Targeted Therapy of Colorectal Cancer - Simple...

ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2008

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 336

Targeted Therapy of ColorectalCancer

Preclinical Evaluation of a Radiolabelled Antibody

YLVA ALMQVIST

ISSN 1651-6206ISBN 978-91-554-7171-2urn:nbn:se:uu:diva-8657

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List of papers

This thesis is based on the following papers, which will be denoted by their roman numerals when referred to in the text.

I Almqvist Y, Orlova A, Sjöström A, Jensen HJ, Lundqvist H,

Sundin A, and Tolmachev V: In Vitro Characterization of 211At-labeled Antibody A33 – a Potential Therapeutic Agent Against Metastatic Colorectal Carcinoma. Cancer Biotherapy and Radiopharmaceuticals 2005, 20(5):514-23.

II Almqvist Y, Steffen AC, Lundqvist H, Jensen H, Tolmachev V,

and Sundin A: Biodistribution of 211At-labeled Humanized Anti-body A33. Cancer Biotherapy and Radiopharmaceuticals 2007, 22(4):480-7.

III Almqvist Y, Steffen AC, Tolmachev V, Divgi CR, and

Sundin A: In vitro and in vivo characterization of 177Lu-huA33: A radioimmunoconjugate against colorectal cancer. Nuclear Medicine and Biology 2006, 33(8):991-8.

IV Almqvist Y, Tolmachev V, Lundqvist H, and Sundin A: Therapy

of colorectal cancer xenografts in nude mice using 177Lu labelled humanized monoclonal antibody A33. Manuscript

Reprints were made with permission from Mary Ann Liebert Inc. (I and II), and Elsevier Inc. (III).

Contents

1. Introduction.................................................................................................9 1.1 Cancer...................................................................................................9

1.1.1 Colorectal cancer ..........................................................................9 1.1.2 Current treatments ......................................................................10

1.2 Tumour targeting................................................................................10 1.2.1 Targets ........................................................................................10 1.2.2 Targeting agents..........................................................................11 1.2.3 Effector substances .....................................................................15 1.2.4 Radioimmunotherapy .................................................................15 1.2.5 Radioimmunodiagnostics ...........................................................15

1.3 Radionuclides and radioactive decay .................................................16 1.3.1 Radioactive decay.......................................................................16 1.3.2 Radionuclides for therapy...........................................................17 1.3.3 Radionuclides for diagnostics.....................................................18

1.4 Labelling.............................................................................................19 1.4.1 Radiohalogens.............................................................................19 1.4.2 Radiometals ................................................................................19

2. Aim ...........................................................................................................21

3. The present study ......................................................................................22 3.1 211At-labelled huMAb A33 (papers I and II) ......................................22

3.1.1 Binding and retention studies (I) ................................................22 3.1.2 Therapy in vitro (I) .....................................................................24 3.1.3 Biodistribution experiments (II) .................................................25 3.1.4 Comments (I and II)....................................................................28

3.2 177Lu-labelled huMAb A33 (papers III and IV) .................................29 3.2.1 In vitro experiments (III) ............................................................30 3.2.2 Biodistribution and dosimetry (III and IV).................................30 3.2.3 Imaging (IV) ...............................................................................33 3.2.4 Experimental therapy (IV) ..........................................................34 3.2.5 Biodistribution of 177Lu-cU36 (control experiment)...................35 3.2.6 Comments (III and IV) ...............................................................36

4. Summary...................................................................................................39

5. Future studies ............................................................................................41 5.1 Alpha particle therapy. .......................................................................41 5.2 Cellular mechanisms ..........................................................................41 5.3 A33 antibody fragments .....................................................................42 5.4 Experimental 177Lu therapy ................................................................42

6. Sammanfattning på svenska (Summary in Swedish) ................................43 6.1 Mål och bakgrund...............................................................................43 6.2 Astatexperiment .................................................................................43 6.3 Lutetiumexperiment ...........................................................................44 6.4 Slutsatser ............................................................................................45

Acknowledgements.......................................................................................46

References.....................................................................................................48

Abbreviations

177Lu-huA33 The huMAb A33 labelled with 177Lu via the chelator CHX-A’’-DTPA

125I-huA33 The huMAb A33 labelled with 125I via the linker SPMB

211At-huA33 The huMAb A33 labelled with 211At via the linker SPMB

125I-mA33 The mMAb A33 labelled with 125I 177Lu-cU36 The cMAb U36 labelled with 177Lu via the chelator

CHX-A’’-DTPA ADCC Antibody-dependent cell-mediated cytotoxicity ATP Adenosine triphosphate CAT Chloramine-T CDR Complementarity determining region CHX-A’’-DTPA [(R)-2-Amino-3-(4-isothiocyanatophenyl)propyl]-

trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid cMAb Chimeric monoclonal antibody cU36 cMAb U36 Da Dalton DNA Deoxyribonucleic acid DPC Decays per cell EGF Epidermal growth factor EGFR Epidermal growth factor receptor Fab Antigen-binding fragment FAP Familial adenomatous polyposis Fv Variable fragment HAHA Human anti-human antibody HAMA Human anti-mouse antibody HMW High molecular weight huA33 huMAb A33 huMAb Humanized Mab IgG1 Immunoglobulin, class G1 i.v. Intravenously Kd Equilibrium dissociation constant LET Linear energy transfer LMW Low molecular weight MAb Monoclonal antibody

mMAb Murine monoclonal antibody PET Positron emission tomography p.i. Post injection RID Radioimmunodiagnostics RIT Radioimmunotherapy s.c. Subcutaneously scFv Single-chain Fv SD Standard deviation SPMB N-succinimidyl-para-(tri-methylstannyl)benzoate

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1. Introduction

1.1 Cancer Cancer is a major health problem in the western world today. An increasing number of people are diagnosed with cancer each year, and as the mean age of the population continues to increase, there is no reason to expect a decrease in cancer incidence in the near future. Even taking the continuous ageing of the Swedish population into account, there is still an increase in the number of diagnosed cases each year [1]. Even though the survival rate has improved continuously since the 1960s [2], approximately 27 % of all deaths in Sweden are caused by cancer [1].

1.1.1 Colorectal cancer In terms of incidence, colorectal cancer is the third most common type of cancer in the world [3], and it is accountable for about 10 % of cancer deaths in Europe [4] and the USA [5]. Risk factors associated with this disease include hereditary factors, as well as those connected to life-style, such as low levels of exercise, and dietary factors. A high risk diet involves a high intake of alcohol and red meat, and a low intake of vegetables. One of the inherited genetic disorders that lead to colorectal cancer is familial adenomatous polyposis (FAP) [6]. Though FAP is the cause in only a small number of colorectal cancer cases (approximately 1 %), patients with this genetic disposition have a risk of developing colorectal cancer that is almost 100 %. Most colorectal cancers arise from existing adenomas (90 % of colorectal cancers are adenocarcinomas), and FAP patients develop a very large number of adenomatous polyps, sometimes thousands. Since it has been estimated that about 5 % of these polyps turn malignant, the risk of developing cancer dramatically increases with a large increase in adenoma-tous polyp incidence [7].

Colorectal cancer often involves metastatic disease, and at the time of diagnosis many patients have already developed metastases [8]. The liver is the most common site for distant colorectal metastases, another common site being the lung [9]. Usually, the liver is the first site for these recurrences to occur in, and without hepatic metastases present, there is pulmonary involvement in less than 5 % of cases [9, 10]. The frequency of metastatic disease remains a challenge for successful treatment of colorectal cancer.

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1.1.2 Current treatments The only curative treatment for colorectal cancer is surgery, but metastatic spread hampers successful treatment outcome. After resection of the primary tumour, recurrences often appear, both locally and in the form of distant metastases. [9]. About one third of patients treated surgically with curative intent develop recurrences, and the 5-year survival is approximately 65 % [9, 11]. Different strategies have been deployed to reduce the recurrence rate after tumour resection, and/or to increase patient survival. When the disease is found to be at an advanced stage, chemotherapy is now routinely applied as adjuvant treatment [12, 13], and external radiotherapy can be applied for well-defined areas before surgery [14]. Regarding hepatic metastases, they may in some cases also be operable, but the rate of recurrence is high [15, 16]. Thermal ablative techniques, such as radiofre-quency ablation or microwave ablation, are possible methods for treatment of hepatic metastases, either with curative intent, or as palliative treat-ment [17]. There are also a number of catheter-delivered treatments that could be considered, e.g. trans-arterial embolisation, where the intention is to arrest the blood flow to the metastases [17]. Despite efforts to increase the survival of colorectal patients with metastatic disease, there are still a large number of patients who do not survive beyond 5 years [15].

1.2 Tumour targeting Tumour targeting is a concept where one specifically targets tumour tissue present in the body. This is accomplished by molecular recognition of structures that are present in tumours only, or, in reality, are expressed to a much higher extent in tumours than in normal tissue. To locate these structures in the body, a targeting agent, that recognises only the selected structure, is used. To the targeting agent, an effector substance can be coupled. The selection of effector depends on the desired effect, e.g. tumour treatment or visualisation.

1.2.1 Targets Ideally, one would like to identify pure tumour specific structures. In reality, this has not yet been the case. What have been identified, are structures that are overexpressed in tumours, but expressed to a lower extent in normal tissue. The normal tissue expression can also be restricted to a few organs. Structures overexpressed on tumour cells could be certain proteins that will give these cells some advantage. An example of this are growth factor receptors, that when activated could lead to an increase in proliferation and migration, as well as a reduced rate of apoptosis [18]. Structures with a

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restricted normal tissue expression could be necessary for survival and/or specific for the tissue in question [19]. A good targeting structure should have a high expression in tumours, be easily accessible for the targeting agent, and remain in tumour tissue for the duration of the treatment. Examples of targets overexpressed in colorectal cancers are the epidermal growth factor receptor (EGFR) [20], and various tumour associated antigens, such as the carcinoembryonic antigen (CEA), and the tumour-associated glycoprotein-72 (TAG-72) [21-23]. An example of a tissue-specific target for colorectal cancer treatment is the A33 antigen.

The A33 antigen The A33 antigen is a 43 kDa transmembrane glycoprotein that belongs to the immunoglobulin superfamily [24, 25]. The function of this protein is unknown, but it is believed to be involved in cell-cell interactions, due to its homology with junctional proteins, such as the coxsackie adenovirus receptor (CAR), and the junctional adhesion molecules (JAM) [26, 27]. The A33 antigen is expressed in more than 95 % of all colorectal carcinomas, and it is also expressed in many gastric, and pancreatic carcinomas [19, 28]. It is not secreted or shed into the circulation [29], and the normal tissue expression is very restricted [19]. These are favourable characteristics for an RIT target, since it will optimise tumour-to-normal tissue ratios. For metastatic treatment, a consistency in antigen expression is important. There should not be a lower expression of the antigen in metastases than in the primary tumour, a phenomenon that has been observed for certain antigens in certain tumours [30-32]. For the A33 antigen in colorectal cancers, no such loss of expression has been observed. It has the same level of expression in metastases as in the primary tumour [19]. The normal tissue expression of the A33 antigen is almost completely restricted to normal colonic mucosa, with some expression also in the mucosa of the small intestine, and in the salivary glands. The antigen has been reported to internalise [33, 34], something the results in paper I also point towards, but recently, one study has reported that the antigen is perhaps not internalised after all, at least not in all cell lines [35].

1.2.2 Targeting agents The targeting agents can be a wide variety of molecules. To target receptors, natural ligands may be used, e.g. EGF. These ligands may also be modified, or synthetic, such as the somatostatin analogues. Size matters, depending on the situation, different properties might be desirable. For therapy, for example, an intact antibody with a long circulation time in the body could be a good choice, whereas for imaging, a small peptide that is rapidly excreted from the body would be more appropriate.

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Small molecules A small molecule targeting agent may be e.g. a tyrosine kinase inhibitor such as Gefitinib (Iressa) or Erlotinib (Tarceva) that bind selectively to the ATP-binding region of the EGFR [36]. Another example is the aromatase inhibitor Letrozole, which ultimately inhibits estrogen synthesis and has been used in clinical trials in breast cancer patients [37, 38].

Peptides and ligands Perhaps the most commonly used peptides for tumour targeting are the somatostatin analogues [39, 40]. They target the somatostatin receptors, which are often overexpressed in neuroendocrine tumours [39, 41]. Radiolabelled somatostatin analogues have been used primarily for imaging [42, 43], but they have also been studied for therapeutic pur-poses [44-46], and 177Lu-labelled somatostatin analogues are now routinely used in some centres [47]. Other peptides of interest are the bombesin analogues that bind to gastrin releasing peptide (GRP) receptors, overex-pressed in various tumours, such as prostate, ovarian, breast, and head and neck cancer [48, 49]. Other growth factor receptors that are overexpressed in cancer cells (e.g. EGFR), can be targeted using the natural ligands as targeting agents (e.g. EGF) [50, 51].

Affinity proteins A relatively new variety of targeting agents are the non-antibody based affinity proteins. They consist of a protein scaffold that can be modified to create a high-affinity targeting agent [52]. Examples include the Anticalins, using the Lipocalin scaffold [53], and the Affibody molecules, using the Protein A scaffold [54].

Antibodies and antibody fragments Antibodies were the first targeting agents used, and still today they are the most commonly used clinical targeting agents. They have the advantages of being easily produced, and having a high affinity for their target struc-ture [55]. Many monoclonal antibodies used for targeting are murine in origin, and these murine monoclonal antibodies (mMAb) can induce an immune response in patients in the form of human anti-mouse antibodies (HAMA). To overcome this problem, chimeric monoclonal antibodies (cMAb) have been constructed. The cMAb has a human constant region, but the variable fragment (Fv) remains from the original murine antibody. Construction of cMAb’s do not always solve the problem of HAMA, and humanized antibodies have been created, where only the complementarity determining region (CDR) remains from the original mMAb. Even fully human antibodies have been constructed [56].

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Antibodies can be used as therapeutic or diagnostic conjugates labelled with e.g. radionuclides such as 90Y, 131I (for therapy), 111In, or 99mTc (for diagnostics). They can also be used as therapeutic agents in themselves, mainly through actions such as antibody-dependent cell-mediated cytotoxic-ity (ADCC) [57-59]. The ADCC mechanism involves cells with cytotoxic potential that express receptors for the Fc region of the antibody, thus binding the target cell, after which they may mediate cell lysis. Some of the antibodies approved for clinical use in cancer patients are listed in Table 1 [60, 61].

Table 1. Examples of antibodies approved for clinical use in cancer patients

Product Type Target Use Effector

Avastin huMAb VEGF Treatment, metastatic colorectal - Bexxar mMAb CD20 Treatment, non-Hodgkin´s 131I Erbitux cMAb EGFR Treatment, metastatic colorectal - Herceptin huMAb HER-2 Treatment, metastatic breast - MabThera cMAb CD20 Treatment, non-Hodgkin´s - Mylotarg huMAb CD33 Treatment, acute myeloid leukemia Zogamicin OncoScint mMAb TAG-72 Diagnosis, colorectal and ovarian 111In Zevalin mMAb CD20 Treatment, non-Hodgkin´s 90Y

Antibodies can be used as targeting agents in their intact form, or they can be modified, usually to reduce the size of the targeting molecule. An intact antibody, with a molecular weight of about 150 kDa, has slow blood clearance, and usually a poor penetration into tumour tissue [61, 62]. Especially for diagnostic purposes, a smaller targeting agent with fast kinetics, and better tumour penetration, might be desirable. By enzymatic cleavage, Fab’ or F(ab’)2 fragments can be produced. Yet other constructs, such as single chain Fv’s (scFv) or diabodies, can be produced by antibody engineering. Figure 1 shows the schematic structure of intact antibodies, and fragments derived thereof.

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Figure 1. Schematic structure of an intact antibody, and antibody fragments. Light grey areas represent heavy chain regions, dark grey areas represent light chain regions, and white bands represent the CDR´s. Fab and F(ab’)2 fragments can be produced by enzymatic digestion, while the other fragments are produced by antibody engineering.

The A33 antibody The humanized monoclonal antibody A33 (huA33) is an IgG1 antibody that binds to the A33 antigen. Originally developed as a murine antibody, it was humanized by CDR grafting [63] after human anti-mouse antibodies were detected in the blood of patients treated with 131I-labelled mMAb A33 [64]. The HAMA response led to a quick clearance of huA33 after a second injection, with no tumour uptake as a result. The A33 antibody has been shown to have a very good retention in tumour tissue. The murine antibody could still be traced in patients six weeks after injection of 125I-mA33, while it was cleared from normal colon after about one week [65]. The same clearance pattern has been observed for huA33, and it has been hypothesized that this is a result of the turnover of normal colonocytes [66]. The huA33 antibody has been investigated in several clinical studies [66-70]. An excellent tumour uptake has been demonstrated, with the unusual ability of the antibody to penetrate to the centre of tumours [66], and 131I-labelled huA33 has been well tolerated at doses up to 1.85 GBq/m2. Unfortunately,

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even though the antibody has been humanized, an immune response has been triggered in the majority of patients receiving huA33, and human anti-human antibodies (HAHA) have been detected [67, 71]. This has led to the construction of a new humanized A33 antibody that may hopefully circumvent the problem of HAHA-response in patients [72].

1.2.3 Effector substances The targeting agent may in itself be an effector substance. This is the case with the tyrosine kinase inhibitors Iressa, and Tarceva. Antibodies can also be used as both targeting agents and effectors (see above). The effector might also be a chemotherapy agent or a toxin, e.g. zogamicin, effector in the Mylotarg antibody [73], or the pseudomonas exotoxin [74]. One advantage of using a radionuclide as the effector substance, is that any problem of multidrug resistance would be overcome. Another, is that antigen heterogeneity will be less of a problem. A chemotherapy agent, for example, is dependent on reaching all cells for successful treatment, whereas a radionuclide usually emits particles with a path-length of several cell diameters. Thus affecting not only the targeted cell, but also the neighbour-ing ones.

1.2.4 Radioimmunotherapy In radioimmunotherapy (RIT), the targeting agent is an antibody, or antibody fragment, to which a therapeutic radionuclide is coupled. Successful results have been achieved when treating lymphomas with antibodies directed against the CD20 antigen present on B-lymphocytes [75]. Unfortunately, the results for solid tumours have not been very encouraging, and there is a common consensus that the future for RIT with intact antibodies is most likely as an adjuvant treatment modality [61]. In the adjuvant setting, smaller lesions would be targeted, and the effect of poor antibody penetration into tumours would not be so pronounced.

1.2.5 Radioimmunodiagnostics In radioimmunodiagnostics (RID), a diagnostic radionuclide is coupled to the targeting protein, and the aim is to visualise the tumour, and hopefully detect metastases at an early stage. In RID, the most important factor is to get a good enough tumour-to-normal tissue ratio to be able to successfully visualise the tumours, without too much interference from the normal tissue background. For diagnostic purposes, intact antibodies are not the best choice, since they are cleared slowly from the blood. A high concentration of radioactivity in the blood gives a high background, and patients might be forced to wait several days after injection of the RID agent before the

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diagnostic examination is possible. Instead, to achieve a higher tumour-to-background ratio, antibody fragments can be used, since they are more rapidly cleared from normal tissue.

1.3 Radionuclides and radioactive decay Depending on the type of targeting agent used, there are different radionu-clides that are suitable. It is usually appropriate to use a radionuclide with a half-life that approximately matches the biological half-life of the targeting agent [76]. By doing so, an unnecessary loss of activity will be avoided. A too short half-life will lead to a loss of activity by decay before a maximum amount of the targeting agent has reached the tumour. A too long half-life might give a lower dose than possible to the tumour, and a higher dose to normal tissue.

1.3.1 Radioactive decay Radioactive elements have unstable nuclei, and emit ionising radiation when they decay to form a more stable state. This ionising radiation may consist of particles with different qualities depending on the mode of decay. Some of these particles, as well as gamma radiation, will be briefly described below.

Alpha particles The �-particle consists of a monoenergetic helium nucleus (two protons and two neutrons) that is emitted from the nucleus in an �-decay. This particle has a short range, in tissue about 50-100 μm, and a high linear energy transfer (LET). The �-particle will thus transfer a high amount of energy over a short distance, causing a dense ionisation track.

Beta particles The �-particle is an electron emitted from the nucleus in a �- decay. These particles can have a continuous spectrum of energies, up to a maximum value. They are low LET particles with a much longer range than �-particles, the range being dependent on the energy of the particle. The �-particles cause less ionisations per distance than the �-particles.

The positron (�+) particle is the antiparticle of the electron, and is emitted from the nucleus in �+ decays. The positron is annihilated as soon as it encounters an electron, creating two photons with an energy of 511 keV each. These two photons will move in opposite directions from the site of the collision, and this is used in positron emission tomography (PET) to obtain high resolution images.

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Auger electrons Auger electrons are shell electrons emitted as they are given excess energy from rearrangements in the electron shell after a decay has taken place. These electrons have low energy, but are often emitted in abundance (so called Auger cascades). They have a very short range, and deposit their energy within one cell diameter. From a therapeutic point of view, they are described as low LET when present in the cytoplasm, but as high LET when attached to the DNA [77].

Gamma radiation In �-radiation, photons are emitted from an excited nucleus as a release of excess energy. These photons have a very long range, often easily penetrat-ing the body of a patient.

1.3.2 Radionuclides for therapy Deciding on the optimal therapeutic radionuclide is a choice that depends on the situation. Such factors must be taken into account as, size of the tumour, target expression, and ability of the targeting agent to penetrate into the tumour. For RIT to be successful, all tumour cells must be reached by the ionising radiation emitted by the radionuclide used. Hence, if the tumour is rather large, the target expression heterogeneous, and the targeting agent is unable to penetrate into the tumour, one would want a radionuclide emitting rather long-range particles. In that case, there will be a cross-fire effect, exposing even those cells to radiation that are not reached by the targeting agent. A �-emitter of rather high energy might be a suitable choice in such cases. Of course, the risk of damaging normal tissue will increase with the path-length of the emitted particle. Obtaining the best tumour cure must therefore, in each of these cases, be balanced against the risk of damaging normal tissue.

If, on the other hand, small tumour cell clusters, or even single cells, are to be targeted, a shorter path-length is desirable. Otherwise, most of the energy will be deposited outside the targeted cells. A shorter path-length might also be appropriate when the target structure is homogenously expressed, and nearly all tumour cells are reached by the targeting agent. In such cases, an �-emitter might be a good choice.

With Auger electron emitters, the radionuclide should preferably be situated very close to the DNA in order to achieve the desired effect [77]. Hence, they are mainly considered for applications such as two-step targeting, where a primary targeting agent delivers the radionuclide to the cell, and a secondary agent (to which the radionuclide is coupled) targets the DNA upon internalisation of the conjugate [78, 79].

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As de Jong et al. have suggested, the optimal choice of nuclide might, in fact, be a combination of therapeutic nuclides, the so called “cocktail” approach [80]. The rationale for this approach being that tumours of different sizes are optimally cured at different path-lengths, and for successful therapy, all tumours must be optimally treated.

Table 2 lists some radionuclides that can be considered for targeted radiotherapy.

Table 2. Radionuclides of interest for targeted radiotherapy

Radionuclide Half-life Main therapeutic emission Energy/Emax Application

211At 7.2 h � 5.9 MeV Single cells or small clusters � (daughter) 7.5 MeV 212Bi 1 h � 6.1 MeV Single cells or small clusters � (daughter) 8.8 MeV 213Bi 46 min � 5.9 MeV Single cells or small clusters � (daughter) 8.4 MeV 67Cu 2.6 days � 470 keV Small clusters 125I 60 days Auger cascade Single cells 131I 8.0 days � 606 keV Small clusters 111In 2.8 days Auger cascade Single cells 177Lu 6.7 days � 500 keV Small clusters 186Re 3.8 days � 1.1 MeV Intermediate clusters 188Re 17 h � 2.1 MeV Large clusters 90Y 2.7 days � 2.3 MeV Large clusters

1.3.3 Radionuclides for diagnostics When choosing a radionuclide for diagnostics, the diagnostic method is of course of utmost importance. If a PET camera is to be used, one must naturally use a positron emitter. For SPECT (single photon emission computer tomography), a photon emitter of suitable energy should be used. Table 3 lists some radionuclides that might be used for imaging.

Table 3. Radionuclides of interest for imaging

Radionuclide Half-life Emission Main application 11C 20 min �+ PET 76Br 16 h �+, � PET 18F 110 min �+ PET 123I 13 h � SPECT 124I 4.2 days �+, � PET 111In 2.8 days � SPECT 99mTc 6 h � SPECT

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1.4 Labelling By using different labelling techniques, the properties of the targeting conjugate can be altered. For example, the cellular retention might be increased, or the biodistribution may be altered.

1.4.1 Radiohalogens When labelling a targeting agent with a radiohalogen, several methods may be used. The most simple way is to use a direct labelling method, where the radiohalogen is attached directly to the protein. An example of this is the Chloramine-T (CAT) method, by which the radioactive label becomes covalently bound to tyrosine residues in the protein. However, this method has its disadvantages. If the binding site is rich in tyrosine residues, the binding capacity of the antibody might be destroyed, or weakened, by CAT labelling. Also, if an internalising targeting agent is used, a rapid excretion of the halogen from the cell can be expected when using a direct labelling method [81].

Indirect labelling methods involve the use of a linker molecule between the radiohalogen and the antibody. The linker thus consists of one part that becomes attached to the protein, and one part that binds the radiolabel. By varying the linker, different properties might be conferred to the targeting conjugate. For example, the cellular retention might be increased by conferring a charge to the catabolite, thus trapping the radionuclide inside the cell. The labelling method used in the present study for radiohalogen labelling is indirect labelling via SPMB (N-succinimidyl-para-(tri-methylstannyl)benzoate), Figure 2, by which the radionuclide is attached to lysine residues in the protein.

Figure 2. Structure of SPMB.

1.4.2 Radiometals Radiometal chelation is also possible to perform via direct labelling, but commonly an indirect labelling method is used. There are several chelators that can be used for radiometal labelling of proteins, e.g. DOTA and DTPA

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(and a number of derivatives thereof) [82]. In this thesis, CHX-A’’-DTPA (Figure 3) is used for labelling of huA33 with 177Lu. This chelator is a modified form of the DTPA molecule, where a cyclohexane group has been added to the DTPA backbone in order to get a more rigid structure. This modification makes the chelated compound more stable [61, 82].

Figure 3. Structure of CHX-A’’-DTPA.

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2. Aim

The aim of this thesis was to investigate the potential of the humanized monoclonal antibody A33 as an agent for targeted radiotherapy of colorectal cancer. The A33 antibody was to be labelled with therapeutic nuclides, and the in vitro, and in vivo characteristics and effects of the resulting therapeutic conjugates were to be determined in a preclinical setting.

The specific aims are listed below:

� To investigate whether huA33 could be labelled with the therapeutic alpha-emitter 211At with retained in vitro specificity and affinity.

� To confirm the in vitro cytotoxicity of 211At-huA33. � To study the biodistribution and in vivo specificity of 211At-huA33 in

tumour-bearing nude mice. � To investigate whether huA33 could be labelled with the therapeutic beta-

emitter 177Lu with retained in vitro specificity and affinity. � To study the biodistribution and in vivo specificity of 177Lu-huA33 in

tumour-bearing nude mice. � To confirm the possibility of gamma camera imaging with the therapeutic

177Lu-huA33 conjugate. � To determine the therapeutic potential of 177Lu-huA33 in vivo in tumour-

bearing mice.

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3. The present study

3.1 211At-labelled huMAb A33 (papers I and II) Targeted radiotherapy using �-emitters could potentially be very successful. The short range of the �-particle in tissue means that there should be minimal damage to normal tissue, and the high LET of the � radiation means that the targeted cell will be subjected to many ionising events.

In the present study, the huA33 antibody was labelled with 211At via the linker SPMB. The in vitro stability of the 211At-huA33 conjugate was confirmed in paper I, and the result from the stability test is shown in Table 4.

Table 4. In vitro stability of 211At-huA33

Incubation solution Incubation temperature, °C Incubation time, h Radioactivity in

LMW fraction, % Calf serum 37 3.6 2.1 ± 0.1 Calf serum 37 7.2 2.7 ± 0.3 Calf serum 37 22 1.5 ± 0.3 Ethanol, 30 % 20 3.6 6.5 ± 0.4 NaI, 1 M 20 3.6 5.7 ± 0.5 Urea, 8 M 20 3.6 5.6 ± 1.5

3.1.1 Binding and retention studies (I) For targeted radiotherapy to be successful, not only must the radiolabel stay attached to the targeting agent under physiological conditions, but the targeting agent must also stay bound to the tumour cell for as long as possible. The affinity of the targeting agent for the tumour cell in question is thus of utmost importance. A saturation assay was performed to determine the equilibrium dissociation constant (Kd) that was used as a measure of affinity in the present study. A low Kd value indicates a high affinity. The Kd value of 211At-huA33 was 1.7 ± 0.2 nM, a low value that means the conjugate has a high affinity for colorectal tumour cells. There might be some concern that the radiolabel could have a detrimental effect on the affinity of the targeting agent. When using a potent radionuclide such as 211At, radiolysis might, for example, be a problem in the labelling proce-dure [83]. For that reason, a comparative study was made with 125I-labelled

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huA33, a conjugate that has been studied earlier [84, 85]. This was done in order to determine whether the affinity of huA33 for SW1222 cells was lowered using 211At as label, when compared to labelling with 125I. The Kd value of 125I-huA33 was 1.8 ± 0.1 nM, thus almost exactly the same as that of 211At-huA33. It could also be shown that both conjugates bound specifically to tumour cells in vitro (Figure 4).

Figure 4. Specific binding of 125I-huA33 and 211At-huA33 to SW1222 cells in vitro. An excess amount of non-labelled huA33 was added to the blocked samples. Error bars represent standard deviation (n=3).

The A33 antibody has been reported to have excellent retention in tumour tissue. We wanted to confirm this in the current setting, with the radiolabels used in this study. As can be seen in Figure 5, the retention of 211At-huA33 and 125I-huA33 was very good for the duration of the experiment. Approxi-mately 70 % of the radioactivity remained cell associated after 21 h for both conjugates.

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Figure 5. The cellular retention of 211At (a) and 125I (b) in SW1222 cells after interrupted incubation with 211At-huA33 and 125I-huA33. Error bars represent standard deviation (n = 3).

3.1.2 Therapy in vitro (I) In paper I, we also wanted to confirm the cytotoxic effect of the 211At-huA33 conjugate. Cells were incubated with 211At-huA33 for 24 h, after which they were washed, and the cell growth was then followed for 48 days. Some samples were blocked with an excess amount of non-labelled huA33, in order to determine whether any cytotoxic effect came from cell associated 211At, or whether it might be due to unspecific radiation from 211At present in the culture media. The number of decays per cell (DPC) was estimated by an uptake experiment done parallel to the 211At-huA33 incubation.

Figure 6. The growth (a) and survival (b) of SW1222 cells after interrupted incubation with 211At-huA33. Error bars in (a) represent standard deviation (n = 3), error bars in (b) represent accumulated error in relation with control.

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After cells had resumed exponential growth, the results were analysed by non-linear regression, and the growth curve was extrapolated back to day zero, giving the number of cells responsible for re-growth (Figure 6a). From this, the survival of the different groups was determined (Figure 6b). There was a clear dose dependent cytotoxic effect of 211At-huA33 that could be blocked by adding an excess amount of non-labelled huA33 (Figure 6).

3.1.3 Biodistribution experiments (II) In paper I, the in vitro specificity and cytotoxicity of 211At-huA33 was demonstrated. For a radiotherapy agent to be successful it must, of course, also be able to specifically find its target in vivo. Further, it must show a favourable biodistribution in order to cause minimal damage to normal tissue.

A biodistribution experiment with 211At-huA33 was performed in nude mice with human colon cancer xenografts. Female Balb/c nu/nu mice were injected subcutaneously (s.c.) with 5 · 106 SW1222 cells two weeks prior to the start of the experiment, leading to tumours with a mean weight of 0.35 g (range: 0.1-0.98 g). The 211At-huA33 conjugate was then injected intrave-nously (i.v.), and mice were subsequently sacrificed at 1, 2, 4, 8, and 21 h post injection (p.i.).

Figure 7. Uptake of 211At-huA33 in blocked and unblocked SW1222 tumours.

The 211At-huA33 conjugate bound specifically to colorectal tumours in vivo, as can be seen in Figure 7. By injecting an excess amount of non-labelled huA33 prior to the injection of 211At-huA33, the tumour uptake was reduced from 24 ± 6 %ID/g to 4 ± 0.4 %ID/g, a level comparable to the normal tissue uptake.

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Figure 8. Biodistribution of 211At-huA33 in tumour-bearing nude mice. The inset shows uptake of 211At-huA33 in thyroid. A standard weight of 5 mg was used as an approximation of thyroid weight. Results are presented as percent injected dose per gram (%ID/g) tissue. Error bars represent standard deviation (n=4).

As shown in Figure 8, the tumour uptake was high, at all time points higher than in most organs. Already at one hour p.i., the tumour uptake was at least twice as high as that of most organs, the exceptions being kidney, lung, spleen, thyroid, and heart, the latter probably due mainly to blood contami-nation. Four hours after injection of activity, the tumour had a radioactivity uptake of 15 ± 8 %ID/g, and the only organs with a higher uptake at that time point were kidneys and thyroid. At 8 h p.i., no organ, apart from thyroid, had a higher radioactivity uptake than the tumour.

The radioactivity concentration in blood was naturally rather high, since the targeting agent used in this study was an intact antibody with slow blood clearance. The tumour-to-blood ratio of 211At-huA33 (Figure 9) increased continuously during the course of the study, reaching approximately 2.5 after 21 h.

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Figure 9. Tumour-to-blood ratio of 211At-huA33. Error bars represent standard deviation (n=4).

Dosimetry calculations were performed based on data from the biodistribu-tion. The dose to the tumour was twice as high as that delivered to most organs, the exceptions being kidney, lung and thyroid (Table 5). The thyroid received the highest dose, but the tumour dose was higher than the dose to both kidney and lung. Apart from thyroid, only blood received a higher dose than the tumour.

Table 5. Absorbed dose after injection of 211At-huA33

Organ Absorbed dose (Gy/MBq) ± SD

Tumour-to-organ ratios

Blood 8.95 ± 1.30 0.69 ± 0.70 Bone 0.84 ± 1.34 7.34 ± 1.81 Brain 0.20 ± 0.01 31.4 ± 29.6 Heart 2.84 ± 0.28 2.18 ± 2.11 Kidney 3.59 ± 0.23 1.72 ± 1.61 Large intestine 1.36 ± 0.29 4.54 ± 4.93 Liver 2.49 ± 0.21 2.49 ± 2.38 Lung 3.96 ± 0.31 1.56 ± 1.49 Muscle 0.53 ± 0.23 11.8 ± 15.5 Pancreas 0.87 ± 0.10 7.16 ± 7.10 Salivary glands 2.85 ± 0.26 2.17 ± 2.09 Skin 1.77 ± 0.15 3.49 ± 3.33 Small intestine 1.31 ± 0.37 4.73 ± 5.46 Spleen 2.70 ± 0.28 2.29 ± 2.23 Stomach 2.21 ± 0.18 2.80 ± 2.67 Thyroid 26.5 ± 13.4 0.23 ± 0.32 Tumour 6.19 ± 6.30

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3.1.4 Comments (I and II) Alpha emitters are nuclides of high interest for RIT. The short path-length and high LET of the �-particle would mean minimal damage to normal tissue, and maximum damage to tumour cell DNA. The biggest problem associated with �-emitters is probably finding one with suitable characteris-tics for therapy. The main �-emitters used today, all have short half-lives compared to the biological half-life of an intact antibody (see Table 2). A too short half-life also means difficulties with transport and labelling. Other �-emitters, with longer half-lives, have been considered for therapy [86, 87], but they possess complex decay schemes, which increases the risk of damaging normal tissue [88].

Astatine-211 is the �-emitter most extensively used today that has a half-life most compatible for use together with antibodies, and also the one used in the present study. It has been demonstrated that it is possible to produce sufficient quantities of an 211At-antibody conjugate for therapeutic purposes, and that such a conjugate is safe to use in patients with malignant brain tumours [89, 90].

The in vitro experiments gave very promising results, with high specific-ity and affinity (in the optimal nanomolar range), excellent tumour cell retention, and a dose dependent cytotoxic effect of the 211At-huA33 conjugate. In the in vitro therapy experiment, there was an unexpectedly low survival seen in the blocked group. The reason for this is unclear, since in a repeated experiment, the survival in the blocked group was as high as 95 %. There was a slight uptake of radioactivity in the blocked cells, corresponding to approximately 2 DPC, that might explain the apparent reduced survival compared to the control group. However, no significant decrease in survival for the blocked group, compared to the control, could be stated. The effect of 211At-huA33 seen in the treated groups is thus most likely due to cell associated 211At, and not a result of 211At present in the medium.

The promising in vitro results made us continue with 211At-huA33 to an in vivo setting, where the in vivo specificity was confirmed, and the biodistribu-tion of the conjugate determined. There was a substantial accumulation of 211At-huA33 in tumour, the tumour-to-blood ratio increased from 0.17 ± 0.04 one hour p.i. to 2.58 ± 0.88 after 21 h, a 15-fold increase. Though some accumulation was seen in several organs, no organ had a larger increase in tissue-to-blood ratio than the tumour during the time studied. In the majority of organs, there was no, or only a slight, accumulation of activity during the time period studied. No major accumulation of 211At in stomach and lung, organs known to accumulate free 211At [91], indicate a high stability of 211At-huA33 in vivo. Further, the in vivo plasma stability of 211At-huA33 has been demonstrated in an earlier study [85]. There was, however, a high uptake of 211At in thyroid, indicating some release of 211At from the huA33 antibody in vivo.

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The dosimetry calculations showed that the thyroid received the highest dose, and this is, of course, somewhat discouraging from a therapeutic perspective. It would, however, probably be possible to block thyroid uptake by using e.g. iodine [90, 92]. Other therapeutic aspects to consider, are the normal tissue expression of the A33 antigen, and the excellent retention of huA33 in tumour tissue, warranting the use of a radionuclide with a much longer half-life. It has been shown that 131I-labelled huA33 is well tolerated by patients at therapeutic activities [69], but there might be a toxicity problem to address when using a more potent �-emitter. This is something that must be thoroughly investigated before proceeding to a clinical setting.

The relatively short half-life of 211At might also be a problem. Even though huA33 localizes unusually quick to tumours for an intact anti-body [33], there is still a high amount of activity in blood at the earlier time points. Eight hours after injection, the tumour-to-blood ratio is still less than 1, and at that point, more than half of the radioactivity is lost. However, the targets for therapy with �-emitters are single cells and small cell clusters, which are probably located close to capillaries. In that situation, targeting might be more effective than in the current experimental setting with developed tumour xenografts. Also, a clinical study with an 211At-labelled antibody for therapy of brain tumours shows encouraging results in terms of patient survival [90].

One major advantage of using alpha-emitters for therapy, is that, due to the short range of the �-particle, the bone marrow will probably not be damaged by circulating activity. In a similar situation, (see above, and paper I), the cytotoxic effect of 211At-huA33 was confirmed to be due to cell associated 211At-huA33 only, with no (or very little) contribution from 211At-huA33 present in the medium.

In conclusion, huA33 could be labelled with the therapeutic �-emitting nuclide 211At, with retained specificity and affinity for colorectal tumour cells in vitro. The cytotoxicity of 211At-huA33 was confirmed in vitro, and the conjugate had a favourable biodistribution in tumour-bearing nude mice. There was a high and specific tumour uptake, and a low uptake in most normal organs. Dosimetry calculations showed that the tumour received a higher dose than all organs apart from thyroid. Therefore, 211At-huA33 shows promise as a potential conjugate for adjuvant RIT of colorectal cancer.

3.2 177Lu-labelled huMAb A33 (papers III and IV) Alpha-emitters are appropriate for eradicating single tumour cells and very small tumour cell clusters. For treatment of slightly larger clusters (or micrometastases), a low-energy �-emitter might be a better choice. As discussed in the introduction, it might, in fact, be optimal for RIT to use a

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“cocktail” of radionuclides with different properties. As mentioned earlier, huA33 has a very good retention in tumour tissue, but is released more rapidly from normal cells. A therapeutic nuclide with a considerably longer half-life than 211At would better take advantage of this antibody characteris-tic in order to maximise the tumour-to-normal tissue dose ratio.

The �-emitter 177Lu (Emax = 497 keV) has a half-life of about one week, and should thus be well suited for therapeutic use together with huA33. Lutetium-177 is a promising therapeutic nuclide that is commercially available, and has a �-emission suitable for imaging.

In the present study, huA33 was successfully labelled with 177Lu using the linker CHX-A’’-DTPA.

3.2.1 In vitro experiments (III) The 177Lu-huA33 conjugate bound specifically to SW1222 cells in vitro, with a Kd value of 2.3 ± 0.3 nM (determined as described above), showing a similar affinity to these cells as 211At- and 125I-huA33.

Figure 10. Continuous uptake of 177Lu-huA33 in SW1222 cells. Error bars represent standard error of mean (n = 3).

The continuous uptake of 177Lu-huA33 in SW1222 cells is shown in Figure 10. The uptake was found to increase throughout the duration of the study, indicating an accumulation of 177Lu in tumour cells in vitro.

3.2.2 Biodistribution and dosimetry (III and IV) The in vivo specificity of 177Lu-huA33 is shown in Figure 11. The uptake was clearly specific, since the uptake of 177Lu-huA33 in SW1222 tumours

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was significantly lower after injection of non-labelled huA33 (blocked group).

Figure 11. Uptake of 177Lu-huA33 in blocked and unblocked SW1222 tumours.

A biodistribution study was performed in a similar model as described above (II). The tumour uptake was high, peaking at 72 h p.i. when it reached 134 ± 21 %ID/g (Figure 12). The radioactivity concentration was signifi-cantly higher in tumour than in blood at all time points studied (8 h to 10 days).

Figure 12. Concentration of 177Lu in blood and tumour after i.v. injection of 177Lu-huA33. Error bars represent standard deviation (n = 4).

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Normal tissue uptake of 177Lu-huA33 is shown in Figure 13. The uptake of 177Lu-huA33 was much higher in tumour than in any other organ (cf. Figure 12). In all organs, apart from skin, the radioactivity uptake decreased between 8 and 72 h p.i. During this time period, the tumour uptake continued to increase. The tumour-to-organ ratio increased continuously throughout the study for most organs, the exceptions being liver, spleen, kidney, and bone. In these organs, there was a slight decrease in the tumour-to-organ ratio between 6 and 10 days p.i. For liver, there was also a small decrease between 3 and 10 days p.i.

Figure 13. Normal tissue distribution of 177Lu-huA33 in nude mice bearing SW1222 tumour xenografts. Error bars represent standard deviation (n = 4).

For a maximum effect of the targeting agent on tumours, and a minimal effect on normal tissue, it is important that the targeting conjugate remains stable in vivo. To determine the in vivo stability of 177Lu-huA33, samples of blood plasma were collected at each time point in the biodistribution study. These samples were then analysed by size exclusion chromatography (cut off 5 kDa), and the amount of radioactivity in the high- and low molecular weight (H/LMW) fractions were determined. At all time points, more than 95 % of the radioactivity was found in the HMW fraction (range: 95 – 99 %), indicating a very high stability of the conjugate in vivo.

Dosimetry calculations were performed, based on data from the biodis-tribution experiment. The dose absorbed by the tumour was at least 20 times

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higher than the dose absorbed by any organ (Table 6). Compared to blood, the tumour dose was more than 12 times higher.

Table 6. Absorbed dose after injection of 177Lu-huA33

Organ Absorbed dose (Gy/MBq) ± SD

Tumour-to-organ ratios

Blood 1.08 ± 0.08 12.6 ± 2.2 Bone 0.28 ± 0.09 49.1 ± 20.3 Brain 0.02 ± 0.00 550 ± 10.3 Heart 0.33 ± 0.05 41.1 ± 10.0 Kidney 0.56 ± 0.06 24.4 ± 5.0 Large intestine 0.18 ± 0.02 75.5 ± 15.2 Liver 0.51 ± 0.05 26.4 ± 5.1 Lung 0.52 ± 0.04 26.4 ± 4.9 Muscle 0.14 ± 0.01 97.1 ± 17.6 Pancreas 0.15 ± 0.01 92.3 ± 6.2 Salivary glands 0.38 ± 0.23 35.5 ± 25.4 Skin 0.64 ± 0.04 21.1 ± 3.6 Small intestine 0.20 ± 0.02 67.9 ± 15.0 Spleen 0.67 ± 0.36 20.4 ± 13.1 Stomach 0.16 ± 0.02 83.1 ± 17.2 Tumour 13.5 ± 1.38

3.2.3 Imaging (IV) During targeted radiotherapy it is an advantage if the targeting agent can be traced in vivo. Using 177Lu as therapeutic nuclide, it should be possible to visualise the tumour during treatment with radiolabelled huA33. This was confirmed in an imaging experiment, where 177Lu-huA33 was injected in tumour-bearing nude mice. In one animal, the tumour uptake of 177Lu-huA33 was blocked by co-injection of non-labelled huA33. As can be seen in Figure 14, the tumours were clearly visualised. The blocked tumour had a radioactivity uptake similar to the low normal tissue background, and could not be visualised.

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Figure 14. Gamma camera images of tumour-bearing mice injected i.v. with 177Lu-huA33 (above). In one mouse, the tumour uptake was blocked by co-injection of an excess amount of non-labelled huA33 (below).

3.2.4 Experimental therapy (IV) The excellent tumour uptake of 177Lu-huA33 is very encouraging from a therapeutic perspective. With a tumour-to-blood absorbed dose ratio of 13 (see Table 6), there should be a high probability of tumour cure, while not exposing the sensitive bone marrow to too high a dose.

An experimental therapy study was set up in a nude mouse xenograft model. Bone marrow was assumed to be the dose limiting organ, and a maximum absorbed dose of 1 Gy was set as the upper dose limit for blood. It was therefore decided to inject a maximum activity of 10 MBq, based on an absorbed dose of 1 Gy/MBq for blood (see Table 6). The animals were kept in groups of 6, and the groups were randomized to 5 different treatments as outlined in Table 7. The cMAb U36, which binds to the CD44v6 antigen, was used as unspecific control, and non-labelled huA33 was used as non-radioactive control.

Table 7. Treatment groups in the therapy study

Treatment Initial number of animals

Animals excluded

7 MBq 177Lu-huA33 12 1 10 MBq 177Lu-huA33 12 2 10 MBq 177Lu-cU36 6 2 huA33 6 0 PBS 12 0

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Treatments were given as a single i.v. dose, 4 days after s.c. injection of tumour cells. The animals were followed for a period of 140 days, during which the tumours were measured regularly by staff with no access to information on treatment for each group (until the end of the study). A Kaplan-Meier plot constructed from the data is shown in Figure 15. The end-point for the analysis was either a tumour size exceeding 1 cm3, or a tumour diameter exceeding 15 mm (i.e. a too large tumour size).

Figure 15. Kaplan-Meier plot showing tumour progression after treatment as indicated in the figure.

There was a significant increase in survival for groups treated with 177Lu-huA33, compared to non-radioactive controls (P < 0.0001). There also seemed to be a slight increase in survival for the 10 MBq 177Lu-huA33 treatment group when compared to the lower activity treatment and the unspecific control, but the increase was not significant. The survival of the non-radioactive controls were 0 %, the unspecific controls had a survival of 50 %, the 7 MBq treatment group a survival of 45 %, and the 10 MBq treatment group a survival of 58 %.

3.2.5 Biodistribution of 177Lu-cU36 (control experiment) The unexpectedly high therapeutic effect of the unspecific antibody cMAb U36, led us to speculate if there might be an expression of the CD44v6 antigen in vivo, even though a lack of expression had been confirmed in vitro. A limited biodistribution study of 177Lu-labelled cMAb U36 (177Lu-cU36) was therefore performed. Four nude mice, with established SW1222 xenografts, were injected intraperitoneally with 20 μg of 177Lu-

36

labelled cU36 in 100 μl PBS. Two days later, the mice were sacrificed and dissected, and tumours and selected organs were collected and measured for radioactivity content in an automated gamma counter. Statistical analysis was performed using the GraphPad Prism 4.0 software.

Figure 16. Biodistribution of 177Lu-cU36 in nude mice with SW1222 tumour xenografts, 48 h after injection. Error bars represent standard deviation.

There was a substantial uptake of 177Lu-cU36 in tumours, significantly higher than in all normal organs included in the experiment, but about the same radioactivity concentration was seen in blood as in tumour (Figure 16). Thus, there is probably no specific uptake of 177Lu-cU36 in SW1222 tumour cells in vivo. The tumour-to-normal tissue ratio seen in this experiment can probably be contributed to the enhanced permeability and retention (EPR) effect in tumour tissue [93], leading to an enhanced uptake and retention of macromolecules in tumours.

3.2.6 Comments (III and IV) The therapeutic nuclide 177Lu might be a good choice for RIT with huA33. It is a low energy �-emitter suitable for therapy of small clusters of tumour cells. In other studies, huA33 has been labelled with the �-emitters 131I [69], and 90Y [63], but these nuclides might not be the best suited in an adjuvant setting. Yttrium-90 emits long-range � particles, and if targeting small cell clusters, these particles will deposit most of their energy outside the targeted tumour. Also, bone-marrow toxicity will be a major problem when a high energy �-emitter is used. Further, 90Y is a pure �-emitter and will not be

37

traceable in a patient after injection. Iodine-131, on the other hand, does emit gamma, but of rather high energy and abundance, thus submitting both patients and personnel to background radiation. Lutetium-177 has a gamma emission suitable for imaging, while not contributing substantially to indiscriminate radiation to patients or personnel. Also, as mentioned above, it might be more appropriate to use a lower energy �-emitter, such as 177Lu, when the target is as homogenously expressed as the A33 antigen.

The results from the in vitro experiments were consistent with the results obtained with 211At-huA33. The 177Lu-huA33 conjugate showed a high specificity and affinity for tumour cells, and the tumour cell uptake of 177Lu was continuous during 177Lu-huA33 incubation, indicating an accumulation of 177Lu.

The in vivo experiments showed very favourable results. There was a clear in vivo specificity of 177Lu-huA33, and the biodistribution study showed impressively high tumour uptake of 177Lu. The normal tissue uptake was low in all organs, especially at the later time points. Compared to other biodistribution studies using 177Lu-labelled antibodies [94-96], tumour-to-normal tissue ratios were consistently higher in the present study.

Free 177Lu is known to accumulate in bone, and it is therefore interesting to note the low uptake of 177Lu in bone in the present study. The amount of 177Lu in bone was less than 4 %ID/g at 8 h p.i. (the first time point studied), and it decreased throughout the study. At ten days p.i., the tumour-to-bone ratio was about 70. This low uptake of 177Lu in bone, together with the high amount of 177Lu in the HMW fraction after size exclusion chromatography, indicate a high stability of 177Lu-huA33 in vivo.

The excellent results from the in vivo experiments led us to plan an experimental therapy study in tumour-bearing nude mice. Since we believe bone marrow to be the dose limiting organ, we decided on a maximum dose of 1 Gy to blood. Dosimetry calculations gave a tumour-to-blood absorbed dose ratio of 13, and with a blood-to-marrow ratio of 0.36 [97], this gives a tumour-to-marrow ratio of about 35 for the fraction of bone marrow dose that can be contributed to blood radioactivity. With such a high tumour-to-marrow ratio, there should be a high probability of obtaining therapeutic results without damaging the radiosensitive bone marrow. Especially with such a low uptake of 177Lu in bone as in the present study.

Since the intended therapeutic use of 177Lu-huA33 is as an adjuvant, to be used in combination with other treatment modalities, mainly surgery, the aim would be to target disseminated cells and small tumour cell clusters. With this in mind, the xenograft model is perhaps not the best model to mimic an imagined clinical situation. We nevertheless chose this model since it gives the advantage of having easily measurable tumours that can be monitored continuously throughout the study. Because of the limitation to our model, our intention was to inject 177Lu-huA33 at an early stage, as soon as there were palpable tumours present, but before measurable tumours had formed.

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This turned out to be highly problematic with the fast growing SW1222 cells used in the present study. It was impossible to assess whether tumours had formed before they were visible. The time for treatment was therefore chosen based on earlier results with this xenograft model, at a time when we believed tumour take to be established, but before visible tumours were formed.

Tumours were measured regularly during the therapy study, and there was an initial increase in tumour growth for the majority of animals. Almost all treated animals were then tumour free at some time point during the study. A few animals never developed tumours, and these were excluded from the study. Unfortunately, two of these animals were found in the already rather small cU36 control group, leaving it with only 4 animals. This was a drawback of the study design; since the treatment substances were injected before tumours were formed, we could not be sure of tumour take in animals never developing tumours.

There was an unexpectedly large therapeutic effect of 177Lu-cU36 in the therapy study. In another therapy study where 177Lu-cU36 was used as unspecific control, there was significantly less effect of 177Lu-cU36 compared to the specific antibody labelled with the same activity [98]. However, the amount of activity administered via cU36 was higher in the present study (10 MBq compared to 7 MBq). We were concerned that the reason for the therapeutic effect of 177Lu-cU36 was due to an unexpected acquired expression of the CD44v6 antigen in vivo. A control experiment gave no such indication though. The reason for the effect of 177Lu-cU36 remains uncertain, but could be an effect of the high amount of activity administered in combination with SW1222 being a relatively radiosensitive cell line [99]. It would have been interesting to have had an extra 7 MBq unspecific control group, but for ethical reasons we tried to keep the number of animals as low as possible. This also led to the small number of animals left in the cU36 control group after exclusion of animals without tumour take, resulting in a poor statistical basis for this group.

To summarize, huA33 could be labelled with the therapeutic �-emitter 177Lu with retained affinity and specificity, both in vitro and in vivo. The 177Lu-huA33 conjugate showed a very favourable biodistribution in tumour-bearing nude mice, with a very high tumour uptake and a low uptake in normal tissue. Dosimetry calculations gave an absorbed dose tumour-to-blood ratio of 12.6 ± 2.2. Two days after i.v. injection of 177Lu-huA33 in tumour-bearing mice, the tumours could be clearly visualised by gamma camera imaging, with very low interference from normal tissue radioactivity. In an experimental therapy study, there was an excellent therapeutic effect of 177Lu-huA33, although there was also an unexpectedly large therapeutic effect of the unspecific control.

In conclusion, 177Lu-huA33 shows excellent potential as a therapeutic agent for adjuvant RIT of colorectal cancer.

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4. Summary

This work has focused on the preclinical evaluation of the humanized antibody A33 (huA33) as a radioimmunotherapy (RIT) agent for adjuvant therapy of colorectal cancer. The A33 antigen, against which huA33 is directed, is expressed in more than 95 % of all colorectal carcinomas, and is therefore well suited as an RIT target.

HuA33 was labelled with the �-emitting therapeutic radionuclide 211At (T1/2 = 7.2 h), via the linker SPMB, with retained specificity and affinity (Kd = 1.7 ± 0.2 nM). The cellular retention of 211At-huA33, in the colon cancer cell line SW1222, was very good. Twenty one hours after interrupted incubation, 70 % of the cell associated radioactivity was still remaining.

A dose dependent cytotoxicity of 211At-huA33 was demonstrated in vitro by a growth assay. Cells exposed to 56 and 150 decays per cell (DPC) had a resulting survival of 5 % and 0.3 %, respectively, when compared to unexposed controls. The effect 211At-huA33 could be blocked by adding an excess amount of non-labelled huA33.

The 211At-huA33 conjugate also showed specific binding to human colon cancer xenografts in nude mice. A biodistribution study showed that the uptake of radioactivity in tumour was, at all time points, higher than in most normal organs. At 8 h post injection (p.i.), the tumour had a higher radioactivity concentration than any organ, apart from thyroid. Compared to blood, the tumour had a higher radioactivity concentration only at the last time point studied (21 h p.i.).

Dosimetry calculations, based on data from the biodistribution study, showed, as was to be expected, that the highest dose was delivered to the thyroid. Apart from thyroid, the tumour received a higher dose than any organ. The blood absorbed a somewhat higher dose than the tumour, but the tumour absorbed at least twice as high dose as that absorbed by most other organs, the exceptions being lung and kidney.

Because of the excellent retention of huA33 in tumour tissue, a therapeu-tic radionuclide with a considerably longer half-life than 211At was also considered. The choice fell on the �-emitter 177Lu (T1/2 = 6.7 d), which have the advantages of being commercially available and possible to visualise during treatment.

HuA33 was labelled with 177Lu using the chelator CHX-A’’-DTPA, and 177Lu-huA33 bound specifically and with high affinity to SW1222 cells in vitro (Kd = 2.3 ± 0.3 nM). The in vivo specificity of 177Lu-huA33 was also

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confirmed, by the specific binding of 177Lu-huA33 to SW1222 xenografts in nude mice.

The biodistribution of 177Lu-huA33, in a nude mouse xenograft model, was very promising from a therapeutic perspective, with a very high tumour uptake, and low uptake in normal organs. The radioactivity concentration in tumour was, at all time points studied (8 h to 10 days p.i.), significantly higher than the radioactivity concentration in blood and normal organs. Dosimetry calculations showed that the absorbed dose tumour-to-organ ratio was at least 20 for all organs. The absorbed dose tumour-to-blood ratio was about 13.

The biodistribution results warranted an experimental therapy study. Mice subcutaneously injected with SW1222 cells, were treated with 7 MBq 177Lu-huA33, 10 MBq 177Lu-huA33, 10 MBq 177Lu-cU36 (unspecific control), non-labelled huA33, or vehicle only (PBS). Animals treated with 177Lu-huA33 showed significantly increased survival compared to non-radioactive controls. Surprisingly, the unspecific control had a similar effect as the huA33 conjugates.

To conclude, huA33 could be labelled with both 211At and 177Lu with retained affinity and specificity, both in vitro and in vivo. Both therapeutic conjugates had a favourable biodistribution in tumour-bearing nude mice, with high tumour uptake and a low uptake in normal organs (with the exception of the expected thyroid uptake of 211At). After injection of 177Lu-huA33 in tumour-bearing mice, tumours were clearly visualised by gamma camera imaging, and the conjugate had a large therapeutic effect in an experimental therapy study. Both 211At-huA33 and 177Lu-huA33 thus show promise for use in radioimmunotherapy of colorectal cancer.

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5. Future studies

The deeper you investigate something, the more you become aware of how little you really know, and for every question answered, there seem to be ten new posed. The work in this thesis has dealt with two possible RIT conjugates based on the A33 antibody, and some further studies related to this PhD-project are described below.

5.1 Alpha particle therapy. It would be interesting to conduct an in vivo therapy study with 211At-huA33. A colorectal cancer liver metastases model was set up in nude mice, and it would be most interesting to continue with experimental therapy in this setting using 211At-huA33. It would also be interesting to look into the possibility of using alpha emitters with a longer half-life than 211At, for example 225Ac. As an extended study, the efficacy of 211At-huA33 could be compared to that of 177Lu-huA33 in the same setting, and a cocktail approach could also be considered.

5.2 Cellular mechanisms The retention of the A33 antibody is remarkable, and the reason for it is still uncertain. Further investigations into the mechanisms of cellular binding and processing could shed some light on this matter. The cell line used in all experiments in this thesis (SW1222) does not handle being placed on ice very well. Because of this, some control experiments have been difficult to perform. It would therefore be interesting to expand the study to include several other cell lines. The question of antigen internalisation, would be one of the most interesting to address. Does it, in fact, not internalise, or are perhaps the discrepancies between experiments a result of different antibodies being used.

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5.3 A33 antibody fragments In this thesis, the focus has been RIT, but it should, of course, also be possible to use this antigen system for detection of colorectal cancer metastases. Because of the expression of the A33 antigen in normal colon, huA33 is perhaps even better suited for diagnostics than for therapy. As a smaller sized targeting agent might be better for diagnostic purposes, it would be of interest to study A33 antibody fragments with diagnostic applications in mind.

5.4 Experimental 177Lu therapy The experimental therapy study with 177Lu-huA33 showed an excellent therapeutic effect of this conjugate. The fact that the same therapeutic effect was achieved with the unspecific antibody used, is something that should be further investigated. It would be interesting to repeat the experiment with additional controls, and perhaps also with another cell line. A higher activity might probably also be administered, since there was no signs of toxicity in the present study. As noted above, it would also be interesting to move the experimental therapy to another setting, where liver metastases are targeted.

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6. Sammanfattning på svenska (Summary in Swedish)

6.1 Mål och bakgrund Cancer är ett enormt problem i dagens samhälle, antalet nya fall ökar varje år och ca 27 % av alla dödsfall i Sverige orsakas av cancer. En av de vanligaste tumörsjukdomarna är kolorektalcancer, som är orsaken i ca 10 % av de dödsfall som kan härledas till cancer. Kolorektalcancer är en behandlingsbar sjukdom, med en mycket bra prognos vid tidig diagnos, men ett stort problem är att det ofta bildas metastaser som gör att sjukdomen blir svårbehandlad.

En möjlig behandlingsmetod för att förbättra prognosen för patienter med metastaserande kolorektalcancer är att använda målsökande terapiformer. Vid målsökande terapi använder man sig av molekyler (målsökare) som binder specifikt till strukturer som i idealfallet endast uttrycks på tumörceller och inte på normala celler. Till dessa målsökare kan man sedan koppla en cytotoxisk substans. Tanken är att man på det sättet ska kunna behandla tumörer utan att skada normalvävnad, något som är ett stort problem både vid kemoterapi och extern strålbehandling. Som cytotoxisk substans kan t.ex. radioaktiva nuklider användas. Dessa nuklider avger joniserande strålning som orsakar DNA-skador i levande vävnad.

A33 är ett antigen som uttrycks i över 95 % av alla kolorektala tumörer, både primärtumörer och metastaser. Normalvävnadsuttrycket däremot, är mycket begränsat.

I den här avhandlingen har den humaniserade antikroppen A33 (huA33) använts som målsökare och märkts med de terapeutiska radionukliderna 211At och 177Lu. De resulterande konjugaten, 211At-huA33 och 177Lu-huA33 har sedan studerats i prekliniska försök. Målet med avhandlingsarbetet har varit att bedöma konjugatens potential som målsökare vid adjuvant terapi av kolorektalcancer.

6.2 Astatexperiment 211At har en halveringstid på 7,2 h och sönderfaller genom att avge alfapartiklar, som består av heliumkärnor (2 protoner och 2 neutroner).

44

Dessa partiklar har hög energi och räckvidden i vävnad är upp till ca 100 μm, vilket motsvarar några få celldiametrar. Detta ger goda förutsätt-ningar för att orsaka stor skada på tumörceller utan att förstöra normal vävnad

Vid målsökande terapi är det viktigt att målsökaren binder till den mål-sökta strukturen och att den fortsätter vara starkt bunden så länge som möjligt. 211At-huA33 band starkt (med hög affinitet) och specifikt till odlade tumörceller (in vitro). 211At-huA33 hade också en mycket god retention i cellerna. 70 % av den cellbundna radioaktiviteten fanns fortfarande kvar på cellerna 21 h efter avbruten inkubation med 211At-huA33. Ett terapiförsök in vitro visade på en dosberoende cytotoxisk effekt av 211At-huA33, där tumörceller behandlade med en högre dos fick en lägre överlevnad. Effekten av 211At-huA33 kunde visas bero på 211At bundet till celler, eftersom effekten minskade vid tillsats av omärkt huA33. Den omärkta antikroppen blockerade antigenet och hindrade på så sätt märkt huA33 från att binda.

Målsökaren måste naturligtvis även binda till sin målstruktur in vivo och det är också viktigt att målsökaren inte tas upp av normalvävnad till alltför stor del. Därför undersöktes biodistributionen av 211At-huA33 i tumörbäran-de möss (in vivo). 211At-huA33 band specifikt till humana kolorektala tumörer in vivo. 8 h efter injektion av 211At-huA33 var tumörupptaget högre än upptaget i normala organ, förutom sköldkörteln, som hade ett högre upptag än tumören. Dosimetriberäkningar visade att sköldkörteln som väntat fick högst dos, men att tumören fick en dubbelt så hög dos som andra normala organ, förutom lungor och njurar. Blodet absorberade dock en något högre dos än tumören.

6.3 Lutetiumexperiment Eftersom huA33 har en väldigt bra retention i tumörvävnad, kan det vara fördelaktigt att använda en nuklid med längre halveringstid än 211At. Vi bestämde oss för att använda 177Lu som har en halveringstid på ca 7 dagar och som, till skillnad från astat, finns tillgänglig kommersiellt. När 177Lu sönderfaller avges betapartiklar (som består av en elektron) med en räckvidd i vävnad på ca 2 mm.

Precis som 211At-huA33 band också 177Lu-huA33 starkt och specifikt till tumörceller in vitro. Den specifika bindingen till tumörer in vivo kunde också bekräftas. Biodistributionen av 177Lu-huA33 i tumörbärande möss var mycket lovande ur ett terapeutiskt perspektiv. Radioaktivitets-koncentrationen var högre i tumör än i både blod och normala organ under hela studien (8 h till 10 dagar efter injektion). Dosimetriberäkningar visade att tumören absorberade en minst 20 gånger så hög dos som normala organ absorberade. Jämfört med blod absorberade tumören en 13 gånger så hög dos.

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De lovande biodistributionsresultaten fick oss att utföra en terapistudie i tumörbärande möss. Mössen injicerades subkutant (under huden) med tumörceller och 4 dagar senare injicerades antingen 177Lu-huA33 eller olika kontrollsubstanser intravenöst. Behandlade djur hade en betydligt bättre överlevnad än de icke-radioaktiva kontrollerna, men den ospecifika kontrollantikroppen (märkt med 177Lu, men som inte binder till de här tumörcellerna) hade en lika stor effekt som 177Lu-huA33.

6.4 Slutsatser HuA33 kan märkas med både 211At och 177Lu med bibehållen specificitet och affinitet. I båda fallen var biodistributionerna fördelaktiga för terapi, kanske främst i lutetiumfallet. Experimentell terapi visade på goda resultat med 177Lu-huA33, men ytterligare studier behövs för att utreda de oväntade effekterna av den ospecifika antikroppen.

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Acknowledgements

This thesis has been produced at the Unit of Biomedical Radiation Sciences (BMS) at Uppsala University. It is the result of a collaboration between the Unit of Radiology and BMS that gave me the opportunity to do preclinical research though belonging to a clinical unit. I would like to thank everybody who has contributed in different ways to this thesis, in particular:

My main supervisor Anders Sundin for encouraging me to do all the experiments I wanted, for reading through my manuscripts with great care, and for always having a positive attitude. Also for being a very patient teacher at the animal facility. My co-supervisor Vladimir Tolmachev for all the labellings and measure-ments, for always coming with ideas and suggestions, and for always being ready to lend a helping hand. My other co-supervisor Hans Lundqvist for dosimetry calculations, and for always taking time to explain anything I may have wanted to ask about. Chaitanya Divgi for supplying me with enough antibody to last an entire PhD-project (and more). Also for interesting and rewarding scientific conversations, and for being an excellent co-author. All my co-authors, who have all had a hand in the making of this thesis.

Alla BMS:are, gamla som nya, för att det är roligt att komma till jobbet, för engagemang i filmer och fester, och för de traditionella BMS-aktiviteterna. Speciellt skulle jag vilja tacka:

Jörgen Carlsson för att jag har fått göra mitt avhandlingsarbete på BMS, och för den fantastiska stämningen där som gör att man känner sig hemma.

Veronika, utan dig skulle det inte ha blivit någon avhandling i år. Tack för alla försök du har hjälpt mig med, och all tid du har ägnat åt mina möss!

Lars och Amelie för ovärderlig hjälp med mina djurförsök. Ann-Charlott för skidresor och fjällvandringar, Englandsresor, fredagsöl,

träning och för att du alltid hade tid att prata en stund på morgonen (det är ju så lååångt till fikat ibland). Tack också för all hjälp med mina försök!

47

Åsa för att du tog dig an mig som exjobbare, tålmodigt svarade på alla mina frågor, och direkt fick mig att känna mig som en i gänget. Jag har nog aldrig skrattat så mycket på cellab som under våra gemensamma försök.

Marika för alla fantastiska filmer och underhållande historier, för att du fick med mig i jämställdhetskommittén, och för att du är den bästa rums/tält-kompisen man kan ha.

Mina rumskompisar Lina och Erika för att jag kan prata med er om allt och för att AmaZone fortsätter vara det bästa rummet. Tack också Lina för att du lyckas få iväg mig och träna ibland.

Thuy och Erika för en toppenvecka i Aten! Micke för att du alltid har ställt upp och hjälpt till med allt från celler till

datorer. Shirin för att du kanske är den mest generösa människa jag känner och för

att du alltid gör det roligt att vara på jobbet. Johanna för att du frågade om jag ville vara kvar på BMS. Det har jag

aldrig ångrat!

Alla mina vänner som gör livet värt att leva. Ett speciellt tack till “moster” Emma för att du kom försent till den där kemilabben. Ett stort tack också till min familj:

Mamma och pappa som alltid uppmuntrade mig att studera vidare och som alltid fick mig att känna mig fantastisk oavsett hur, eller vad, jag presterade.

Kalle, som jag alltid har roligt tillsammans med och som har ställt upp, stöttat och uppmuntrat genom hela avhandlingsarbetet, och Nora, för att du finns. Jag älskar er över allt annat!

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 336

Editor: The Dean of the Faculty of Medicine

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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