered with [111In](ZERFR:955)2. The liver uptake could be blocked by administering an excess of (ZEGFR:955)2, while this was not the case for kidney uptake. The kidney uptake of 111In was also high for the irrelevant affibody molecule (see Table 2).

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Table 2. Uptake in liver and kidneys, 4 h post-injection, of 111In delivered as [111In](ZEGFR:955)2 with or without blocking amounts of non-labelled (ZEGFR:955)2 Data are also given for the control affibody molecule [111In](Zabeta-c28s)2

Substance Liver uptake (%ID/g tissue)

Kidney uptake (%ID/g tissue)

[111In](ZEGFR:955)2 16.5 ± 2.2 86.4 ± 14.4 [111In](ZEGFR:955)2 + (ZEGFR:955)2 1.8 ± 0.1 165.2 ± 28.3 [111In](Zabeta3-C28S)2 0.45 ± 0.0 153.9 ± 15.2

A high kidney uptake is probably a consequence of kidney clearance and tubular reabsorption which is common for small proteins [6]. The high liver uptake is most likely due to the normal expression of EGFR in liver tissue [111] and cross-reactivity of [111In](ZEGFR:955)2 with murine EGFR. This is supported by the fact that the liver uptake could be blocked by a factor of ten by administering of an excess of (ZEGFR:955)2, and that the non-specific affi-body molecule did not accumulate in the liver. Uptake in the kidneys could not be inhibited in this way. Note that the kidney uptake of 111In increased when the liver uptake was blocked.

Gamma camera imaging The tumours in the right foreleg of the mice were clearly visualized in a gamma camera, even though they were quite close to the kidneys and liver, which also gave strong signals. An excess amount of (ZEGFR:955)2 could block the [111In](ZEGFR:955)2 mediated gamma camera signal in tumour, which sup-ported the biodistribution results regarding receptor specificity (see Figure 19).

In conclusion, the results presented in this work show that [111In](ZEGFR:955)2 is a promising candidate for imaging of EGFR expression in tumours and metastases. It could potentially also be used to identify patients with tumours that are suitable for treatment with EGFR-targeting agents, e.g. treatment of non-small cell lung cancer with gefitinib and for follow-up of such therapy to ascertain if receptors are up- or down-regulated. (ZEGFR:955)2 could also be of therapeutic interest; however due to the high liver and kid-ney uptake it might only be of use by locoregional administration, e.g. for intravesical treatment of urinary bladder carcinoma [112], intracavitary treatment of gliomas [113] or intraperitoneal treatment of peritoneal carci-nomatosis [114].

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Figure 19. Gamma camera image after injection of [111In](ZEGFR:955)2 in tumour-bearing mice. The image was taken 4 hours after injection. The tumour areas are marked T. The receptors in the mouse to the right were blocked with an excess of (ZEGFR:955)2.

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Summary

In the first part of this thesis, studies were undertaken to improve our under-standing of the EGFR system and thereby modulate the uptake of radionu-clides in tumour cells. In the second part a new EGFR targeting affibody molecule (ZEGFR:955)2 was selected and characterized, EGF and cetuximab were used as reference molecules in some of these studies. Some conclu-sions that can be drawn from these studies are listed below: � EGF could modify the uptake of 125I when delivered as [125I]trastuzumab

in A431 cells. � The affibody molecule (ZEGFR:955)2 binds specifically to EGFR with an

affinity of ~50 nM in biosensor binding studies and ~1 nM in cellular saturation studies.

� Fluorescence-labelled (ZEGFR:955)2 seemed to be internalized into A431 cells.

� Retention studies using [125I](ZEGFR:955)2 showed that a fraction (20–25%) of the 125I delivered with the (ZEGFR:955)2 still was associated with A431 cells for at least 48 h.

� Cellular blocking studies indicated that the three substances (ZEGFR:955)2, EGF and cetuximab competed for the same binding site on domain III of EGFR.

� In signalling studies, no auto-phosphorylation of EGFR could be detected when A431 and U343 cells were treated with (ZEGFR:955)2. EGF gave a strong phosphorylation of EGFR, while cetuximab seemed to give a weak phosphorylation.

� Phosphorylation of Erk was induced by (ZEGFR:955)2, EGF and cetuximab in both U343 and A431 cells.

� Akt was constitutively phosphorylated in U343 cells. � Phosphorylation of Akt was induced by treatment with (ZEGFR:955)2, EGF

and cetuximab in A431 cells. � Retention studies on [111In](ZEGFR:955)2 demonstrated that a fraction

(~20%) of 111In delivered with the (ZEGFR:955)2 was still associated to the cells after 48 hours in A431 cells.

� (ZEGFR:955)2 binds specifically to tumour cells in vivo and could be visual-ized with a gamma camera.

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� The biodistribution study revealed a tumour-specific 111In uptake of ~3.8% injected dose per gram tissue, 4 hours post-injection, at which time the tumour to blood ratio was 9:1.

The results reported in paper I suggest that one can modify the uptake of radionuclides in tumour cells by applying a ligand to a receptor other than the one that was targeted. This may at least be the case when heterodimer receptors can form. It also indicates that it can be more effective to target two receptors than one. In papers II-V we evaluated the potential for the EGFR-targeting affibody molecule (ZEGFR:955)2 to be a candidate for imaging and therapy applications. The results showed that [111In](ZEGFR:955)2 is prom-ising for EGFR targeted imaging and can potentially also be used to identify patients with tumours suitable for EGFR-targeting and for follow-up of such therapy to ascertain if receptors are up- or down-regulated. Our results also suggest that the signalling pattern induced by (ZEGFR:955)2 is similar to that induced by cetuximab. This makes the affibody molecule also a potentially interesting candidate for EGFR-targeted therapy.

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Future studies

In paper I, we reported that it was possible to modulate the cellular uptake of [125I]trastuzumab by adding EGF. This study needs to be expanded by using additional cell-lines and possibly also transfect cells to express different levels of EGFR and HER2 receptors. This will allow more detailed investi-gation of the possibility to modify radionuclide uptake.

Another phenomenon also found in paper I was that the affibody mole-cule and EGF seemed to have different numbers of binding-sites. This phe-nomenon should be further investigated to ascertain if it is possible for the dimeric affibody molecule to bind two EGFR at the same time. This type of study would also be of value for characterization of bi-specific affibody molecules. A bi-specific affibody molecule directed against both EGFRs and HER2 has recently been developed by my colleague Mikaela Friedman (per-sonal communication).

In paper III, we made some internalization studies which indicated that (ZEGFR:955)2, EGF and cetuximab could have different intracellular routing patterns. It is believed that the slow internalization rate of HER2-containing heterodimers result in a more potent signalling. If we can increase our under-standing of cellular routing patterns it ought to be possible to change it and in that way hopefully to modulate the signalling abilities of the receptors.

When the results from papers II, III and V where summarized we found that the affibody molecule (ZEGFR:955)2 and cetuximab had several characteris-tics in common. It would therefore be interesting to study if (ZEGFR:955)2 also has the same radiation sensitizing capacity as cetuximab have.

We should also extend our signalling studies. The fact that (ZEGFR:955)2 did not activate the autophosphorylation of EGFR but could activate the down-stream signalling pathways Erk and Akt is remarkable. It would therefore be of interest to ascertain if other receptors are involved and via interaction or dimerization with EGFR, can initiate such activation. The study should pref-erably also be expanded with more cell-lines expressing different numbers of receptors. It would also be of interest to apply TKIs such as gefitinib and lapatinib to see how these influence the affibody induced signalling patterns.

The field of using affibody molecules in EGFR family tumour targeting is exciting and new interesting affibody molecules, both monofunctional and bifunctional, are already in progress. If possible, these candidates will be included for further studies.

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Acknowledgments

Detta arbete har utförts på avdelningen för Biomedicinsk Strålningsveten-skap vid Uppsala Universitet. Mitt arbete har finansierats av cancerfonden och Vinnova. Det är många människor som har gjort den här avhandlingen möjlig och jag vill speciellt tacka: Min handledare Jörgen Carlsson för att du gav mig chansen och för att du alltid har uppmuntrat och trott på mig, för alla gånger du lagt allt annat åt sidan för att diskutera märkliga fenomen eller på annat sätt ägnat värdefullt tid för min skull.

Min andra handledare Bengt Glimelius för att du har gjort det här arbetet möjligt och för ditt breda kunnande och intressanta idéer som du delat med dig utav. Mina biträdande handledare: Lars Gedda för att du alltid ställt upp när jag stött på problem och för all hjälp med avhandlingen. Bo Stenerlöv för intres-santa diskussioner och att du med ditt glada humör höjer stämningen på av-delningen. Vladimir Tolmachev för att du ställt upp med din expertis inom radiokemin. Hans Lundqvist för din värdefulla kompetens. Alla medförfattare från: KTH – Mikaela Friedman, utan dig så hade jag inte haft någon affibody molekyl att karakterisera, tack för ett jättebra samarbete! Stefan Ståhl för all din positiva energi som du delat med dig av på våra möten. Hjalmar Brismar för all hjälp med konfokal och internaliseringsstudierna. Affibody AB – Lovisa Göstring för de snygga konfokalbilderna som du gjorde. Ingmarie Höidén-Guthenberg för all hjälp och goda råd! Fredrik Nilsson för ditt engagemang och brinnande intresse! Fox Chase Cancer Center – Gregory Adams for supply of the extracellular domain of the EGFR and for all help and positive feedback! Anna Orlova som lärt mig allt jag kan om möss, utan dig hade det inte blivit någon djurstudie. Johan Lennartson för alla värdefulla idéer som du bidragit med i signale-ringsstudien!

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Veronika Asplund för hjälp med det mesta på labbet och att du alltid finns där när någon panikartad situation dyker upp. Maria Östh-Eklind för all administrativ hjälp. Jan Gravé för att du ställt upp med ditt breda kunnande av allt som rör kon-fokalen och FACSen. Mattias Sandström för hjälp med gammakameran. Mina nuvarande rumskompisar – Marika för både hårt arbetande stunder i Gryt och alla skratt som du framkallat under åren. Ylva för alla tips jag fått med skrivandet av avhandlingen och allt kul vi haft! Lina för att du introdu-cerat mig i signaleringsvärldens opålitliga men intressanta värld! Mina gamla rumskompisar – Thuy som alltid ställt upp när jag behövt hjälp och för alla intressanta diskussioner på rummet. Shirin för alla tankar och din underbara humor som du så osjälviskt delar med dig av! Mikael för all hjälp, (speciellt med datorn och nej jag får nog erkänna att det tyvärr inte alltid var datorns fel) och för att du ”överraskat” mig så många gånger, någon dag ska du nog få igen, mohahahahaa som Shirin skulle ha sagt. Anna för att du stått ut med all min städnoja och för all hjälp jag fått under åren! Alla andra gamla och nya BMSare – Ann-Charlott för att du tog dig an mig när jag var ny och introducerade mig för alla traditioner/fester i Uppsala och för alla trevliga stunder under våra fjällvandringar. Åsa för all din uppmunt-ran och goda humör, du skrev i din avhandling som du gav till mig att man blir färdig snabbare än man tror, ville bara säga att du hade rätt, tiden går fort när man har roligt! Karin för allt kul vi haft, jag kommer aldrig att glömma ”the bula”, gör aldrig om det där! Amelie för all hjälp och din sprudlande livsenergi som att du delar med dig utav. Irina för alla lugnande ord inför disputationen. Kalle för att du fanns där jag var lite ur balans och påminde mig om de vitala sakerna i livet, mat och sömn. Per för att du visade vad underbart det kan vara att vandra i fjällen, Norge ska inte få skrämma bort mig! Ulrika och Henrik B för alla trevliga och ibland udda diskussioner kring fikabordet. Sen vill jag även tacka det nya gänget på BMS, Sara H för att du kämpat med mina cellförsök när jag haft det lite stressigt på slutet, Hanna, Sara A, Helena, Camilla, Ann-Sofie och John som jag önskar att jag kommer att lära känna lite bättre för ni rookisar verkar vara ett bra gäng! Min högstadielärare Torbjörn Enetjärn för att du introducerade mig för de naturvetenskapliga ämnena och genom det fick mig att välja rätt gymnasie-inriktning. Utan dig hade jag kanske inte alls vågat satsa och varit där jag är i dag. Tack!

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Dorthy och Kaj för att ni alltid är så måna om mig och för alla fina kort som ni har skickat under åren som jag har varit så dålig på att tacka för! Barbro och Lasse för att ni härdade ut med mig och alla mina påhitt när jag var liten och för allt kul ni hittade på! Mina vänner – Lena för att du var den bästa bästisen när vi växte upp. Gäng-et från gymnasiet, Siri, Fia och Matilda för att ni gjorde gymnasietiden till en härlig tid. Marie för att du är en grym kompis, jag kommer alltid att ha ett leende på läpparna när jag tänker tillbaka på Umeå tiden. E-type rocks! Sist av allt vill jag tacka min underbara, mest fantastiska superfamilj: Mamma och Pappa för allt stöd som ni gav mig under min studietid och att jag fått växa upp i en miljö med varma bullar och saft, massor av uppmärk-samhet och oändligt med kärlek. Mina syskon för att ni alltid har tagit hand om mig och alltid uppmuntrat mig när jag behöver det som bäst. Ni är de bästa förebilderna en lillasyster kan ha, jag älskar er oändligt mycket! Mina syskonbarn Oscar, David, Frida och Jesper för att ni ger mig så mycket skoj och värme. Erik, mitt livs kärlek. För att du är så underbar och stått ut med mig den här tiden när jag ibland har varit mindre lugn och harmonisk. Tack för att du alltid finns där och gör mig så lycklig!

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 350

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A doctoral dissertation from the Faculty of Medicine, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofMedicine. (Prior to January, 2005, the series was publishedunder the title “Comprehensive Summaries of UppsalaDissertations from the Faculty of Medicine”.)

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