A novel 212Pb-labelled PSMA-targeting ligand for alpha therapy of metastatic prostate cancer

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A novel

²¹²

Pb-labelled PSMA-targeting ligand for alpha therapy of metastatic prostate cancer

Thesis for the Degree of Philosophiae Doctor by Vilde Yuli Stenberg

Institute of Clinical Medicine, University of Oslo

Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital

Nucligen AS

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© Vilde Yuli Stenberg, 2023

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

ISBN 978-82-348-0148-8

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

Print production: Graphics Center, University of Oslo.

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Abstract

Patients with metastatic castration-resistant prostate cancer (mCRPC) often experience bone and extraskeletal metastases, resulting in poor prognoses and reduced quality of life. Currently available therapies are not curative. Thus, there is a significant unmet need for novel treatments that can overcome therapeutic resistance. Late-stage mCRPC often overexpresses the prostate- specific membrane antigen (PSMA) that can be targeted by radionuclide therapy. In recent years, several small molecular PSMA-targeting ligands have been developed and used as tumour-targeting vectors for radionuclides. The small radioligands offer potential advantages over monoclonal antibodies, including rapid tumour accumulation and rapid clearance from both circulation and non-targeted tissues, but similarly ensures the delivery of a high dose of therapeutic radiation to cancer cells while minimising exposure to normal cells. PSMA-targeted therapy with alpha emitters may deliver potent and local radiation more selectively to cancer cells than beta emitters and has recently gained remarkable interest for the treatment of mCRPC.

The most clinically studied radionuclides for PSMA-targeted alpha therapy (TAT) are ²²⁵Ac and ²¹³Bi. However, several limitations are associated with the use of these radionuclides, including limited availability, logistics related to the short half-life of ²¹³Bi (t1/2 ≈ 46 min) and salivary gland toxicity caused by ²²⁵Ac. Another suitable radionuclide is the beta emitter ²¹²Pb (t1/2 ≈ 10.6 hours), which acts as an in vivo generator for its alpha-emitting progenies ²¹²Bi and

212Po, generating one alpha particle per ²¹²Pb decay. The radionuclide can be produced in industrial scale from ²²⁸Th/²²⁴Ra-based generators, and has been well tolerated when employed in TAT clinical trials (²¹²Pb-trastuzumab for HER-2 expressing malignancies, and ²¹²Pb- DOTAMTATE and ²¹²Pb-VMT-α-NET for neuroendocrine tumours). This thesis includes preclinical studies investigating the therapeutic potential and safety of the novel PSMA- targeting radioligand ²¹²Pb-NG001 in various models of prostate cancer. The feasibility of a

224Ra/²¹²Pb liquid generator for efficient ²¹²Pb-labelling of the PSMA ligand was demonstrated.

The generator allowed the simple preparation of a radiopharmaceutical solution with dual alpha targeting properties: the natural bone-seeking ²²⁴Ra which targets osteoblastic bone metastases

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and the ²¹²Pb-NG001 which targets PSMA-expressing circulating cancer cells and metastases.

The accumulation of ²²⁴Ra in bones and ²¹²Pb-NG001 in tumour sites was verified in tumour- bearing mice.

The radioligand could also be prepared as a purified product by simple desalting gel exclusion separation. The purified ²¹²Pb-NG001 showed tumour-targeting ability and therapeutic efficacy in in vitro and in vivo models with different PSMA expressions: C4-2 (PSMA+) and PC-3 PIP (PSMA+++). The increase in therapeutic efficacy of the radioligand in the PC-3 PIP compared to the C4-2 model was modest (< 1.8-fold better) considering the 10-fold to 20-fold higher PSMA expression. The lower cellular internalisation and less blood-rich stroma in PC-3 PIP xenografts indicate that these factors also influence the anti-tumour activity of the radioligand.

The ²¹²Pb-NG001 demonstrated high tolerability with no short- or long-term radiotoxicity observed at therapeutic relevant doses (0.3 to 0.4 MBq per mouse). At high activity doses (0.7 and 1.5 MBq), the kidneys were identified as the dose-limiting organ. Reductions in red blood cells and haemoglobin levels were observed, whereas no toxicity was detected in other tissues.

The results presented in this thesis demonstrate that ²¹²Pb-NG001 is a promising candidate for PSMA-TAT of mCRPC and warrant further exploration in early-phase clinical studies in patients. In fact, Nucligen AS is currently initiating a phase 0 study to explore the biodistribution and clearance of the purified ²¹²Pb-NG001 in late-stage mCRPC patients, potentially progressing to a phase I study to investigate the safety and tolerability of the radioligand.

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Sammendrag

Pasienter med metastatisk kastrasjonsresistens prostatakreft har ofte spredning til skjelett, lymfeknuter og lever eller lunger, som fører til redusert livskvalitet og høy dødelighet.

Tilgjengelige behandlinger kan ikke kurere metastatisk prostatakreft. Derfor er det et stort behov for nye, mer målrettede behandlinger. Metastatisk prostatakreft overuttrykker proteinet prostata-spesifikt membranantigen (PSMA) på overflaten av celler, som kan brukes som mål for radionuklidterapi. De siste årene har flere små molekylære PSMA-søkende ligander blitt utviklet og brukt som tumorsøkende vektorer for radionuklider. Små radioligander har flere fordeler over monoklonale antistoffer, inkludert rask akkumulering i tumor og rask utskillelse fra sirkulasjon og andre organer. PSMA-målrettet terapi med alfaemittere gir mer toksisk og lokalisert stråling selektivt til kreftceller sammenlignet med betaemittere. Derfor er det en økende interesse for bruk av alfaemittere i behandlingen av metastatisk kastrasjonsresistens prostatakreft.

Radionuklidene ²²⁵Ac og ²¹³Bi er de mest studerte i kliniske studier for PSMA-rettet alfaterapi.

Utfordringer ved bruk av disse radionuklidene inkluderer begrenset tilgjengelighet, logistikk knyttet til den korte halveringstiden til ²¹³Bi (t1/2 ≈ 46 min) og toksisitet i spyttkjertel forårsaket av ²²⁵Ac. En annen passende radionuklide er betaemitteren ²¹²Pb (t1/2 ≈ 10.6 t) som genererer alfapartikler via dens radioaktive datterisotoper ²¹²Bi og ²¹²Po. Bly-212 kan produseres i industriell skala fra ²²⁸Th/²²⁴Ra-baserte generatorer, og gir lav toksisitet i kliniske studier med målsøkende ligander (²¹²Pb-trastuzumab for HER-2 uttrykkende krefttyper, og ²¹²Pb- DOTAMTATE og ²¹²Pb-VMT-α-NET for neuroendokrine tumorer). Denne avhandlingen inkluderer prekliniske studier som undersøker terapeutisk potensial og toksisitet av den nye PSMA-søkende radioliganden ²¹²Pb-NG001 i forskjellige prostatakreftmodeller. En ²²⁴Ra/²¹²Pb væske-generator ble brukt for effektiv ²¹²Pb-merking av PSMA liganden. Generatoren tillot enkel framstilling av en radiofarmasøytisk løsning med to målrettede komponenter: naturlig

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bensøkende ²²⁴Ra vil angripe benmetastaser, og ²¹²Pb-NG001 vil rettes mot sirkulerende kreftceller og mikrometastaser som uttrykker PSMA. Akkumulering av ²²⁴Ra i skjelett og ²¹²Pb- NG001 i tumor ble bekreftet i mus med PSMA-positive tumorer.

Radioliganden kan også framstilles som et renset produkt uten ²²⁴Ra i løsningen ved hjelp av enkel eksklusjonskromatografi. Renset ²¹²Pb-NG001 viste god tumorsøkende evne og terapeutisk effekt i in vitro- og in vivo-modeller med forskjellig PSMA-ekspresjon: C4-2 (PSMA+) og PC-3 PIP (PSMA+++). Tumoropptak og terapeutisk effekt av radioliganden i PC- 3 PIP modellen var kun 1.8 ganger bedre sammenlignet med C4-2 modellen, til tross for 10–20 ganger høyere PSMA-uttrykk. Lavere cellulær internalisering og mindre blodrik stroma i PC-3 PIP tumorer tyder på at disse faktorene også påvirker anti-tumor aktiviteten til radioliganden.

212Pb-NG001 induserte ingen kortsiktig eller langsiktig radiotoksisitet ved terapeutisk relevante doser (0.3 til 0.4 MBq per mus). Ved høye aktivitetsdoser (0.7 og 1.5 MBq) ble nyrene identifisert som det dosebegrensende organet. Det ble observert en reduksjon i røde blodceller og hemoglobin, mens ingen toksisitet ble observert i andre organer.

Resultatene presentert i denne avhandlingen har vist at ²¹²Pb-NG001 er en lovende kandidat for PSMA-rettet alfaterapi av metastatisk kastrasjonsresistens prostatakreft. Nucligen AS har fått godkjenning til å initiere en fase 0 studie for å se på biodistribusjon og ekskresjon av renset

212Pb-NG001 i pasienter med metastatisk prostata kreft i sent stadium.

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Acknowledgements

The work presented in this thesis was performed at the Department of Radiation Biology, The Norwegian Radium Hospital, Oslo University Hospital and at Nucligen AS. The financial support from Nucligen AS and The Norwegian Research Council is greatly acknowledged.

First, I would like to thank my supervisors Asta Juzeniene, Roy Larsen and Øyvind Bruland for the opportunity to work on this fascinating project with such talented and experienced researchers. I am very grateful for all the guidance and support I have received from you over the past four years. I have learnt so much from working with you – thank you for sharing all your knowledge and expertise. A special thanks to Asta, whom I have worked closely with in the office, lab and at the animal department – your hard work, ambition and enthusiasm are truly inspiring.

I want to acknowledge current and former members of the research group at the Norwegian Radium Hospital for all your help and support in the lab, at the animal facility and during the writing process: Ma, Sivan, Andrea, Petras, Peng, Mantas and Alfonso. Thanks to my PhD partners Anna Julie and Ruth for the great collaboration and nice company in the lab and the office. Thanks to everyone in the R&D department at Oncoinvent for a friendly and inspiring working environment: Marion, Kim, Elisa, Ruth, Tina, Sara, Ida Sofie and Carina. A special thanks to Marion, Kim and Elisa for scientific and non-scientific conversations during hikes, kayak trips and movie nights.

Further, I would like to thank all the co-authors for their great collaborations and invaluable contributions to the papers. Thanks to current and former colleagues in Nucligen AS and Radforsk for interesting discussions and helpful insights: Eva, Anders and Jonas.

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Finally, I would like to thank my family, Vis and my friends for all their support. A special thanks to Mamma, Pappa and Thea, for their continuous love and encouragement, and for always being there for me.

Oslo, June 2022

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Declaration of Interests

The work presented in this thesis was financially supported by the Norwegian Research Council (Industrial PhD grant number 290639), the South-Eastern Norway Regional Health Authority (grant number 2020028) and Nucligen AS.

Vilde Stenberg, Roy Larsen (Sciencons AS) and Øyvind Bruland (Blaahaugen AS) are shareholders in Nucligen AS. Roy Larsen is the chairman of the board of Nucligen AS.

Sciencons AS holds intellectual property rights of the presented dual alpha technology platform and the novel PSMA radioligand (US Patent No. 9433690B1 and 10377778B2). Anna Julie Tornes is employed as an industrial PhD student in Nucligen AS and is financially supported by the Norwegian Research Council (grant number 329538). There are no conflicts of interest to disclose for the co-authors not mentioned above.

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List of Abbreviations

2Nal 2-naphtyl-L-alanine

ACTH Adrenocorticotropic hormone ADT Androgen deprivation therapy

AMCH trans-4-(aminomethyl)-cyclohexanecarboxylic acid

AR Androgen receptor

BSA Bovine serum albumin Bn-SCN Isothiocyanatobenzyl

CRH Corticotropin-releasing hormone CRPC Castration-resistant prostate cancer DHEA Dehydroepiandrosterone

DHT Dihydrotestosterone

DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid DTPA Diethylenetriaminepentaacetic acid

EDTA Ethylenediaminetetraacetic acid EMA European Medicines Agency FBS Fetal bovine serum

FDA U.S. Food and Drug Administration H&E Hematoxylin and eosin

HSA Human serum albumin IHC Immunohistochemistry LET Linear energy transfer LH Luteinising hormone

LHRH Luteinising hormone-releasing hormone mAb Monoclonal antibody

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mCRPC Metastatic castration-resistant prostate cancer NAAG N-acetyl aspartyl glutamate

NSG Non-obese diabetic (NOD) – severe combined immunodeficient (SCID) gamma PARP Poly ADP ribose polymerase

PBS Phosphate-buffered saline PET Positron emission tomography PSA Prostate-specific antigen

PSMA Prostate-specific membrane antigen RCP Radiochemical purity

TAT Targeted alpha therapy

TCMC 1,4,7,10-tetraaza-1,4,7,10-tetra(2-carbamoylmethyl)cyclododecane TI Therapeutic index

TLC Thin layer chromatography TRT Targeted radionuclide therapy

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List of Publications

The following peer-reviewed publications comprise this thesis:

Paper I

In situ Generated ²¹²Pb-PSMA Ligand in a ²²⁴Ra-solution for Dual Targeting of Prostate Cancer Sclerotic Stroma and PSMA-Positive Cells

Stenberg, V.Y.; Juzeniene, A.; Bruland, Ø.S.; Larsen, R.H. Current Radiopharmaceuticals.

2020, 313, 130-141.

Paper II

Preparation of the alpha-emitting prostate-specific membrane antigen targeted radioligand [²¹²Pb]Pb-NG001 for prostate cancer

Stenberg, V.Y.; Juzeniene, A.; Chen, Q.; Yang, X.; Bruland, Ø.S., Larsen, R.H. Journal of Labelled Compounds and Radiopharmaceuticals. 2020, 63, 129-143.

Paper III

Evaluation of the PSMA-Binding Ligand ²¹²Pb-NG001 in Multicellular Tumour Spheroid and Mouse Models of Prostate Cancer

Stenberg, V.Y.; Larsen, R.H.; Ma, L.W.; Peng, Q.; Juzenas, P.; Bruland, Ø.S.; Juzeniene, A.

International Journal of Molecular Sciences. 2021, 22, 4815.

Paper IV

Factors influencing the therapeutic efficacy of the PSMA-targeting radioligand ²¹²Pb- NG001

Stenberg, V.Y.; Tornes, A.J.K.; Nilsen, H.R.; Revheim, M.E.; Bruland, Ø.S.; Larsen, R.H.;

Juzeniene, A. Cancers. 2022, 14, 2784.

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In addition, I have contributed to the following papers during the PhD project period:

Calibration of sodium iodide detectors and reentrant ionisation chambers for ²¹²Pb activity in different geometries by HPGe activity determined samples

Napoli, E.; Stenberg, V.Y.; Juzeniene, A.; Hjellum, G.E.; Bruland, Ø.S.; Larsen, R.H. Applied Radiation and Isotopes. 2020, 166, 109362

Preclinical and Clinical Status of PSMA-Targeted Alpha Therapy for Metastatic Castration-Resistant Prostate Cancer

Juzeniene, A.; Stenberg, V.Y.; Bruland, Ø.S.; Larsen, R.H. Cancers. 2020, 13, 779

A Novel Experimental Generator for Production of High Purity Lead-212 for Use in Radiopharmaceuticals

Li, R.G.; Stenberg, V.Y.; Larsen, R.H. Manuscript submitted for publication. 2022.

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1 Introduction

1.1 Prostate cancer

Prostate cancer is the second most common cancer in men worldwide with an estimated incidence of 1.4 million new cases and 375 000 associated deaths yearly [1]. The incidence and mortality of prostate cancer increases with age, and the average age at the time of diagnosis is 66 years [2]. The majority of patients have localised disease (80%) or loco-regional disease (12%), while less than 5% have metastatic disease at the time of diagnosis. Disseminated disease is the leading cause of prostate cancer related deaths with a 5-year survival rate of only 31% [3,4].

Initially, prostate cancer is hormone-sensitive and tumour growth is dependent on androgens, such as testosterone and its derivative dihydrotestosterone (DHT). The majority of testosterone is synthesised in the testes (> 90%), while the remaining is mainly produced in the adrenal glands [5]. The luteinising hormone-releasing hormone (LHRH) and the corticotropin-releasing hormone (CRH) produced by the hypothalamus stimulate the release of the luteinising hormone (LH) and the adrenocorticotropic hormone (ACTH), respectively, from the anterior pituitary gland into the blood (Figure 1.1). The LH activates the production of testosterone by Leydig cells of the testes while ACTH triggers the production of testosterone and the other androgen dehydroepiandrosterone (DHEA) by the adrenal glands. A negative feedback loop regulates hypothalamic and pituitary hormone secretion [5,6].

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Introduction

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Figure 1.1. Hormonal regulation of androgen production. This figure was inspired by Shore et al. [6], and created using illustrations from Servier Medical Art (smart.servier.com) and BioRender (biorender.com). LHRH: luteinising hormone-releasing hormone; CRH: corticotropin-releasing hormone; LH: luteinising hormone; ACTH: adrenocorticotropic hormone; DHEA:

dehydroepiandrosterone.

The activity of the androgens is mediated via the androgen receptor (AR), which is expressed both in the prostate luminal epithelial cells and stromal cells [7]. Once inside the prostatic cells, testosterone is converted into the more potent AR ligand, DHT, by the enzyme 5-alpha reductase. The AR binds the androgens in the cytoplasm and translocates into the nucleus where it, in complex with other coregulator proteins, induces or maintains the transcription of AR- targeted genes, including genes involved in cell growth, proliferation and survival as well as genes encoding seminal proteins, such as the prostate-specific antigen (PSA) [5,8]. Cancerous prostate cells exhibit excess activation of the androgen signalling pathway, resulting in an uncontrolled proliferation of tumour cells [8]. The increased PSA levels that can be detected in the serum of prostate cancer patients is believed to result from disruptions of the prostatic cell and basement membranes followed by increased leakage of PSA into circulation [9]. Therefore, serum PSA is used as a diagnostic marker for prostate cancer and as a measure of treatment effectiveness in patients with active disease (Figure 1.2). The PSA is not a specific biomarker for prostate cancer and can also be detected in patients experiencing for example benign prostatic hyperplasia and infection or inflammation of the prostate [9].

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Introduction

3 Figure 1.2. Progression of prostate cancer and available treatments. This figure was modified from Abou et al. [10] and created using illustrations from BioRender. PSA: prostate-specific antigen; ADT:

androgen deprivation therapy.

Patients with localised primary prostate cancer have several treatment options including active surveillance, radical prostatectomy, external beam therapy and prostate brachytherapy (Figure 1.2) [5]. The first line of therapy for hormone-sensitive prostate cancer is androgen deprivation therapy (ADT), which involves chemical castration with LHRH agonists or antagonists.

Chemical castration lowers testicular testosterone production and thus reduces androgen levels in the blood, inhibiting AR activation and thereby the growth and proliferation of prostate cancer cells. The castrate level of serum testosterone is defined as < 0.5 ng/mL [5,8]. Castration does not affect the production of CRH and ACTH, and consequently, androgens produced by adrenal glands and prostate can still fuel cancer growth. Additional suppression of prostate cancer cell growth could be achieved with androgen synthesis inhibitors that target the testosterone biosynthesis and AR blockers, or anti-androgens, that prevent binding of androgens to the AR [5,8].

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Introduction

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The majority of prostate cancer patients (80% to 90%) respond to ADT. However, within 5 years, 10% to 20% of patients develop castration-resistant prostate cancer (CRPC; Figure 1.2) [8,11,12]. In CRPC, the cancer cells are stimulated by minimal androgen levels in the blood or grow independently of androgen stimulation. This is achieved through several mechanisms that involve the AR, including adrenal gland and intratumoural production of androgens, gene amplification or overexpression of the AR, constitutively active ligand-independent AR splice variants, and gain-of-function mutations involving the AR ligand-binding domain [8,13].

Metastatic CRPC (mCRPC) can either progress from non-metastatic CRPC or metastatic hormone-sensitive prostate cancer (Figure 1.2). The most common metastatic sites are bone followed by regional lymph nodes, the liver and the lungs (Figure 1.3) [14-20]. Regional lymph node and visceral metastases are seen in 5% to 15% of newly diagnosed mCRPC patients but increase over time to affect almost 50% of patients during the course of their disease [14,18].

mCRPC is associated with decreased quality of life and poor prognoses with median overall survival of 16.3 months in patients with any visceral disease, 21.3 months in those with non- visceral bone metastases and 31.6 months in those with only lymph node metastases [11,20].

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Introduction

5 Figure 1.3. The most common sites of metastases in mCRPC patients. In mCRPC patients, cancer cells from the primary tumour have spread to surrounding or distant tissues via blood or lymphatic vessels (left), most frequently to bone, regional lymph nodes, the liver and the lungs (right). This figure was created using illustrations from Servier Medical Art and is based on data from literature [14-20].

The topoisomerase II inhibitor mitoxantrone was approved by the U.S. Food and Drug Administration (FDA) for palliative treatment of mCRPC in 1996. This drug improved pain and quality of life for patients but showed no survival benefit [21,22]. In 2004, the microtubule inhibitor docetaxel was the first therapy to demonstrate survival benefits for mCRPC patients.

Docetaxel increased median survival by 2.9 months compared to mitoxantrone, and was approved and established as the new standard of care for mCRPC patients (Table 1.1) [22,23].

After docetaxel treatment, mCRPC eventually progresses due to innate or acquired resistance and must subsequently be treated with other treatment options. In the last decade, eight novel agents have demonstrated survival benefit in mCRPC patients (Table 1.1) and have been approved by the FDA: the chemotherapy cabazitaxel, the hormone therapies abiraterone acetate and enzalutamide, the autologous immunotherapy sipuleucel-T, the targeted radionuclide therapies (TRT) ²²³Ra dichloride and ¹⁷⁷Lu-PSMA-617, and the poly ADP ribose polymerase (PARP) inhibitor therapies olaparib and rucaparib. The first five have also been approved by the European Medicines Agency (EMA).

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Introduction

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Cabazitaxel improved survival in patients progressing during or after docetaxel-based treatment, whereas abiraterone acetate and enzalutamide have shown promise in both pre- and post-docetaxel settings [24-28]. The other approved treatments are suitable for specific mCRPC indications; Sipuleucel-T is indicated for asymptomatic or minimally symptomatic disease,

223Ra dichloride is suitable for patients with bone metastases but no extraskeletal metastases,

177Lu-PSMA-617 is appropriate for prostate cancers overexpressing the prostate-specific membrane antigen (PSMA) and PARP inhibitor therapy is for patients with homologous recombination DNA repair gene mutations [29-33].

Currently available therapies of mCRPC are not curative and demonstrate modest survival benefit (Table 1.1). Thus, there is a significant unmet need for novel targeted therapies that can overcome therapeutic resistance. Several novel therapies, including AR down regulators, TRT and immune-directed therapies, as well as combinational therapies, are currently under investigation in clinical trials [11,34-38].

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Introduction

7 Table 1.1. Overview of approved treatments for metastatic castration-resistant prostate cancer (mCRPC). FDA: U.S. Food and Drug Administration; EMA: European Medicines Agency; NR: not reached; AR: androgen receptor; PARP: poly ADP ribose polymerase; HRR: homologous recombination DNA repair; NA: not available; PSMA: prostate-specific membrane antigen.

*pre-docetaxel

**post-docetaxel

***objective response rate used as endpoint Treatment

(approved by FDA/EMA)

Mechanism of

action Indication Control

Median overall survival benefit vs

control (months) Docetaxel

(2004/2007)

Microtubule inhibitor

(inhibits mitosis) mCRPC Mitoxantrone 19.2 vs 16.3 [23]

Cabazitaxel (2010/2011)

Microtubule inhibitor

(inhibits mitosis) mCRPC, post-docetaxel Mitoxantrone 15.1 vs 12.7 [24]

Sipuleucel-T (2010/2013)

Autologous immunotherapy

Asymptomatic or minimally

symptomatic mCRPC Placebo 25.8 vs 21.7 [29]

Abiraterone acetate (2011/2011)

Androgen synthesis inhibitor (by cytochrome P450

inhibition)

mCRPC, pre- or post- docetaxel

Prednisone NR vs 21.7 [25]^* 15.8 vs 11.2 [26]^**

Enzalutamide

(2012/2013) AR inhibitor mCRPC, pre- or post-

docetaxel Placebo 32.4 vs 30.2 [27]^* 18.4 vs 13.6 [28]^**

223Ra dichloride (2013/2013)

Targeted alpha therapy (bone-targeting)

mCRPC with bone but no

visceral metastases Placebo 14.9 vs 11.3 [31]

Olaparib

(2020/-) PARP inhibitor

mCRPC with HRR gene mutations, post- enzalutamide or abiraterone

Enzalutamide/

abiraterone acetate

17.3 vs 14.0 [32]

Rucaparib

(2020/-) PARP inhibitor

mCRPC with HRR gene mutations, post- hormone

and chemotherapy

NA NA^***

177Lu-PSMA- 617 (2022/-)

Targeted radionuclide therapy (PSMA-targeting)

mCRPC, post- docetaxel/cabazitaxel and

post-AR inhibition

Standard care 15.3 vs 11.3 [33]

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Introduction

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1.2 Targeted radionuclide therapy

TRT is based on the targeting of radionuclides to tumour cells, either by natural accumulation in tumour tissue or using cancer-specific carrier molecules, including monoclonal antibodies (mAbs), antibody fragments or small molecular ligands (Figure 1.4). The radiolabelled targeting agent ensures the delivery of a high dose of therapeutic radiation to cancer cells while minimising exposure to normal cells. The specificity of TRT allows the treatment of systemic malignancies, such as bone and extraskeletal metastases, whereas a whole-body irradiation using external beam radiotherapy would not be possible [39,40].

Figure 1.4. Targeted radionuclide therapy ensures the delivery of a therapeutic radiation dose to cancer cells by using carrier molecules, such as monoclonal antibodies or small molecular ligands, that specifically target antigens on the surface of cancer cells. Some antigens are internalised upon ligand binding, leading to increased tumour uptake and retention. This figure was created using illustrations from Servier Medical Art and BioRender.

1.2.1 Selection of target antigen for TRT

An ideal target antigen for TRT should be highly expressed on the surface of tumour cells and have low or negligible expression in normal cells [39,41]. Several tumour-associated antigens, including PSMA, prostate stem cell antigen, human epidermal growth factor receptor 2, mucin 1 and the gastrin-releasing peptide receptor, have been investigated for targeted therapy of mCRPC (Table 1.2) [40,42,43]. Some cell surface antigens are rapidly internalised after ligand binding, leading to increased tumour uptake and retention [39,44].

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Introduction

9 Table 1.2. Overview of selected investigated tumour-associated antigens for targeted therapy of mCRPC [40,42,43]. PSMA: prostate-specific membrane antigen; NAAG: N-acetyl aspartyl glutamate; PSCA:

prostate stem cell antigen; HER-2: human epidermal growth factor receptor 2; MUC1: mucin 1; GRPR:

gastrin-releasing peptide receptor.

1.2.2 Choice of targeting agent

The targeting molecule of a radiopharmaceutical should have high affinity and specificity for its target. The carrier must have chelation chemistry compatible with the selected radionuclide, either via a suitable chelator or a functional group that allows direct radiohalogenation, and be able to withstand radiolytic degradation under storage conditions prior to administration. In addition, the carrier molecule should not be toxic or immunogenic [39,56]. Earlier, the most widely used targeting agents in TRT were mAbs. However, the high molecular weight (150 kDa of intact mAb) causes long circulatory half-life, typically associated with high bone marrow toxicity and slow clearance from non-targeted tissues (Table 1.3). Antibodies also exhibit slow diffusion into the tumour and accumulate in the liver due to hepatobiliary clearance and Fc receptor-mediated recycling [57].

Target

antigen Function Overexpression in prostate cancer

Expression in normal tissues

PSMA Folate and NAAG hydrolase activity

60% to 94% of primary prostate cancers; increases at advanced

disease stages [45-48]

Prostate, kidneys, intestines, brain and

salivary glands;

significantly lower than in cancer cells

PSCA Unknown

88% to 94% of primary prostate cancers; increases at advanced

disease stages [49-51]

Prostate, stomach, intestines and brain;

HER-2

Activates pathways which promote cell growth and proliferation

6% to 68% of primary cancers, 42% to 78% of metastatic

tumours [52,53]

Epithelial cells of skin, gastrointestinal, respiratory, reproductive and urinary tract systems;

significantly lower than in cancer cells MUC-1 Protects and lubricates

epithelial surfaces

60% of primary prostate cancers; increases at advanced

stages (90% of lymph node metastases) [42]

Prostate, lungs and intestines; significantly lower than in cancer cells

GRPR

Regulates numerous functions of the gastrointestinal and central nervous systems

63% to 100% of primary prostate cancers; inversely

correlated with disease progression [54,55]

Pancreas and stomach;

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Smaller targeting vehicles, such as small molecular ligands, offer potential advantages over mAbs, including easy synthesis and modification, rapid clearance from circulation and non- targeted tissues, and rapid accumulation in tumour (Table 1.3). On the other hand, smaller molecules tend to have a shorter retention time in tumours and are rapidly cleared via the renal route, typically resulting in relatively higher kidney doses than obtained with larger molecules [56-58].

Table 1.3. Characteristics of small molecular ligands, antibody fragments and antibodies [40,58-61].

1.2.3 Radionuclide selection

Therapeutic radionuclides can be divided into three subgroups: alpha, beta and Auger emitters (Table 1.4). A range of different radionuclides with a variety of half-lives, particle ranges and chemical properties allows for the selection of the most suitable radionuclide for a specific therapeutic application [56,62].

Characteristics Small molecular

ligands Antibody fragments Antibodies Molecular weight < 1.5 kDa 15 kDa to 110 kDa ~ 150 kDa

Structure One peptide chain One to four shorter

polypeptide chains Four polypeptide chains

Production Easy Difficult Difficult

In vivo half-life Few hours 0.5 h to 30 h > 3 d to 7 d Pharmacokinetics

Rapid clearance from blood, rapid tissue

penetration

Rapid to intermediate clearance from blood, rapid to intermediate

tissue penetration

Long circulatory half- life, slow tissue

penetration Excretion Renal clearance

Renal clearance of antibody fragments

< 60 kDa

Hepatobiliary clearance and Fc receptor- mediated recycling

Immunogenicity Low Low to moderate Expected

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Introduction

11 Table 1.4. Characteristics of therapeutic radionuclides [39,44,61-63].

Characteristics Alpha emitters Beta emitters Auger emitters Emission per decay 1 helium nucleus 1 high-energetic

electron

5−30 low-energetic electrons Energy 5 MeV to 9 MeV^* 0.05 MeV to 2.3

MeV^** A few eV to 1 keV^* Range 40 µm to 100 µm 50 µm to 12 000 µm 0.002 µm to 0.5 µm Linear energy transfer 80 keV/µm to

100 keV/µm ~ 0.2 keV/µm 4 keV/µm to

26 keV/µm Ionisations Dense ionisations Sparse ionisations Dense ionisations DNA damage

Poorly repairable (clusters of DNA

damage)

Easily repairable (individual DNA

lesions)

Poorly repairable (multiple DNA

lesions) Number of DNA hits to

kill a cell < 10 100−1000 < 10

Requisite for cell killing Binding to cancer cell

Within the targeted tissue

Translocation into the nucleus

Oxygen effect Low High Low (nuclear), high

(extranuclear)

Bystander effect Yes Yes Yes

Tumour cross-fire effect Yes Yes No

*monoenergetic

**average; continuous spectrum of energies

Beta emitters have been the most widely used radionuclides for TRT. The linear energy transfer (LET) of beta particles is low (~ 0.2 keV/µm), producing sparse ionisations and individual DNA lesions that are easily repairable [62,64]. The long range of beta particles in tissue (0.05 mm to 12 mm), corresponding to up to several hundred cell diameters, allow the damage of tumour cells adjacent to the targeted cells (Figure 1.5). This energy deposition in adjacent cells which are not themselves specifically targeted, called cross-fire irradiation, is particularly desirable when not all tumour cells can be reached by the radiolabelled molecules due to heterogenous antigen expression or a poor distribution of molecules in the tumour tissue. Therefore, beta emitters are suitable for the treatment of bulky, heterogenous tumours, whereas they are inefficient for treating single cancer cells or micrometastases (< 2.0 mm) since most of the emitted energy will be absorbed in the surrounding healthy tissue [44,56,64,65].

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Introduction

12

Figure 1.5. Tissue range of alpha particles, beta particles and Auger electrons relative to a cell diameter.

The path length corresponds to less than a cell diameter for Auger electrons, a few cell diameters for alpha particles and up to several hundred cell diameters for beta particles, depending on their energies.

This figure was modified from Pouget et al. [64] and created using illustrations from Servier Medical Art and BioRender. α: alpha particle; β: beta particle.

In contrast, alpha emitters have high LET (~ 80 keV/µm to 100 keV/µm) and short range in tissue (< 0.1 mm, corresponding to a few cell diameters), making them suitable for the treatment of microscopic or small-volume disease due to the more efficient and specific killing of tumour cells while sparing surrounding normal tissue [56,66]. The high LET of alpha particles produces clusters of DNA damage that are difficult to repair, leading to high cytotoxicity that is independent of dose rate and local oxygen concentration. Since alpha emitters have short range, and therefore less cross-fire ability than beta emitters, actual binding of radiolabelled molecules to the cancer cells is often required for the therapy to be effective [64].

Low-energy Auger electrons are emitted during electron capture or internal conversion (average 5−30 Auger electrons). They have relatively dense ionisations with high toxicity, but their translocation into the nucleus, and preferably incorporation into the DNA, is necessary for effective cell killing due to the very short range of the radiation (2 nm to 500 nm). Therefore, Auger emitters may be suitable for the treatment of micrometastases that abundantly express internalising antigens [44,62].

Other considerations regarding radionuclide selection are physical half-life and co-emission of imageable photons as well as availability and production mode of the nuclide. To achieve optimal therapeutic efficacy, the physical half-life of the radionuclide should match the

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Introduction

13 biological half-life of the targeting molecule [44]. An antibody exerting slow tumour penetration and long circulatory half-life should not be labelled with a radionuclide with a too short half-life. This would cause a large part of the radionuclides to decay when the conjugate is still outside the tumour, possibly contributing to the irradiation of healthy tissue. On the other hand, if the physical half-life of the radionuclide is too long compared to the biological half-life of the targeting molecule, a large amount of the radiation dose will be delivered when the carrier has started to clear from the tumour tissue and are being metabolised or excreted, increasing exposure to normal tissues and organs responsible for clearance from the body [44]. In addition, the half-life of the radionuclide should preferably be more than a few hours to allow sufficient time for production and transportation but should avoid extended radiation effects after administration in patients.

To be suitable for routine clinical use, the radionuclides should be readily available and possible to produce in a cost-efficient way. The radiochemical properties of the radionuclide should enable high-yield labelling of targeting molecules with a suitable specific activity. The decay of a radionuclide is often accompanied by the emission of photons (gamma or X-rays). An abundance of high-energy photons is undesirable since it would lead to whole-body irradiation, while low-energy photons (100 keV to 200 keV) are advantageous for imaging and therapy monitoring [44].

1.2.4 Radiolabelling of targeting molecules and radiolysis

Radioactive isotopes of halogens, e.g., fluorine, iodine and astatine, can be directly integrated into a molecule by electrophilic or nucleophilic substitution, typically at tyrosine residues, or indirectly via a prosthetic group. Prosthetic groups contain two functional moieties: one enables efficient radiohalogenation and the other enables rapid coupling to functional groups available on proteins and peptides, such as the amino group of a lysine residue or at the N-terminus, or the thiol group of a cysteine residue [44,67]. In contrast, radioactive metal ions have reduced reactivity compared to halogens and are typically incapable of forming stable covalent bonds with elements present in proteins and peptides. Thus, labelling with radioactive metals is preferably performed with the use of chelators, which form non-covalent complexes with the metal, called chelates. The chelator could be bi-functional, i.e., contain functional moieties for both the chelation of the radionuclide and incorporation into the protein or peptide, or be incorporated during the synthesis of, for example, a smaller molecular ligand [44,67].

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Introduction

14

The stability of the chelator should be high with a slow dissociation rate in vivo since a number of blood plasma proteins also have chelating properties and could cause trans-chelation of the radionuclides from the radiolabelled molecule [44]. Furthermore, radionuclide loss from a radiopharmaceutical could be followed by an undesirable accumulation in healthy tissues. For example, actinium and lead accumulate in bone and liver tissue, and bismuth and leadlocalise in kidneys [68-71].

Radiolytic degradation, or radiolysis, of the targeting molecule could be a potential problem during the labelling, storage and distribution of the radiopharmaceutical. The greatest challenge is radiolysis during labelling since the radioactivity concentration is highest at this point. It has been reported that the addition of the radioprotectants ascorbic acid and human serum albumin efficiently protects antibodies during labelling with radiometals without interfering with metal chelation [44,72,73]. Generally, the prevention of radiolysis is simpler during storage since lower radioactivity concentrations can be used. Dilution of the radiolabelled conjugate or addition of ascorbic acid and/or human serum albumin appreciably reduces radiolysis [44,72,73].

1.2.5 Radiobiology

TRT delivers therapeutic doses of ionising radiation to specific disease sites and induces biological effects by 1) the direct interaction of ionising radiation with DNA, leading to damage, including single-strand and double-strand breaks, DNA base damage and DNA-protein crosslinks, or 2) the interaction of ionising radiation with water, generating reactive oxygen that can react with and damage biomolecules, including DNA, RNA, proteins and phospholipids (Figure 1.6) [56,74].

DNA damage activates the serine/threonine protein kinases ataxia-telangiectasia mutated (ATM), ATM- and RAD3-related (ATR) and DNA-dependent protein kinase, which, in turn, activate proteins involved in cell cycle control and DNA repair. Depending on the extent of the damage and the repair capability of the cells, the outcome is either cell death (apoptosis, autophagy or necrosis) or survival (Figure 1.6) [64].

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Introduction

15 As mentioned, low LET beta particles produce sparse ionisations and individual DNA lesions that are easily repairable (Table 1.4), and 100−1000 beta particles are needed to induce cell death. In comparison, high LET alpha particles produce clusters of DNA damage that are difficult to repair, and only 1–5 alpha particles hitting the DNA are needed to kill a single cell [39,62,64]. Unrepaired or erroneously repaired double-strand breaks lead to cell death or a surviving cell with altered genome, where chromosomal translocations or deletions may affect tumour suppressor genes and oncogenes [64,75].

Figure 1.6. Direct and indirect interaction of ionising radiation with DNA. Depending on the extent of the resulting DNA damage and the repair capability of cells, the outcome is either cell survival, carcinogenesis or cell death. This figure was created using Servier Medical Art. α: alpha particle; β: beta particle; ROS: reactive oxygen species.

For beta particles and extranuclear Auger electrons, the probability of direct ionisation of DNA is low, and cell death mechanisms mainly rely on indirect effects, in particular, the formation of reactive oxygen species. This effect is supressed in hypoxic environments due to lowered oxygen density and is a mechanism of radioresistance in hypoxic tumours. In contrast, the cytotoxic efficacy of alpha particles is less dependent on local oxygen concentration since most biological effects would result from the direct ionisation of target molecules [64,76]. Besides DNA, radiation-sensitive targets in cells include the membrane lipids and mitochondria, and damage to these subcellular targets may activate cell death pathways [64,74,75].

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Introduction

16

Absorbed dose in gray [Gy] is defined as the amount of ionising radiation absorbed per unit mass of material [J kg^-1]. The absorbed dose needed to achieve a given level of biological damage varies for different kinds of radiation. High LET radiation is generally more damaging to a biological system per unit dose than low LET radiation [64,77]. The relative biological effectiveness – the ratio of the absorbed tumour doses of the radionuclides that produce the same biological effect – has been reported to be 2−7.2 for TRT with alpha versus beta emitters [66,78]. Biological effects depend not only on the total dose to the tissue but also on the rate at which the dose is received, measured as the absorbed dose rate [Gy s^-1]. Repair mechanisms make it possible for cells to recover if they have not been too severely damaged, provided that the dose is supplied at a sufficiently slow rate [64,77]. Because high LET damage is not easily repaired, and can be produced by a single hit in the DNA, no dose rate effect is expected after alpha particle irradiation [62,64]. The repair capacity of the cells is significantly higher for low LET versus high LET radiation at low radiation doses. This difference in repair capacity diminishes at higher doses, so the relative biological effectiveness might be lower at clinically relevant absorbed radiation doses [78].

Biological effects after irradiation can also arise in cells that are in proximity to the irradiated cells. In this process, called the radiation-induced bystander effect, irradiated cells send signals to neighbouring cells, either by release of soluble factors or by direct cell communication via gap junctions, leading to a variety of responses, including DNA damage and cell death. This radiation-induced bystander effect has been reported to contribute to up to 30% of cell killing after alpha irradiation [74,76,79]. Furthermore, abscopal bystander responses, believed to arise from an upregulated immune response to radiotherapy, could result in anti-tumour effects in remote lesions [56,76].

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Introduction

17

1.3 PSMA-TRT of mCRPC

PSMA is a well characterised target antigen in prostate cancer and has been widely used as a molecular target for imaging diagnostics and TRT. Several PSMA-targeting agents and radionuclides have been evaluated for PSMA-TRT.

1.3.1 PSMA

PSMA, also known as folate hydrolase 1 or glutamate carboxypeptidase II, is a transmembrane glycoprotein with a molecular weight of about 100 kDa. The enzymatically active form of PSMA is a symmetric dimer with each polypeptide chain containing three domains: an intracellular domain (amino acids 1−18) responsible for internalisation, a transmembrane domain (amino acids 19−43), and a large extracellular domain (amino acids 44−750) responsible for enzymatic function (Figure 1.7) [80-83].

Figure 1.7. Schematic representation of PSMA. Binding sites for PSMA-targeting molecules are presented. This figure was inspired by Maurer et al. [84] and created using illustrations from Servier Medical Art. mAb: monoclonal antibody; Gly: glycine; Pro: proline.

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Introduction

18

PSMA is highly overexpressed in prostate cancers, and this expression increases with tumour aggressiveness, androgen independence and metastatic disease [46,85-87]. In addition, PSMA is overexpressed in the neovascular endothelium of most solid tumours, including lung, colon, breast and renal cancers, but is not expressed in normal vasculature [88]. PSMA is expressed at low levels (100-fold to 1000-fold lower) in healthy prostate, kidney, intestine, brain, and lacrimal and salivary glands [48,89,90].

The catalytic domain of PSMA executes enzymatic activities including the hydrolysis of polyglutamated folate and N-acetyl aspartyl glutamate (NAAG) by releasing the terminal glutamate residue of the substrate molecules. It has been suggested that elevated PSMA activity in prostate cancer cells increases cell folate uptake and proliferation, facilitating prostate carcinogenesis and progression [82,91]. The substrate binding cavity of PSMA consists of the active site containing two zinc ions, the S1’ glutamate recognition pocket and an entrance funnel connecting the active site to the external surface of PSMA (Figure 1.8). The S1’ pocket is restricted in size and shape, while the entrance funnel is more spacious and can accommodate functional groups of different sizes and physicochemical characteristics. The entrance funnel contains a binding site for aromatic hydrocarbons (an arene-binding site) and an S1 accessory hydrophobic pocket that includes an arginine-rich region [81,92-94].

Figure 1.8. The substrate binding cavity of PSMA. The binding of a small molecule PSMA inhibitor (PSMA-1007) is presented. This figure was adapted from Fakiri et al. [94].

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Introduction

19 PSMA continuously undergoes internalisation which may reflect the recycling of a structural protein or may be mediated by the binding of a ligand. The internalisation rate is enhanced up to 5-fold by the binding of PSMA-specific mAbs and radioligands [95,96]. Upon binding, the antigen-carrier complex is transported to the lysosomal compartment where the PSMA receptor undergoes degradation or is recycled intact back to the plasma membrane via the recycling endosomal compartment. The carrier molecule undergoes degradation, and radiocatabolites of radiometal-labelled ligands will be retained within the cell, increasing anti-cancer effects [44,97,98].

1.3.2 PSMA-targeting molecules

Several approaches have been used for targeting PSMA; mAbs and antibody-based molecules are developed to selectively and specifically bind to intracellular or extracellular epitopes of PSMA, while small molecule inhibitors exploit the enzymatic activity of PSMA and bind to the substrate binding cavity of the receptor (Figures 1.7 and 1.8) [40,81].

The first anti-PSMA antibody, murine mAb 7E11-C5.3, targeted an intracellular epitope of PSMA and was therefore not able to bind to viable cells. This limitation led to the development of a number of second-generation mAbs specific to extracellular epitopes of PSMA. Several of these mAbs and their fragments are being evaluated for TRT in preclinical and clinical studies, including J591, D2B, 107-1A4, J415 and PSMA-TTC [40,99-103]. Although the mAbs and their fragments have demonstrated promising results in diagnostic and therapeutic radiopharmaceuticals, small molecule inhibitors offer potential advantages over antibodies (Table 1.3) and have recently gained remarkable interest as vehicles in PSMA-TRT.

Small molecule PSMA inhibitors generally contain a strong zinc binding group to interact with the active site and a glutamate moiety to bind to the S1’ pocket of the enzyme (Figure 1.8). The PSMA inhibitors are classified into three groups: phosphorous- (including phosphonate, phosphate and phosphoramidate), urea- and thiol-based compounds (Figure 1.9) [83].

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Introduction

20

Figure 1.9. Classes of PSMA inhibitors: phosphorus-, urea- and thiol-based compounds. Phosphorus- based inhibitors are transition state analogues which mimic the hydrolysation state of the N-acetyl aspartyl glutamate (NAAG) substrate (yellow), while urea-based inhibitors are substrate analogues which mimic the peptide bond of NAAG (red). PSMA inhibitors typically contain a glutamate moiety (blue). This figure was modified from Wüstemann et al. [83].

Phosphorus-based inhibitors are transition state analogues, where the phosphorus-based motif mimics the transition state of the hydrolysation reaction of the natural NAAG substrate (Figure 1.9) [83]. The first reported potent PSMA inhibitor was the phosphorus-based compound 2- (phosphonomethyl)pentanedioic acid (2-PMPA). The 2-PMPA has high affinity and selectivity and has been used as a template in structure-activity relationship studies to yield other potent PSMA inhibitors, including GPI 5232, VA-033 and phenylalkylphosphonamidates. However, the phosphorus-based compounds are highly polar and have relatively poor pharmacokinetic profile, which limits their clinical applications [82,92,104]. To reduce the polarity of the inhibitors, the phosphorus-containing groups were replaced by thiol groups, leading to the development of several thiol-based inhibitors (Figure 1.9), including the most potent 2-(3- mercaptopropyl)pentanedioic acid (2-MPPA) among other analogues. These inhibitors demonstrated improved pharmacokinetic profile, but their metabolic stability and selectivity were not adequate for clinical application [82,105,106].

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Introduction

21 Currently, the most explored and advanced class of PSMA inhibitors are the urea-based compounds. Urea-based inhibitors are substrate analogues, where the urea motif mimics the peptide bond of the natural NAAG substrate (Figure 1.9) [83]. The inhibitors usually consist of three components: a PSMA-binding motif, a linker and a radiolabel-bearing moiety. In general, there are four urea-based PSMA-binding motifs: glutamate-urea-lysine (Glu-urea-Lys), glutamate-urea-glutamate (Glu-urea-Glu), glutamate-urea-cysteine (Glu-urea-Cys) and glutamate-urea-tyrosine (Glu-urea-Tyr). The Glu-urea-Lys scaffold is the most widely used binding motif, in which the lysine moiety forms hydrogen bonds with arginine and asparagine residues in the S1 pocket of PSMA (Figure 1.8) [107,108]. The linker region connects the binding motif to the radiolabel-bearing moiety and is essential for positioning the bulky radiolabel-bearing entity outside the active site in the spacious entrance funnel so it will not interfere with binding to the active site. The linker is also used to adjust molecular size and polarity to impact the pharmacokinetic characteristics of the ligand [108-110].

Urea-based PSMA inhibitors were first reported by Kozikowski et al. in 2001 [111]. The first radiolabelled inhibitors (¹¹C-DCMC and ¹²⁵I-DCIT) were developed in 2002 [112], while the first inhibitors for prostate cancer imaging (¹²³I-MIP-1072 and ¹²³I-MIP-1095) were introduced into the clinic in 2008 [113-115]. Later, a variety of radiolabelled urea-based PSMA ligands were investigated in clinical trials, including diagnostic ^99mTc-MIP-1404/-1405, ⁶⁸Ga-PSMA- 11, ¹⁸F-DCFBC, ¹⁸F-DCFPyl and ¹⁸F-PSMA-1007, and theragnostic/therapeutic ¹²³I/ ¹²⁴I/¹³¹I- MIP-1095, ⁶⁸Ga/¹¹¹In/¹⁷⁷Lu/²²⁵Ac-PSMA-I&T and ⁶⁸Ga/⁴⁴Sc/¹¹¹In/¹⁷⁷Lu/²²⁵Ac/²¹³Bi-PSMA- 617 (Table 1.5) [93,109]. PSMA theragnostics enable a highly personalised approach using PSMA-positron emission tomography (PET) and computed tomography to image and quantify PSMA expression to select patients most likely to benefit from PSMA-TRT [116-118].

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Introduction

22

Table 1.5. Overview of urea-based PSMA inhibitors investigated in clinical studies for diagnostics and TRT of prostate cancer. NA: not applicable; SPECT: single photon-emission computed tomography;

PET: positron emission tomography.

*Radioiodination by iododestannylation of trimethylstannyl precursors

1.3.3 Linker

Modifications in the linker region affect the molecular size, hydrophobicity and polarity of the ligands and can significantly affect the binding, internalisation and pharmacokinetic characteristics of PSMA ligands. Benešová et al. developed and evaluated 18 different linkers with the same 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator and Glu-urea-Lys PSMA-binding domain [110]. It was observed that at least one aromatic moiety with a rigid conformation in the linker region was required for high binding affinity and sufficient internalisation ratios in vitro. PSMA-617 with the aromatic moieties 2-naphtyl-L- alanine (2Nal) and trans-4-(aminomethyl)-cyclohexanecarboxylic acid (AMCH) in the linker (Figure 1.10) showed the most favourable characteristics both in vitro and in vivo. In the series of inhibitors, the only other structural modification with potential comprised a substitution of the AMCH with the aromatic moiety 4-(aminomethyl)benzoic acid. The change resulted in higher binding affinity but also a lower clearance from the kidneys compared to PSMA-617, possibly due to higher lipophilicity [110].

PSMA ligand

PSMA- binding motif

Radiolabel- bearing

moiety

Radionuclide Class References

MIP-1072 Glu-urea-Lys NA^* ¹²³I Diagnostic

SPECT [113-115]

MIP-1095 Glu-urea-Lys NA^* ¹²³I/¹²⁴I/¹³¹I Theragnostic [113- 115,119,120]

MIP-1404 Glu-urea-Glu TIM ^99mTc Diagnostic

SPECT [121,122]

MIP-1405 Glu-urea-Lys CIM ^99mTc Diagnostic

SPECT [121,122]

PSMA-11 Glu-urea-Lys HBED-CC ⁶⁸Ga Diagnostic PET [123,124]

DCFBC Glu-urea-Cys 4-[¹⁸F]FBB ¹⁸F Diagnostic PET [125-127]

DCFPyl Glu-urea-Lys [¹⁸F]TFPFN ¹⁸F Diagnostic PET [128,129]

PSMA-1007 Glu-urea-Lys [¹⁸F]F-Py-TFP ¹⁸F Diagnostic PET [130,131]

PSMA-I&T Glu-urea-Lys DOTAGA ⁶⁸Ga/¹¹¹In/¹⁷⁷Lu/²²⁵Ac Theragnostic [132-135]

PSMA-617 Glu-urea-Lys DOTA

68Ga/⁴⁴Sc/¹¹¹In/¹⁷⁷Lu/

225Ac/²¹³Bi Theragnostic [33,34,116,136 -143]

A novel 212Pb-labelled PSMA-targeting ligand for alpha therapy of metastatic prostate cancer

A novel

Pb-labelled PSMA-targeting ligand for alpha therapy of metastatic prostate cancer

Thesis for the Degree of Philosophiae Doctor by Vilde Yuli Stenberg

Institute of Clinical Medicine, University of Oslo

Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital

Nucligen AS

Abstract

Sammendrag

Acknowledgements

Declaration of Interests

Table of Contents

List of Abbreviations

List of Publications

1 Introduction

1.1 Prostate cancer

1.2 Targeted radionuclide therapy

1.3 PSMA-TRT of mCRPC