Evaluation of Carrier Compounds for Systemic and Intracavitary α-Radionuclide Therapy of Cancer

Systemic and Intracavitary

α -Radionuclide Therapy of Cancer

Thesis for the Degree of Philosophiae Doctor by

Sara Westrøm

Oncoinvent AS

Institute of Clinical Medicine, Faculty of Medicine, University of Oslo

Department of Tumor Biology, Institute for Cancer Research,

Norwegian Radium Hospital, Oslo University Hospital

© Sara Westrøm, 2019

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8377-477-1

All rights reserved. No part of this publication may be

reproduced or transmitted, in any form or by any means, without permission.

Cover: Hanne Baadsgaard Utigard.

Photo cover: Ine Eriksen, UiO.

Print production: Reprosentralen, University of Oslo.

Cancer is a leading cause of death worldwide, with metastatic disease responsible for the majority of mortalities. Radionuclide therapy utilizing radioactive elements, either alone or combined with a carrier compound, is an emerging therapeutic strategy. The aim is for the carrier to transport or maintain its payload to the tumor site and ensure it remains there for a sufficient time for the radionuclide to deliver a toxic radiation dose. Ideally, this results in a treatment that can cure, mitigate or control the disease, while normal tissues are spared. A range of therapeutic radionuclides and carrier compounds exists. These two components should be selected after careful consideration of the compatibility of their properties with each other, the disease situation and the mode of administration (localvs.

systemic).

The long-term goal with the studies performed in this Ph.D project is to develop a novel radionuclide therapy platform for improved treatment of metastatic cancer. Patients with some cancers, such as osteosarcoma and ovarian cancer, often present with micrometas- tases. For treatment of micrometastases, it can be argued that the high linear energy transfer and short path length ofα-emitting radionuclides are advantageous toβ- andγ- emitters in terms of more effective cancer cell inactivation and less damage to surrounding normal tissues. In this thesis, the potential of theα-emitter ²²⁴Ra and its progeny ²¹²Pb has been explored. Radium-224 has attractive properties forα-therapy as it produces four α-particles per complete decay and has a convenient 3.6 days half-life. Amongst the rel- atively few α-emitting radionuclides of clinical interest, ²²⁴Ra was considered a sound candidate to pursue. The supply from its parent radionuclide,²²⁸Th, should be sufficient to sustain commercialization of a product, which is not necessarily the case for several of the other clinically relevantα-emitters.

Two different carrier compounds have been evaluated. The first, an anti-CD146 mon- oclonal antibody (mAb), named OI-3, was intended for cell-specific targeting following systemic administration. The OI-3 mAb showed selective binding to human osteosarcoma cell linesin vitroand was able to target CD146 expressing tumors after intravenous injec- tion in mice. In contrast to²²⁴Ra, which cannot be stably bound to a targeting molecule,

solution of ²²⁴Ra was used directly for labeling, eliminating the need to prepare a pure solution of ²¹²Pb. This proposed method may be operationally simpler and less time- consuming for the end user compared to the procedure which requires preparation of a pure²¹²Pb-solution, as cumbersome steps involving handling and evaporation of concen- trated acids with high radioactivity are avoided.

The other examined carrier compound, inorganic calcium carbonate microparticles, was intended for local treatment of cavitary cancers. The microparticles were labeled with

224Ra in an efficient procedure, resulting in high yields of both²²⁴Ra and daughter²¹²Pb, and a high degree of retention of these radionuclides for at least one weekin vitro. Biodis- tribution studies in mice showed that the microparticles retained its radioactive payload to a large extent alsoin vivo, as the radioactivity mainly remained in the peritoneal cav- ity after intraperitoneal (IP) injection. In two murine models of human ovarian cancer growing IP, the ²²⁴Ra-microparticles gave significant antitumor effect after IP adminis- tration, with either considerably reduced tumor volume or a survival benefit. No signs of acute or subacute toxicity, neither locally nor systemic, has been observed at thera- peutically active doses, indicating that the IP ²²⁴Ra-microparticle treatment is safe and well-tolerated.

Working with this thesis has been a wonderful journey - educational every step of the way - ranging from deep frustrations to moments of complete satisfaction. I am grateful to Roy H. Larsen and Tina B. Bønsdorff for giving me the opportunity to join the Oncoinvent team and share this experience with them.

First, I would like to thank my supervisor Tina. You have given me the necessary guid- ance, and your enthusiasm and optimism is contagious. You are a superhuman, with seemingly unlimited resources of time, advice and encouragements. You have faced all challenges coming our way head-on, and found solutions to practical problems when I did not see any. Thank you for always listening and for numerous late evening talks in the office about everything and nothing.

I want to express my gratitude to co-supervisor Øyvind S. Bruland, you have also been essential for this project. You have kept track on my progress and always made yourself available to me. You are a source of inspiration and your way of explaining complicated matters so everyone can understand it is something I admire and try to continuously learn from. You have helped me improve, sharing your knowledge and expertise by providing sound advice and constructive feedback.

I would also like to thank my co-supervisor, Gunhild M. Mælandsmo, for your contribu- tions. In particular for your part in providing me access to valuable research facilities at the Radium Hospital.

A special appreciation goes to Roy. You have jokingly given yourself the title "official misleader" of my Ph.D project. In reality, the situation is quite the opposite. Your support and immense experience in the field of radiotherapy have steered me back on the right path plenty of times during these years. Thank you for having faith in my abilities and motivating me by constantly challenging my reasoning, methods and conclusions. Your creativity and eagerness to move forward have been indispensable for our research.

I would like to acknowledge my Oncoinvent colleagues. I was fortunate to experience

Sofie Jorstad and former colleague Marion Malenge. You have been exquisite company in the lab and at the animal department. I have enjoyed and benefited from our discus- sions on all subjects ranging from writing, experiment planning and analysis of results.

Furthermore, I want to thank co-authors Thora J. Jonasdottir and Nasir Abbas for their contributions.

I appreciated working alongside all of you in Nordic Nanovector’s R&D department when we shared laboratory facilities. Thank you for creating a nice atmosphere and your good humor during the numerous hours we shared in the lab. I am especially grateful to co- author Roman Generalov, I truly enjoyed our time working together. Your diligence in the lab and our discussions about results and improvement of the manuscript have been valuable.

Thank you Stein Waagene for being a wonderful teacher of animal laboratory skills. You are my role model for handling of mice. Your patience and calm manner both with the animals and your "students", like me, are exemplary. In addition to being educational, it has been fun to work together. You are a gifted story-teller and hearing about your previous experiences has been a real pleasure.

Our collaborators at the Radium Hospital, Asta Juzeniene and Vladimir Iani deserve recognition for assistance with animal experiments and contributing with interesting ideas and suggestions for future studies.

The unit for comparative medicine at the Radium Hospital is acknowledged for care of animals in all the in vivoexperiments. Thanks to the dedicated staff for being excellent mice-caretakers, always happy to help and offer advice.

Lastly, I want to thank my husband, parents and siblings for their continuous support and endless patience. Thank you for always being there and knowing what to say at the right moments. Particularly during the last year, after baby-Ida arrived, which made the final writing process slightly more challenging. To all other family members and my best friend Gry, who helped out with babysitting from time to time, your efforts have been invaluable for me to finalize the thesis.

The work presented in this thesis was financially supported by The Research Council of Norway as an Industrial Ph.D grant (grant number 237661) and a BIA project (grant number 235531), both received by the privately owned Norwegian company Oncoinvent AS, which also has funded the work. Oncoinvent AS has an approved patent (US Patent 9 539 346 and European Patent 3111959) that relates to the combined use of particles and anα-emitting radionuclide in the treatment of cancer and a patent (US Patent 9 782 500) that relates to a novel anti-CD146 antibody and derivatives thereof.

Regarding the role of authors in the publications included in this thesis: Sara Westrøm, Tina B. Bønsdorff, Ida Sofie Jorstad and Elisa Napoli are employed at Oncoinvent AS.

Nasir Abbas, Thora J. Jonasdottir and Marion Malenge were employed at Oncoinvent AS at the time they contributed to the original publications comprising this thesis. Sara Westrøm, Tina B. Bønsdorff, Ida Sofie Jorstad, Elisa Napoli, Thora J. Jonasdottir, Marion Malenge and Roy H. Larsen are also shareholders in Oncoinvent AS. Roy H. Larsen is affiliated with Sciencons AS, which is a shareholder of Oncoinvent AS. Øyvind S. Bruland is affiliated with Blaahaugen AS, which is a shareholder and has a consultancy contract with Oncoinvent AS. Roy H. Larsen is chairman of the board of Oncoinvent AS and Thora J. Jonasdottir is a board member.

Abstract i

Acknowledgments iii

Declaration of Interests v

List of Abbreviations ix

List of Publications xi

1 Introduction 1

1.1 Metastatic Cancer . . . 1

1.2 Radionuclide Therapy of Cancer . . . 3

1.2.1 Choice of Radionuclide . . . 3

1.2.2 Carrier Compounds . . . 4

1.2.3 Radiolabeling of Carrier Compounds . . . 7

1.3 Monoclonal Antibodies as Radionuclide Carriers . . . 7

1.4 Particles as Radionuclide Carriers . . . 8

1.5 Radiobiology of High and Low-LET Radiation . . . 9

1.6 Alpha-Emitting Radionuclides of Potential Clinical Interest . . . 12

1.6.1 Actinium-225 . . . 12

1.6.2 Astatine-211 . . . 13

1.6.3 Bismuth-212 and 213 . . . 15

1.6.4 Lead-212 . . . 16

1.6.5 Radium-223, 224 and 225 . . . 17

1.6.6 Thorium-227 . . . 19

1.7 Intraperitoneal Radionuclide Therapy in Peritoneal Carcinomatosis . . . . 19

2 Objectives 25

3.1.1 Radioactivity Measurements . . . 29

3.2 Radionuclide Carriers . . . 30

3.2.1 OI-3 - A Novel Antibody Targeting CD146 . . . 30

3.2.2 Calcium Carbonate Microparticles . . . 30

3.3 Radiolabeling andIn VitroCharacterization of Radiolabeled Compounds . 31 3.3.1 Radiolabeled Monoclonal Antibodies . . . 31

3.3.2 Ra-224 Labeled Calcium Carbonate Microparticles . . . 33

3.4 In VivoExperiments . . . 34

3.4.1 Biodistribution Studies of Radiolabeled Compounds . . . 34

3.4.2 Therapy Studies . . . 35

3.4.3 Toxicity Assessments . . . 37

4 Summary of Results 39 4.1 Radiolabeled Monoclonal Antibodies . . . 39

4.1.1 Targeting of CD146 by the OI-3 Monoclonal Antibodies . . . 39

4.1.2 Feasibility of Novel Method for ²¹²Pb-Labeling of Monoclonal Antibodies . . . 40

4.2 Ra-224 Labeled Calcium Carbonate Microparticles . . . 41

4.2.1 In VitroCharacterization . . . 41

4.2.2 Biodistribution Studies . . . 41

4.2.3 Therapy Studies . . . 43

4.2.4 Toxicity Assessments . . . 45

5 Discussion and Future Outlook 51

6 Conclusions 65

References 67

Publications 93

% ID/g Percent injected dose per gram ALP Alkaline phosphatase

ALT Alanine aminotransferase AST Aspartate aminotransferase BSA Bovine serum albumine CaCO₃ Calcium carbonate

CHOI-3.1 Chimeric variant of the OI-3 monoclonal antibody with human immunoglobulinG1 Fc portion

CHOI-3.3 Chimeric variant of the OI-3 monoclonal antibody with human immunoglobulinG3 Fc portion

DOTA 1,4,7,10-tetraazacyclododecane tetraacetic acid DPBS Dulbecco’s phosphate buffered saline

DTPA Diethylenetriaminepentaacetic acid D50 Volume-based median diameter

EDTMP Ethylenediamine tetra(methylene phosphonic acid) FDA U.S. Food and Drug Administration

Ig Immunoglobulin

IP Intraperitoneal

IRF Immunoreactive fraction

IV Intravenous

LET Linear energy transfer mAb Monoclonal antibody

OS Osteosarcoma

PARP Poly(ADP-ribose) polymerase PC Peritoneal carcinomatosis

PSMA Prostate-specific membrane antigen PSA Peritoneal surface area

TCMC 2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10- tetra-(2-carbamoyl methyl)-cyclododecane

The following peer-reviewed publications comprise this thesis:

I Evaluation of CD146 as Target for Radioimmunotherapy against Osteosar- coma.

Westrøm S, Bønsdorff TB, Abbas N, Bruland ØS, Jonasdottir TJ, Mælandsmo GM, Larsen, RH.

PLoS One. 2016;11:e0165382.

II Preparation of ²¹²Pb-labeled monoclonal antibody using a novel ²²⁴Ra-based generator solution.

Westrøm S, Generalov R, Bønsdorff TB, Larsen RH.

Nuclear Medicine and Biology. 2017;51:1-9.

III Ra-224 Labeling of Calcium Carbonate Microparticles for Internalα-Therapy:

Preparation, Stability and Biodistribution in Mice.

Westrøm S, Malenge M, Jorstad IS, Napoli E, Bruland ØS, Bønsdorff TB, Larsen RH.

Journal of Labelled Compounds and Radiopharmaceuticals. 2018;61:472-486.

IV Therapeutic Effect ofα-Emitting²²⁴Ra-Labeled Calcium Carbonate Micropar- ticles in Mice with Intraperitoneal Ovarian Cancer.

Westrøm S, Bønsdorff TB, Bruland ØS, Larsen, RH.

Translational Oncology. 2018;11:259-267.

1.1 Metastatic Cancer

Cancer comprises a wide group of diseases which can develop in nearly every tissue in the body. The predicted global cancer burden is expected to exceed 20 million new cases an- nually by 2025 [1]. Despite a constant increase in our understanding of cancer, it remains a difficult disease to cure. Treatment is dictated by the cancer type, disease stage, and the general health condition of the patient. In addition, the size and location of both primary and metastatic tumors are determining factors. A vast array of therapies exists, which can be systemic or local in nature. Surgery and external-beam radiotherapy are examples of local treatments that are usually efficient against primary tumors and regional spread to draining lymph nodes. It is, however, metastatic disease that is responsible for the ma- jority of mortalities [2, 3]. Depending on the metastasis pattern of the cancer, local and systemic approaches, or a combination of the two, may have a curative therapeutic aim.

Nevertheless, the approximately eight million cancer-related deaths reported worldwide in 2012 [1] makes it evident that curative treatment options are still lacking and sorely needed for a large number of patients.

Metastatic cancer develops when cells from the primary tumor spread to surrounding or distant parts of the body where it can settle and form new tumors (Figure 1.1). Most commonly, cancer cells disseminate by entering the circulation system either via blood or lymphatic vessels, but some types of primary tumors can metastasize by direct exten- sion into nearby body cavities (transcoelomic spread), such as the peritoneal, pleural or pericardial cavity.

Micrometastases can be single cells or clusters of cells smaller than 2mm [4]. They are subclinical and beyond the detection ability of available radiological diagnostic modali- ties, and therefore often contribute to treatment failure [5]. Osteosarcoma (OS) and ovar- ian cancer are examples of cancers where patients often present with micrometastatic spread. As with most tumor types, they have an organ specific metastasis pattern [3, 6–

8]. OS is the most common malignant primary tumor of bone [9] and primarily spread hematogenously and cause micrometastases in the lungs. The most typical feature of ovar- ian cancer on the other hand, is a transcoelomic spread of tumor cells into the peritoneal cavity where they establish micrometastatic sites on the visceral and parietal peritoneal lining [10–12].

Primary tumor

Metastatic site

Figure 1.1: The metastatic process involves escape of cancer cells from a primary tu- mor, invasion into the surrounding stroma and further dissemination by hematogenous, lymphatic or transcoelomic spread [13]. Migration into the circulation is most common and depicted in the figure. The tumor cells can then be trapped in the capillaries in a new organ, extravasate into the surrounding tissue to establish a micrometastatic site [7]

and finally initiate and maintain growth by adaption of the new microenvironment [3, 14], which amongst other things include vascularization of the metastatic tumor. The figure was created using illustrations from Servier Medical Art (smart.servier.com).

1.2 Radionuclide Therapy of Cancer

Radionuclide therapy of cancer is based on the use of radioactive isotopes of various elements, either alone or combined with a carrier substance, to irradiate tumors. The intent is for the ionizing radiation to inactivate and prevent proliferation of cancer cells, while the carrier contributes to deliver the radiation in the target location. Ideally, the sum of these two is a treatment that can cure, control or mitigate the disease, while normal tissues are spared.

1.2.1 Choice of Radionuclide

Upon choice of radionuclide for a given therapeutic application, several factors including its emission type, physical half-life, chemical properties, availability and ease of produc- tion should be considered [15, 16]. The existence of radionuclides with a vast array of physical characteristics makes it possible to match the radionuclide properties with a spe- cific tumor type or disease pattern in addition to the chemical relation andin vivobehavior of the intended carrier.

For therapeutic purposes, radionuclides withα,β⁻and Auger electron emissions are con- sidered, and their general characteristics are shown in table 1.1. Of these,α-particles and Auger electrons are monoenergetic, whereas the negatively chargedβ-electrons are emit- ted with a spectrum of energies. Due to variations in mass and energy of these emitted particles, their range in tissue and thereby also their linear energy transfer (LET), which is defined as the energy transferred to matter per unit length, differs significantly. In general, low-LETβ-emitters are believed to be more suitable for treatment of larger tumors than the high-LETα- and Auger-emitters, which are preferred for treatment of micrometas- tases and single cell diseases [15]. The decay of a radionuclide is often accompanied by emission of photons in the form of γ or X-rays. This type of emission allows external imaging if the energy and intensity are within the diagnostic range, which can be useful for monitoring the distribution and to perform dosimetry estimates.

The physical half-life of a radionuclide will influence both practical and therapeutic as- pects. A short half-life, typically in the range of a few hours, imposes time constraints to the application of the radionuclide. Efficient production and purification procedures are heavily relied on, and they must be performed, at least partly, at the hospital site. With half-lives of several days and upwards, the flexibility is greater and would usually allow centralized production before shipment to the end user. However, a too long half-life can also be impractical if patients need to be hospitalized for extended time periods.

To achieve optimal therapeutic efficacy and minimize radiation exposure to normal tis-

Table 1.1: General characteristics of radionuclide emission relevant for therapeutic use.

Table adapted from reference [17].

Decay Particle(s) Energy Range in tissue LET^*

(keV/µm)

α Helium-nucleus 5-9MeV 40-100µm 80-100

β⁻ Energetic electron 50-2300keV 0.05-12mm 0.2-0.5 Auger 5-30 nonenergetic electrons 1eV-100keV 2-500nm 4-26

*Linear energy transfer

sues, the half-life of the radionuclide should match thein vivo pharmacokinetics of the carrier compound. If the physical half-life is too short compared to the carrier distribu- tion time, much of the decay will occur prior to the agent reaching its target, reducing the deposited dose in the tumor. On the other hand, if the half-life is too long compared to the biological half-life of the carrier, a significant amount of the product may be metabo- lized and/or excreted while still being highly radioactive. This can both reduce the dose delivered in the target location and increase the exposure to normal tissues and organs responsible for clearing the agent from the body.

For translation of a radionuclide therapy into clinical use, the radionuclide of choice or its precursor should be available in sufficient quantities, either by being processed from natural mineral sources or from a synthetically produced source, e.g. made by cyclotron or nuclear reactor production. A feasible extraction process resulting in high purity should also exist. Some of the radionuclides that have beneficial properties for therapy will be challenging to develop for routine clinical use today due to lack of or limited production sites [18], high production costs [15, 19] or difficult production logistics because of short half-lives.

Several radionuclides are already being used routinely in the clinic or have been evaluated in clinical trials. An overview of these, together with their physical properties are listed in table 1.2.

1.2.2 Carrier Compounds

The purpose of the carrier compound is to transport or maintain its payload to the disease site and ensure it remains there for an amount of time sufficient to deliver a relevant therapeutic radiation dose. As a wide range of disease situations exists, it is obvious that different carrier properties are needed depending on the clinical application. There are however some commonalities between the carrier compounds in radionuclide therapy.

They are required to withstand radiolytic degradation within a relevant time frame, they

Table 1.2: Radionuclides that have been investigated clinically and their physical proper- ties.

Radionuclide Daughter(s) Half-life^* Emission Energy^† (MeV/Bq-s)

32P ³²S 14.3 days 1β⁻ 0.695

89Sr ⁸⁹Y 50.6 days 1β⁻ 0.587

90Y ⁹⁰Zr 2.7 days 1β⁻ 0.934

131I ¹³¹Xe 8.0 days 1β⁻ 0.573

153Sm ¹⁵³Eu 1.9 days 1β⁻ 0.328

166Ho ¹⁶⁶Er 1.1 days 1β⁻ 0.723

177Lu ¹⁷⁷Hf 6.7 days 1β⁻ 0.131

186Re ¹⁸⁶Os 3.7 days 1β⁻ 0.035

188Re ¹⁸⁸Os 17.0 h 1β⁻ 0.836

198Au ¹⁹⁸Hg 2.7 days 1β⁻ 0.731

211At ²⁰⁷Bi/²¹¹Po,²⁰⁷Pb 7.2 h 1α, 1 EC^‡ 7.647

212Pb ²¹²Bi,²⁰⁸Tl/²¹²Po,²⁰⁸Pb 10.6 h 1α, 2β⁻ 10.25

213Bi ²⁰⁹Tl/²¹³Po,²⁰⁹Pb,²⁰⁹Bi 45.6 min 1α, 2β⁻ 12.31

223Ra ²¹⁹Rn,²¹⁵Po,²¹¹Pb,²¹¹Bi,

207Tl/²¹¹Po,²⁰⁷Pb

11.4 days 4α, 2β⁻ 27.94

224Ra ²²⁰Rn,²¹⁶Po,²¹²Pb,²¹²Bi,

208Tl/²¹²Po,²⁰⁸Pb

3.6 days 4α, 2β⁻ 29.24

225Ac ²²¹Fr,²¹⁷At,²¹³Bi,

209Tl/²¹³Po,²⁰⁹Pb,²⁰⁹Bi

10.0 days 4α, 2β⁻ 31.67

227Th ²²³Ra,²¹⁹Rn,²¹⁵Po,²¹¹Pb,

211Bi,²⁰⁷Tl/²¹¹Po,²⁰⁷Pb

18.7 days 5α, 2β⁻ 33.95

*From Decay Data Evaluation Project (http://www.nucleide.org/DDEP_WG/DDEPdata.htm).

†From ENSDF decay data in MIRD format (www.nndc.bnl.gov/mird). Listedα,β,γand X-rays for the radionuclide and, if applicable, progeny combined.

‡Electron capture

should be readily available or easy to produce and have a chemistry compatible with the radionuclide of choice [16, 19]. In addition, the carrier should not by itself be toxic or harmful for normal cells or tissues in the body.

A carrier can fulfill its purpose by several mechanisms, which all have been found useful in certain applications. In the simplest situation, the radionuclide itself functions as a car- rier. This is the case when the radionuclide has chemical properties that naturally ensure localization in the target area after administration in the body. Examples are concentration of radioactive iodine in the thyroid glands used for treatment of well-differentiated thyroid cancer [20, 21] and accumulation of radioactive strontium [22, 23] or radium [24, 25] in bones for pain palliation and treatment of skeletal metastases.

Another way, commonly referred to as targeted radionuclide therapy, is to use small molecules which specifically bind to receptors or proteins expressed on cell surfaces as carriers. Examples of such targeting moieties can be peptides, monoclonal antibodies (mAbs) or fragments thereof. The main advantages with this type of carrier is the specific targeting of cancer cells while at the same time minimizing radiation exposure to normal cells. In many cases, the targeting molecule with its radionuclide payload can also be internalized, thereby increasing the proximity to the cell’s DNA, a critical target for the biological effects of radiation. The cell-specific targeting may be a limitation as well, since the treatment only can be used on patients whose tumors express the antigen. It can also be challenging to select an antigen which is truly unique to the tumor cells or expressed in a very low amount in normal cells.

A third possibility is to utilize particles, either nano- or microsized, as carriers for radionu- clides. For systemic therapy, nanoparticles are generally favored to avoid rapid and/or nonspecific clearance from the circulation and embolic phenomena [26], in addition to exploiting the enhanced permeability and retention effect for passive targeting to the tu- mor [27, 28]. For local treatment applications, the mechanism of action is that the particle size ensures regional retention at the target site for an amount of time sufficient to deliver the radiation dose.

Radioactive implants, in the form of seeds, pellets, tubes, wires or needles [29], can also be reckoned as a type of carrier compound. Such implants are placed either intracavitary in close proximity to a tumor or interstitially within the tumor itself [29, 30], in a local treatment application termed brachytherapy. There are two types of brachytherapy im- plants: Permanent, where the radioactive source remains in the tissue, and temporary, in which the source is removed after the radiation dose has been delivered.

1.2.3 Radiolabeling of Carrier Compounds

Radiolabeling of carrier compounds can be performed by direct or indirect methods, where the latter require a bifunctional linker to connect the carrier and the radionuclide.

Regardless of which carrier to be radiolabeled, the method should fulfill some general requirements [16, 31]. Primarily, the procedure should result in as high as possible la- beling yields. This condition is especially important for radionuclides with high cost and/or limited availability. Radionuclides with short half-lives require rapid and efficient labeling protocols to minimize loss due to decay, but simple and efficient procedures are beneficial in all cases, both to reduce the radiation exposure to the worker and to the car- rier compound to avoid radiolysis. A simple procedure also facilitates implementation of automated methods with minimal manual handling. This is a benefit when working with high radioactivity levels, which are needed for clinical use of some radionuclides.

The stability of the radiolabeled carrier should be within acceptable limits during storage, transportation and after administration to the patient. This can be particularly challenging for radionuclides with α-emitting daughters because their recoil energy upon decay are larger than the binding energy of most chemical bonds [32]. Lastly, if the carrier com- pound has biological activity, such as binding to an antigen site, this property should be preserved during the radiolabeling.

1.3 Monoclonal Antibodies as Radionuclide Carriers

Antibodies, also known as immunoglobulins (Ig), constitute a part of the immune sys- tem. By their ability to specifically bind antigens, they function to recognize and remove pathogens before they become harmful to the host. The antibodies are relatively large proteins of around 150 kDa and are composed of pairs of heavy and light polypeptide chains which form a Y-shaped structure [33]. They can exist as a single unit (IgD, IgE, IgG), dimer (IgA) or pentamer (IgM) in humans. In the 1970s it became possible to pro- duce mAbs, antibodies of a single specificity, in vitro after the invention of hybridoma technology [34]. This development soon lead to a recognition of the therapeutic potential of this class of molecules as generation of mAbs against tumor specific antigens could be achieved on a large scale.

The first mAbs evaluated for targeted cancer therapy were of murine origin. Their use is limited due to immunogenicity and modifications by protein and genetic engineering to replace murine sequences with human components have been developed. In this way, chimeric, humanized and even fully human mAbs have been created, hence significantly broadening the clinical usefulness of mAbs. Today, antibody-based therapeutics include both naked mAbs and mAbs conjugated to different payloads such as drugs, toxins or

radionuclides [35], with mAbs of the IgG type being the most common form.

In the context as carrier compounds for radionuclides, mAbs are considered to be one of the most suitable types and is perhaps the most extensively studied carrier to date. One mAb conjugated to⁹⁰Y (Zevalin®) is currently in use for treatment of patients with non- Hodgkin’s lymphoma, and numerous others are being examined in clinical trials, both against hematological and solid malignancies [33, 36–39]. For solid cancers, radiolabeled antibodies are being investigated as a systemic therapy, but there is also interest in its use as a locoregional therapy, such as for intracavitary administration into the brain, pleural, pericardial, and peritoneal compartments [40].

The efficacy of mAbs as radionuclide carriers is limited by insufficient penetration into solid tumors [19] due to common barriers to drug delivery in tumor tissue such as abnor- mal blood vessel network, accumulated solid stress, elevated interstitial fluid pressure and a dense interstitial structure [41]. A heterogeneous expression of the antigen on the tumor cell population may be an additional limitation. Both these factors can lead to uneven dose deposition. With the development of molecular techniques, the possibility of engineering smaller antibody derived molecules has emerged. The use of such smaller molecules, like antibody fragments, minibodies and affibodies, can increase the tumor penetration to some degree, but it comes at the expense of a more rapid clearance from the circulation which again may cause a smaller amount of the carrier to reach the target site. Targeting agents based on smaller molecules than mAbs are therefore more suited for use together with short-lived radionuclides.

Radiolabeling of mAbs typically requires the use of a bifunctional chelator for conjugation to a radionuclide, the exception being direct iodination of tyrosine residues which are part of the antibody structure. The choice of radionuclide is therefore limited to iodine isotopes and those nuclides where a bifunctional chelator that creates a stable complex in vivois available. Conditions during the labeling procedure also need to be compatible with mAbs to preserve their biological function,i.e. their immunoreactivity, and prevent aggregation and/or denaturation of the protein. IgGs can e.g. be sensitive to elevated temperatures.

1.4 Particles as Radionuclide Carriers

In the past few decades, design of nano- and microcarriers to function as drug delivery systems in the treatment of various cancers have captured the interest of the scientific community [26]. The majority of particulate drug delivery systems are developed to im- prove the performance and safety of traditional drugs already on the market, but it also includes novel applications such as delivery of peptides, proteins, vaccines, gene therapy

and radiation [42–46]. This wide field of applications is largely owing to the versatility of particle carriers as they can be produced in a range of materials and sizes. The seem- ingly endless options of properties enable tailoring of the particles for their intended use.

Examples of particle carriers studied for therapeutic purposes include, but are not lim- ited to, liposomes, micelles, dendrimers, polymeric, inorganic and magnetic nano- and/or microparticles [45, 47].

So far, a small number of particle-based cancer therapies have found their way into routine clinical use. This includes liposomes carrying various chemotherapeutic drugs (Doxil®, DaunoXome®, Marqibo®, ONIVYDE®), albumin nanoparticles loaded with paclitaxel (Abraxane®) [48], as well as radiolabeled microparticles (SIR-Spheres® and TheraS- phere®) [49, 50]. The radiolabeled microparticles are used in an approach termed ra- dioembolization. Microspheres made of glass or resin material with diameters from 20 to 60µmlabeled with⁹⁰Y are injected into the arteries that supply a liver tumor. Here they become trapped due to their size and irradiate cancerous tissue. Particle size can also be used as a means to ensure intracavitary retention in the peritoneal or pleural cavity. This principle was applied by using radioactive colloids (e.g. Phosphocol®) in treatment of patients with malignant effusions and will be further discussed in section 1.7.

Radiolabeling can be performed either during or after preparation of the particles [51].

The latter is generally preferred, especially for radionuclides with short half-lives. Due to various material compositions of particle carriers, a range of labeling strategies can be applied. It is possible to encapsulate or incorporate the radionuclides within the particle matrix, to attach the radionuclides to the particle surface or to label throughout the vol- ume of a porous particle. Diverse labeling chemistry is also available. Particles can be radiolabeled by covalent binding of radionuclides, adsorption processes, ion exchange, or through chelator based processes. The possibility of radiolabeling particle carriers with- out the use of a chelator can make radionuclides with beneficial therapeutic properties available for use which would be inaccessible otherwise due to the lack of a suitable bi- functional ligand for conjugation to other carrier compounds.

1.5 Radiobiology of High and Low-LET Radiation

Radiobiology is defined as the biological response of cells to ionizing radiation and the resulting cellular consequences [38]. The biological effects of radiation are mainly caused by DNA damage, although the cytoplasm and membrane lipids are also radiation sensitive targets in the cell. Different types of DNA damage can be produced, including single- strand breaks, double-strand breaks, base damage and DNA-protein cross-links [38, 52].

A condition with multiple damaged sites may also occur, which refers to several DNA

lesions in close proximity to one another. DNA damage is potentially lethal for cells and subsequent to its occurrence, different repair mechanisms are activated depending on the type of DNA lesion. Most DNA damage is possible to repair, but double-strand breaks and multiple damaged sites are more complex and therefore more likely to cause irreversible changes [53], which eventually can lead to cell death.

Ionizing radiation can cause DNA damage either directly or indirectly. When emitted charged particles hit the DNA molecule, it is a direct interaction. Indirect effects on the other hand, occur when free radicals are produced from radiation interacting with water molecules [54]. The distribution and type of DNA damage produced by direct action de- pends on the physical characteristics of the radionuclide emission (Table 1.1). High-LET α-particles cause dense ionization along its track, whereas the low-LET β-particles only produce sparse ionization events, as illustrated in figure 1.2. Due to these differences, low LET-radiation is more prone to result in individual DNA lesions that may be more easily repaired, such as single-strand breaks, whereas high-LET radiation produces clusters of DNA damage that are more difficult to repair, such as double-strand breaks. Consequently, substantially more hits of the DNA are needed to produce irreversible effects with low- LET radiation. In addition, the mechanism of action for cytotoxic events of high-LET radiation is less dependent on the presence of oxygen in tissue [55], which is beneficial due to many tumors being hypoxic.

The average number ofα-particle nuclear traversals required to kill a cell have been mea- sured to range from a single hit up to 20 [56], which is in stark contrast to the hundreds to thousands ofβ-particles that must traverse a cell nucleus for its inactivation [19, 52].

It can also be exemplified by comparing the number of hits required to obtain the same absorbed dose, defined as the energy deposited per unit mass (Gy = J/kg), to the cell’s nucleus. Goodhead et al. estimated that 1-4 α-particle tracks produced 1 Gy, whereas about 1000β tracks [57] were needed. This implies that a significantly higher dose-rate is required with low-LET radiation to produce sufficient ionization for irreversible DNA damage to occur.

Cells may not only be irradiated by decay that takes place on or inside the cell, but also from decay occurring in nearby cells if the range of the emitted particles in tissue is larger than a typical cell diameter of 10µm. The contribution from this so-called crossfire effect on the absorbed dose, varies with the emission type due to the different range of particles in tissue (Table 1.1). As visualized in figure 1.3, the longer path length ofβ-particles will result in irradiation of a significantly higher portion of neighboring and distant cells com- pared toα-particles, which at their maximum range only will pass through approximately ten cells.

α

β

Figure 1.2: Patterns of DNA damage caused by different LET-radiation illustrated by showing the density of ionizations produced from a high-LETα-particle and a low-LET β-particle traveling through a DNA strand. The figure was created by using an illustration from Servier Medical Art (smart.servier.com).

β α

0 100 µm 12 mm

Figure 1.3: The maximum path length of α- and β-particles relative to a cell diameter of 10µm. The figure was inspired by a figure seen in reference [38] and created using illustrations from Servier Medical Art (smart.servier.com).

Crossfire irradiation can be particularly beneficial in situations with a heterogeneous dis- tribution of carrier in the tumor tissue, as it to some extent can compensate for nonuniform uptake of radionuclides. It may also result in more irradiation of surrounding normal tis- sues, especially when the tumor size is much smaller than the range of the β-particles.

Simulations have shown that when the tumor mass, and thereby also the tumor diameter, is reduced, the gain from crossfire irradiation rapidly decreases as the fraction of energy escaping the tumor increases [58]. It is therefore a general consensus that high-LET radi- ation are optimal for treatment of microscopic and single-cell disease, whereas on larger tumors, the low-LET radiation is more suitable partly due to the larger contribution of crossfire effects.

Biological responses after irradiation can also arise in cells that have not themselves been directly traversed by energetic particles. This phenomenon is called the radiation-induced bystander effect. A variety of bystander responses has been reported in cells neighboring irradiated cells, including cell death, apoptosis, genetic mutations and malignant trans- formations [38, 59]. The main mechanisms behind the bystander effects are believed to be secretion of factors to the surrounding media and direct cell-cell signaling via gap junctions.

1.6 Alpha-Emitting Radionuclides of Potential Clinical In- terest

The potential ofα-emitters for radionuclide cancer therapy is well-known, but relatively few radionuclides are considered to be of clinical interest. In this section, a brief descrip- tion of the properties, chemistry and production of these radionuclides are given, with emphasis on advantages and limitations for translation into clinical use and for commer- cialization.

1.6.1 Actinium-225

The radiometal ²²⁵Ac has a half-life of 10.0 days and generates an average of four α- particles per complete decay to stable²⁰⁹Bi. It can be obtained by radiochemical extraction from²²⁹Th (t_1/2 = 7340 years) or by different accelerator-based methods [60].

The use of²²⁵Ac for radiolabeling of targeting molecules was limited by lack of appropri- ate chelators, both to give sufficient yield and stability [61, 62]. A two-step procedure for complexing ²²⁵Ac to 1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) improved the situation considerably [63]. With this method, a ²²⁵Ac-labeled mAb has shown ad- equate conjugate stability to be examined in clinical trials for systemic therapy of ad-

vanced myeloid leukemias [64, 65]. The treatment was feasible and antileukemic activity has been reported. Regardless, the ²²⁵Ac-complex will be challenged by recoil energy from the multiple α-emissions in the decay chain. This might lead to release of some daughter nuclides which again pose a risk of inflicting normal tissue toxicity. Therefore, careful consideration of carrier compound and applications must be made, for instance,

225Ac might be best suited for internalizing targeting molecules. One example of such is the coupling of ²²⁵Ac to the low molecular weight prostate-specific membrane anti- gen targeting ligand (PSMA-617). In patients with metastatic castration-resistant prostate cancer, this compound has shown promising antitumor activity [66, 67]. Novel strategies to circumvent challenges with release ofα-emitting progeny are also being explored in preclinical studies, such as encapsulation of ²²⁵Ac in liposomes [68–72] or lanthanum phosphate nanoparticles [73, 74].

Both the availability of²²⁵Ac and, to some extent, the labeling chemistry are impediments to translation into clinical use. When it comes to availability,²²⁹Th in large enough quan- tities to produce sufficient amounts of ²²⁵Ac is only available from three stock sources of ²³³U worldwide. The annual supply from these sources combined is estimated to be 63 GBq of ²²⁵Ac [75]. If a patient dose is assumed to be in the range of 5-10 MBq, most likely approximately twice that amount needs to be produced to have material for quality control. In addition, a 25 % loss due to decay during production and shipment to the end user can be reckoned. This amounts to production for 236-4253 patients per year¹. The lower and upper limit assume a 10 % and 90 % labeling yield respectively, where the lower limit accounts for the reported 90 % loss of input²²⁵Ac with the current two-step method of labeling mAbs with ²²⁵Ac [76]. From these numbers it is possible to conclude that the supply of²²⁵Ac from the three ²²⁹Th sources can sustain a limited number of clinical trials. Larger clinical trials and commercialization will necessitate new productions methods together with optimization of labeling protocols to achieve higher yields. Several production routes are being investigated, including irradiation of ²²⁶Ra and²³²Th with various particle beams [75]. Of these, proton irradiation of²²⁶Ra in a cy- clotron for direct formation of²²⁵Ac [77] seems most promising for cost-effective, large scale production [60].

1.6.2 Astatine-211

The halogen ²¹¹At has a 7.2h half-life and generates oneα-particle on average per de- cay, via a branched pathway to stable ²⁰⁷Pb. It is typically produced in a cyclotron by bombarding a bismuth target with a beam ofα-particles via the²⁰⁹Bi(α,2n)²¹¹At nuclear reaction [78]. Thereafter, the produced²¹¹At needs to be separated from the target either

1Lower limit =10 MBq×2÷0.75÷0.1^63×103MBq = 236, Upper limit = 5 MBq×2÷0.75÷0.9^63×103MBq = 4253

by acid treatment followed by solvent extraction or by dry distillation, where the latter is the most common [79].

Despite²¹¹At being a halogen, it also has metallic characteristics, which makes its chem- istry both diverse and complex. For radiolabeling of targeting molecules with ²¹¹At, the standard approach has become to use "activated tin ester" derivatives as the conjugation moiety [80,81]. Although this procedure is considerably faster than previous methods, the approximately 2 hspent is still relatively long compared to the half-life of the radionu- clide [79] and can at high activity levels cause problems with radiolysis of the product.

The resulting conjugates produced with this method are considered to be sufficiently sta- ble in vivowhen using intact mAbs, but there is a concern with reduced stability when smaller, more rapidly metabolized molecules are utilized [79]. Efforts are therefore made to further develop and optimize the labeling chemistry, both to increase stability and to reduce preparation time. Complexation of astatine by a boron cage is one possible method that has shown some promise [82, 83].

The main obstacle with ²¹¹At is its availability [78, 84]. Only a limited number of cy- clotrons able to produce²¹¹At with sufficient purity and activity levels for routine clinical use exist. High intensity of the α-particle beam is required for production of relevant amounts and the incident energy of the beam must be optimal to minimize production of unwanted impurities [84]. In addition, a considerable amount of time will elapse dur- ing separation from the target and subsequent conjugation to targeting molecules, making logistics cumbersome if the cyclotron is not located in relatively close proximity to the hospital due to the short²¹¹At half-life. Commercialization of²¹¹At production by build- ing or purchasing a new cyclotron is claimed not to be likely in the near future for at least two reasons [78]:

• Not many other radionuclides of commercial interest can be produced in the same facility.

• An investment of this magnitude requires a certainty that the demand for²¹¹At will persist and yield a reasonable profit, which is difficult to predict prior to demonstra- tion of clinical efficacy.

On top of that, the geographical area a new cyclotron can serve will be restricted due to the short half-life of²¹¹At.

As of today, ²¹¹At has been evaluated in two human trials. One examined patients with recurrent brain tumors given²¹¹At-labeled mAbs locally into a surgically created resection cavity [85] and the second investigated intraperitoneal (IP) administration of²¹¹At-labeled mAb fragments in ovarian cancer patients [86]. Both trials demonstrated that the ²¹¹At- treatment was tolerable and safe across all dose levels, but only limited data on therapeutic

effect has been reported. A phase I/II trial of intravenous (IV) administration of a²¹¹At conjugated mAb before donor stem cell transplant for treatment of patients with high-risk acute myeloid leukemia, acute lymphoblastic leukemia or myelodysplastic syndrome is ongoing (NCT03128034).

1.6.3 Bismuth-212 and 213

The bismuth isotopes of interest for α-therapy, ²¹²Bi and ²¹³Bi, have short half-lives of 60.6minand 45.6minrespectively. They are the finalα-emitting daughters in the²²⁴Ra- and²²⁵Ac-decay chains and can thus be eluted from generators based on these respective radionuclides. Because of the short half-lives of both isotopes, the production must be performed at the radiopharmacy or nuclear department at the hospital immediately before administration to the patient.

Bismuth is a heavy metal and the chemistry is well-established. Carrier compounds can be stably complexed by common bifunctional chelating agents based on derivatives of diethylenetriaminepentaacetic acid (DTPA) or DOTA. Short labeling times and high yields are important to minimize loss of activity due to decay during preparation, and for that reason DTPA is usually preferred because of faster kinetics [61,62]. Consequently, it is not the labeling chemistry in itself that limits the use bismuth isotopes, but rather challenges related to their their short half-lives.

As of today, human studies have only been performed using the²¹³Bi isotope. Both sys- temic and local approaches have been explored, with different types of carriers. IV in- jections of²¹³Bi conjugated to mAbs have been evaluated in patients with acute myeloid leukemia [87, 88] and metastatic melanoma [89, 90], and in one patient with metastatic castration resistant prostate cancer using PSMA-617 as the targeting agent [91]. Tri- als with local administration include intralesional²¹³Bi-mAbs in patients with metastatic skin melanoma [92], intratumoral²¹³Bi-conjugated peptides in patients with gliomas [93]

and intraarterial infusion of ²¹³Bi-labeled peptides in patients with neuroendocrine tu- mors [94]. These studies have shown that preparation of a patient dose with bismuth will require activity in the GBq range. If a patient dose of 1-2 GBq is to be produced, at least twice the amount of activity is probably needed due to loss during decay and some material needed for quality control of the finished product. For²¹²Bi, this is particularly challenging due to the highly energetic 2.6MeVγ-emission from the²⁰⁸Tl daughter which requires extensive shielding. The use of²¹²Bi has therefore to a large degree been replaced with its mother nuclide²¹²Pb, for feasibility reasons elaborated in the section below. With

213Bi, there are no specific γ-rays of concern, but such activity levels are nevertheless so high that they can be demanding to handle for many institutions and will also lead to a high cost per effective dose [95].

There is also a concern about availability of²¹³Bi due to limited supply of the generator radionuclide ²²⁵Ac. The generators used for production of clinical grade material has been reported to give an average²¹³Bi yield of 76 % [96] and should have a shelf-life of at least one month. Transient equilibrium between the two nuclides is established relatively fast, allowing generator elution every 2-3h[75]. Despite these favorable qualities of the generator, even if all²²⁵Ac currently available worldwide would be used for production of²¹³Bi, it is claimed that only 100-200 patients could be treated annually [84]. This is clearly not a high enough number to sustain commercialization of a product. Accordingly, a translation of²¹³Bi into clinical use is dependent on a substantially increased supply of

225Ac.

1.6.4 Lead-212

Lead-212 has a half-life of 10.6hand decays viaβ-emission to the therapeutically potent α-emitting daughter²¹²Bi. For that reason,²¹²Pb can be used as anin vivogenerator ofα- particles from²¹²Bi, resulting in a virtual prolongation of the²¹²Bi half-life. Lead-212 is available from generators based on²²⁴Ra and the half-life necessitates on-site production.

Elution of ²¹²Pb from the ²²⁴Ra generator column is a somewhat cumbersome process, as it requires handling and evaporation of concentrated acids containing high radioactiv- ity [97].

In terms of ²¹²Pb-radiolabeling, an improvement in protocol came with the develop- ment of the lead-chelator 2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra-(2- carbamoyl methyl)-cyclododecane (TCMC). It creates a more stable complex at low pH and provides more efficient conjugation than its DOTA analog [98]. However, release of some²¹²Bi from the chelate upon²¹²Pb decay might raise toxicity concerns. The first clinical evaluation of a²¹²Pb-conjugated mAb has been performed by intracavitary admin- istration in patients with peritoneal cancers [99, 100]. This reduces the risk for systemic toxicity due to release of ²¹²Bi, because most of the radioactive decay will occur within the peritoneal cavity before redistribution. Regardless, in 2018, a phase I trial of a peptide labeled with²¹²Pb through the DOTA chelator given IV to patients with neuroendocrine tumors was initiated (NCT03466216).

The supply of ²¹²Pb is dependent on the availability of the long-lived ²²⁸Th (t_1/2 = 1.9 years) mother nuclide. Th-228 is a decay product of naturally occurring²³²Th, from where

228Ra (t1/2= 5.8 years) can be extracted and purified, before²²⁸Th is obtained by allowing decay of ²²⁸Ra. In addition, ²²⁸Th can be generated from decay of ²³²U, which can be made available from used nuclear fuel [101, 102]. A third source of²²⁸Th is production through neutron irradiation of natural ²²⁶Ra. It is reported that approximately 3.7 GBq

228Ra is obtained from one ton of 30-year old ²³²Th [103]. Given the vast resources of

naturally occurring ²³²Th, estimated to be in the range of several million tonnes [104], and the existence of other production routes, the supply of ²²⁸Th does not seem to be a concern for commercialization. A determination of actual production costs and yields with the different methods are needed to elaborate on which production route will be most cost-effective and feasible. The Orano Med group has recently initiated the building of a facility for industrial-scale production of ²¹²Pb from a supply of thorium derived from mining activities [105], which supports the view that no principal limitations to produce

228Th exist.

The major drawback with²¹²Pb is the highγ-activity of the²⁰⁸Tl daughter. It is less of a problem with²¹²Pb compared to²¹²Bi due to the lower levels of activity needed, but still extensive shielding is called for. Since²¹²Pb-based products need to be prepared on-site, the shielding requirements are not only applicable at the production plant, but also at the hospital.

1.6.5 Radium-223, 224 and 225

Radium has a long history in treatment of cancer. Shortly after Marie Skłodowska Curie’s discovery of²²⁶Ra in 1898 [106], pioneers of radio-oncology began to use it for treatment of cancer in the form of brachytherapy. The first attempts were performed on tumors located in easily accessible sites and encapsulated radium sources were placed either in close proximity or directly into the tumor. Intracavitary brachytherapy for treatment of cervical cancer was soon regarded as effective and became one of the primary applications of radium [107]. Ra-226 is by itself anα-emitter, but when used in a sealed source it was the highly energeticγ-rays of the progeny that were the effectors. As a consequence of radiation safety concerns with the high energies of these photons, potential leakage of the gaseous²²²Rn daughter from the encapsulated sources and the exceptionally long half-life of 1600 years [24, 106], by now,²²⁶Ra has long ago been replaced by other radionuclides, such as¹⁹²Ir,⁶⁰Co and¹³⁷Cs [106], in the clinic.

Today, more than a century after its discovery, another radium isotope has experienced a renaissance. In 2013, the U.S. Food and Drug Administration (FDA) approved²²³Ra- dichloride (Xofigo®, Bayer) for treatment of patients with symptomatic bone metastases from castration-resistant prostate cancer [108] and it is the first in classα-emitting radio- pharmaceutical.

All radium isotopes are radioactive, but it is only²²³Ra (t_1/2 = 11.4 days),²²⁴Ra (t_1/2 = 3.6 days) and²²⁵Ra (t_1/2 = 14.9 days) that have half-lives compatible for use as therapeutic radionuclides. They all decay via multipleα- andβ-emitting progeny with shorter half- lives than their respective radium parent, with an average of four emittedα-particles per

complete decay. The complete decay of each series releases a high total energy of 28- 29 MeV (see table 1.2), where more than 90 % of the energy is associated with the α- emissions. The three radium isotopes can all be obtained from their long-lived parental radionuclides: ²²³Ra from ²²⁷Ac (t_1/2 = 21.8 years), ²²⁴Ra from ²²⁸Th and ²²⁵Ra from

229Th. In the same way as²²⁸Th,²²⁷Ac can be produced by neutron irradiation of natural

226Ra [103], but it is also available in uranium minerals from decay of²³⁵U [109]. The first production method is most feasible and it has been estimated that one 20 day exposure of 1 cm³ of ²²⁶Ra could meet the demand of Xofigo® in the U.S. for at least five or ten years [110]. The exact yield will be dependent on the flux and energy of the neutrons in the nuclear reactor, but it indicates that the supply through this route should be adequate to sustain commercial use. The production and availability of²²⁸Th and²²⁹Th have been discussed earlier in section 1.6.4 and 1.6.1 respectively.

Radium is an alkaline earth metal, and thereby chemically similar to calcium. Its calcium- mimetic properties cause radium to be naturally targeted to the bones after systemic ad- ministration. The rapid accumulation in sites of new bone formation [24], which coincides with sites of osteoblastic bone metastases, is the property that together with efficient cell kill fromα-particles has made IV injection of²²³Ra-dichloride an effective and relatively safe therapy [111–113]. The bone-seeking property of radium was also exploited med- ically with ²²⁴Ra over many years (1950 [114]-1990 [115] and 2000 [116]-2005 [117]), although not in cancer but as palliative treatment of ankylosing spondylitis, a chronic inflammatory rheumatic disease.

The use of radium isotopes has so far mainly been limited to the above mentioned bone- seeking applications. To direct radium to other tissues than bone, it needs to be transported by a carrier compound. Radium has only one oxidation state (+II) and due to the very al- kaline nature of Ra²⁺ cations in aqueous solutions, radium is not easily complexed and thus most radium compounds are simple ionic salts [118]. This inherent property has made it problematic to couple radium to targeting molecules [119, 120], and no chelating agent that stably binds radium with control of daughter nuclidesin vivoexist to date. New methods to label particle carriers with radium are therefore being explored. Lanthanum phosphate nanoparticles [121], liposomes [68,122], nanozeolites [123,124] and hydroxya- patite nano- and microparticles [125–127] have all been studied. A novel brachytherapy method, termed diffusing α-emitters radiation therapy, using intratumoral ²²⁴Ra-loaded wires is also being researched [128–131]. This work has commenced in an ongoing clinical trial for treatment of squamous cell carcinoma of the skin (NCT03015883 and NCT03353077).

Amongst the three radium isotopes of interest, the supply of²²³Ra and²²⁴Ra through their generator nuclides do not appear to be a concern for routine clinical use, whereas the

availability of²²⁵Ra is scarce and as of today not sufficient. The main obstacle with²²³Ra and²²⁴Ra is thereby not production or availability related, as is the case for several of the otherα-emitting radionuclides, but rather to develop suitable carrier compounds in order to have a utility in cancer management beyond bone targeting applications.

1.6.6 Thorium-227

Thorium-227 has a half-life of 18.7 days and releases fiveα-particles during its decay to stable²⁰⁷Pb. It is the immediate parent radionuclide of ²²³Ra, and highly purified²²⁷Th can be obtained from the same²²⁷Ac generator used for production of²²³Ra [132].

Conjugation of ²²⁷Th to targeting molecules can be performed with the DOTA chelator.

The complex is stable, but as for ²²⁵Ac, it is a two-step procedure with only modest la- beling yields [133]. A octadentate chelator utilizing a 3-hydroxy-N-methyl-2-pyridinone moiety has been developed and reported to give much higher yields and therefore claimed to be superior to DOTA [132]. This chelator is currently being used for coupling of²²⁷Th to two different mAbs in phase I trials of systemic administration in relapsed or refrac- tory non-Hodgkin’s lymphoma (NCT02581878) and advanced epithelioid mesothelioma or serous ovarian cancer (NCT03507452). Similar to²²⁵Ac, the ²²⁷Th-complex will be challenged by recoil energy from theα-emitting daughters that can lead to their release and subsequent redistribution.

The challenges of using²²⁷Th are not related to supply or chemistry, which should be ade- quate and well-established. It is rather the relatively long half-life which can be difficult to match with the pharmacokinetics of a carrier compound. Both the metabolic processing of the compound and recoil energy during decay can lead to release of progeny, which may raise toxicity concerns and limit the dose that can be administered. Decay occur- ring in non-targeted tissues is also a therapeutic disadvantage because the full arsenal of α-particles does not contribute to the radiation dose delivered to the tumor. To optimize retention of daughters in the desired location it is probably advantageous to target inter- nalizing receptors with²²⁷Th.

1.7 Intraperitoneal Radionuclide Therapy in Peritoneal Car- cinomatosis

Peritoneal carcinomatosis (PC) is a condition which occurs when cancer cells from a primary tumor in an adjacent organ spread transcoelomically into the peritoneal cavity and form new tumors. Hallmarks of the condition are IP dissemination of cancer cells not only at diagnosis, but also at relapse. Routine treatment is cytoreductive surgery combined

Table 1.3: Overview of gastrointestinal and gynecological malignancies that can cause peritoneal carcinomatosis (PC), together with the estimated new cases worldwide in 2012 [140] and reported incidences of PC.

Primary cancer site Estimated new cases Incidence of PC at diagnosis

Colorectal cancer 1360000 4-25 % [141, 142]

Gastric cancer 951000 14-40 % [143, 144]

Ovarian cancer 239000 55-60 % [145, 146]

Pancreatic cancer 338000 9 % [147]

with chemotherapy [134–139].

Several of the gastrointestinal and gynecological malignancies can cause PC. As seen in table 1.3, even if the portion of patients presenting with PC at diagnosis varies between the cancer types, the total number of affected patients are high. It is also common that malignant ascites develops as a consequence of the peritoneal metastases. As many as 70 % of patients with PC have been reported to have ascites on computed tomography scans [148]. PC, both with and without the presence of malignant ascites, is regarded as the most common terminal feature of abdominal cancers and generally implies a dismal prognosis [143, 149]. Despite achieving macroscopically complete cytoreductive surgery and giving patients adjuvant chemotherapy, the majority of patients develop recurrent PC.

Recurrent disease is confined to the peritoneal cavity for the majority of ovarian cancer patients, 10-35 % of patients with colorectal cancer and up to 50 % of patients with gastric cancer [143]. As relapse to a large extent occurs in the peritoneal cavity, it emphasizes the need for new adjuvant treatment strategies that eliminate residual microscopic disease after surgery.

Some clinical experience with IP radionuclide therapy of patients with PC, primarily from ovarian carcinoma, exists. Despite α-emitting radionuclides in theory being more suitable for elimination of microsized residual disease in the peritoneal cavity than β- emitters, most clinical experience is with the latter. The investigated compounds are listed in table 1.4, and from here it is seen that particles only have been utilized as car- riers for β-emitting radionuclides, whereas mAbs have been coupled both toα- andβ- emitters.

Already in 1949, Muller performed IP administration of radioactive colloidal gold [150].

The use was associated with a significant incidence of adverse effects, in particular bowel complications [151]. The complications were later ascribed to the γ-component of the radiation in combination with excessive dose (3.7-7.4 GBq) and short physical half-life resulting in high dose-rate. These events lead to a change towards use of colloidal ³²P