ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2018

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1430

Precision medicine and targetedtherapy

Turning the tables on cancer

ANJA C MORTENSEN

ISSN 1651-6206ISBN 978-91-513-0239-3urn:nbn:se:uu:diva-342030

Dissertation presented at Uppsala University to be publicly examined in Rudbecksalen, DagHammarskjölds Väg 20, Uppsala, Friday, 6 April 2018 at 13:00 for the degree of Doctor ofPhilosophy (Faculty of Medicine). The examination will be conducted in English. Facultyexaminer: Ph.D. Gregory Adams (Eleven Biotherapeutics).

AbstractMortensen, A. C. 2018. Precision medicine and targeted therapy. Turning the tables on cancer.Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine1430. 56 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0239-3.

An extended understanding of the molecular characteristics of cancer has led to a revolutionwithin the field of precision medicine. This thesis explores the utilization of two targets forprecision medicine, namely, CD44v6 and murine double-minute 2 and X (MDM2/X).

A novel mini-antibody construct targeting CD44v6 (AbD19384), was assessed for possibleuse in radiodiagnostics, while a recombinant full-length anti-CD44v6 antibody based on thesame construct, AbN44v6, was evaluated for radio-immunotherapy (RIT) following labelingwith 177Lu and 131I. Additionally, normal tissue biodistribution and dosimetry was assessed forradiolabeled AbN44v6. The efficacy and mechanisms behind the observed effects of PM2therapy, a novel stapled peptide that inhibits MDM2/X, were assessed in vitro and in vivo both asmonotherapy and in combination with external beam radiotherapy (EBRT). Lastly, combinationtherapy using RIT (177Lu-AbN44v6) and PM2 was evaluated in an in vitro 3D tumor spheroidmodel.

AbD19384 successfully visualized CD44v6-positive xenografts. Similarly, radiolabeledAbN44v6 bound specifically to CD44v6, and RIT resulted in antigen-dependent, activity-dependent growth inhibition of in vitro 3D tumor spheroids. Biodistribution and dosimetryrevealed low-level accumulation in normal tissues and low total effective doses of 0.1 mSv/MBq of injected radioconjugate. PM2-based therapy increased pro-apoptotic protein levels andcaused growth inhibition of wt p53 cancer cell lines, which was amplified in combinationwith radiotherapy. In vivo studies of wt p53 and p53-knockout xenografts demonstrated thespecificity of PM2 towards wt p53 cancers and established the potency of combining PM2-based therapy with EBRT.

The work presented in this thesis exemplifies the potency of combination treatments basedon a precise understanding of the targeted cancer. Utilizing not one but several of the molecularcharacteristics of a specific cancer will help turn the tables on cancer and improve patientoutcomes in the future.

Keywords: p53 cancers, PM2, radiosensitization, radio-immunotherapy, MDM2/X inhibition,combination therapy, targeted radionuclide therapy, CD44v6

Anja C Mortensen, Department of Immunology, Genetics and Pathology, Medical RadiationScience, Rudbecklaboratoriet, Uppsala University, SE-751 85 Uppsala, Sweden.

© Anja C Mortensen 2018

ISSN 1651-6206ISBN 978-91-513-0239-3urn:nbn:se:uu:diva-342030 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-342030)

Research is what I’m doing when Idon’t know what I’m doing.

- Wernher von Braun

List of Papers

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

I Spiegelberg, D., Mortensen, AC., Selvaraju, RK., Eriksson, O.,

Stenerlöw, B., Nestor, M. (2016) Molecular imaging of EGFR and CD44v6 for prediction and response monitoring of HSP90 inhibition in an in vivo squamous cell carcinoma model. EJNMMI, 43;974-982.

II Mortensen, AC., Spiegelberg, D., Haylock, AK., Lundqvist, H., Nestor, M. (2018) Preclinical evaluation of a novel engineered recombinant human anti-CD44v6 antibody for potential use in radio-immunotherapy. Int J Oncol, (in press).

III Mortensen, AC., Spiegelberg, D., Brown, CJ., Lane, DP., Nes-

tor, M. The stapled peptide PM2 stabilizes p53 levels and radio-sensitizes wild-type p53 cancer cells. Submitted manuscript.

IV *Spiegelberg, D., *Mortensen, AC., Lundsten, S., Brown, CJ.,

Lane, DP., Nestor, M. First in vivo study of the MDM2/X-p53 antagonist PM2 as potentiator of external radiotherapy in wt p53 cancer. Submitted manuscript.

V Mortensen, AC., Morin, E., Brown, CJ., Lane, DP., Nestor, M.

Enhancing therapeutic effects of radio-immunotherapy using the novel stapled peptide MDM2/X-p53 antagonist PM2. Manu-script.

*Contributed equally.

Reprints were made with permission from the respective publishers.

List of Papers not Included in this Thesis

Al-Ramadan, A., Mortensen, AC., Carlsson, J., Nestor, MV. (2018) Analysis of radiation effects in two irradiation tumor spheroid models. Oncol Lett, Mar;15(3):3008-3016.

Haylock, AK., Nilvebrant, J., Mortensen, A., Velikyan, I., Nestor, M., Falk, R. (2017) Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers. Oncotarget, May 18;8(39):65152-65170.

Häggblad Sahlberg, S., Mortensen, AC., Haglöf, J., Engskog, MK., Arvidsson, T., Pettersson, C., Glimelius, B., Stenerlöw, B., Nestor, M. (2017) Different functions of AKT1 and AKT2 in molecular pathways, cell migration and metabolism in colon cancer cells. Int J Oncol, Jan;50(1):5-14.

*Haylock, AK., *Spiegelberg, D., Mortensen, AC., Selvaraju, RK., Nil-vebrant, J., Eriksson, O., Tolmachev, V., Nestor, MV. (2016) Evaluation of a novel type of imaging probe based on a recombinant bivalent mini-antibody construct for detection of CD44v6-expressing squamous cell carcinoma. Int J Oncol, Feb;48(2):461-70.

Spiegelberg, D., Dascalu, A., Mortensen, AC., Abramenkovs, A., Kuku, G., Nestor, M., Stenerlöw, B. (2015) The novel HSP90 inhibitor AT13387 poten-tiates radiation effects in squamous cell carcinoma and adenocarcinoma cells. Oncotarget, Nov 3;6(34):35652-66.

*Contributed equally

Contents

Why one size does not fit all – the rise of precision medicine in cancer ......... 11 Understanding the disease and selecting a strategy ................................ 11 The need for precision ............................................................................. 12 

The role of precision medicine in radiodiagnostics and radiotherapy ........ 12 One test may tell all ................................................................................. 12 An age-old but ever-rising star ................................................................ 13 The established workhorse ...................................................................... 14 

The strategic targets ...................................................................................... 15 An overexpressed cell-surface receptor .................................................. 15 The guardian of the genome .................................................................... 16 The highly conserved feedback loop balancing the scales ..................... 18 

Our entities ................................................................................................... 20 An anti-CD44v6 mini-antibody construct functionally equivalent F(ab’)2 ...................................................................................................... 20 A fully human, full-length anti-CD44v6 antibody ................................. 20 A dual MDM2/X-inhibitor ...................................................................... 21 The importance of radionuclides ............................................................. 21 

The promise of combination therapies ........................................................ 22 Potentiating radiotherapy ........................................................................ 23 PM2 as a radiosensitizer .......................................................................... 24 

Aim .................................................................................................................... 26 

The present study .............................................................................................. 27 Treatment response studied via molecular imaging of two overexpressed antigens (paper I) ................................................................. 27 

Discussion ................................................................................................ 28 Preclinical assessment of an anti-CD44v6 antibody for RIT (paper II) ..... 30 

Discussion ................................................................................................ 31 The potency of PM2 in combination with external beam radiotherapy (papers III & IV) ........................................................................................... 35 

Discussion ................................................................................................ 36 An indication of the benefit of combining two precision medicines (paper V) ....................................................................................................... 39 

Discussion ................................................................................................ 40 

Conclusions ....................................................................................................... 44 

The future at a glance ........................................................................................ 46 Efficacy of PM2....................................................................................... 46 RIT and PM2 in vivo ............................................................................... 46 Targeted drug delivery of PM2 and newer generations of the peptide ...................................................................................................... 47 Combining RIT with HSP90 inhibition .................................................. 47 

Acknowledgements ........................................................................................... 48 

References ......................................................................................................... 51 

Abbreviations

ALL Acute lymphocytic leukemia AML Acute myeloid leukemia AT13387 2,4-dihydroxy-5-isopropyl-phenyl-[5,(4-methyl-piperazin-1-

ylmethyl)-1,3-dihydro-isoindol-2-yl]thanone, 1-lactic acid salt Bcl-2 B-cell lymphoma 2 BH3 Bcl-2 homology 3 CD Cluster of differentiation CDKs cyclin dependent kinases CEA Carcinoembryonic antigen CHOP Cyclophosphamide, doxorubicin hydrochloride, vincristine

sulfate, prednisone CHX-A’’-DTPA [(R)-2-Amin-3-(4-isothiocyanatophenyl)-propyl]-

trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid CLL Chronic lymphocytic leukemia CML Chronic myeloid leukemia CT Computed tomography CVP Cyclophosphamide, vincristine sulfate, prednisone DNA Deoxyribonucleic acid EBRT External beam radiotherapy EGFR Epidermal growth factor receptor Fab Antigen binding fragment FDG Fluorodeoxyglucose GBM Glioblastoma GEP Gastro-enteropancreatic GI Gastrointestinal H&N Head and neck HER2 Human EGFR 2 HLA-DR10 Peptide anchor motif of human leukocyte antigen HPV Human papillomavirus HSP90 Heat shock protein 90 IHC Immuno-histochemistry MDM2 Murine double-minute 2 MDMX Murine double-minute X (MDM4) MOMP Mitochondrial outer membrane permeabilization MUC1 Mucin 1 NET Neuroendocrine tumor

Noxa Phorbol-12-myristate-13-acetate-induced protein 1 NSCLC Non-small cell lung cancer PEM Polymorphic epithelial mucin PET Positron emission tomography PM2 SMTide02 PRRT Peptide receptor radionuclide therapy PSMA Prostate-specific membrane antigen PUMA p53 upregulated modulator of apoptosis RAI Radio-iodinetherapy R/R Relapsed or refractory RIT Radio-immunotherapy SCC Squamous cell carcinoma SCLL Small cell lymphocytic lymphoma SD Standard deviation SSTR Somatostatin receptor TRNT Targeted radionuclide therapy wt wild-type

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Why one size does not fit all – the rise of precision medicine in cancer

Cancer, the collective name for more than two hundred diseases that share a specific set of hallmarks, has existed throughout human history (1,2). How-ever, despite shared characteristics and common features, each cancer is unique and heterogeneous and should ideally be treated as such (3). The het-erogeneity and complexity of the disease as well as the health status and re-sponse to therapy of each unique patient, lower the success rates of standard-ized cancer therapies (4). The importance of identifying the underlying causes and molecular characteristics of individual cancers is becoming increasingly relevant in the field of cancer medicine (5). Once identified, these character-istics can be utilized for more precise therapeutic strategies, which will ulti-mately benefit cancer patients by, hopefully, increasing therapeutic success rates (3,6).

The term “precision medicine” can be defined in a variety of ways, all of which are based on the same underlying principles: to tailor the treatment of each patient based on individual genetic profile, lifestyle, and environment (2). Through utilizing and applying precision medicine principles, cancer pa-tients will hopefully have a greater life expectancy. For this strategy to become a reality, an enhanced arsenal of both precise diagnostic tools and therapeutic compounds is essential.

Understanding the disease and selecting a strategy The therapeutic strategy for any cancer is hypothetically more likely to suc-ceed when based on a collective analysis of the molecular characteristics of the cancer (7). Commonly used diagnostic methods (i.e., [18F]-FDG-PET/CT (FDG-PET) and immuno-histochemistry (IHC) following biopsies) offer val-uable information regarding both diagnosis and prognosis. However, more precise assessments such as genetic screening or targeted molecular imaging can provide additional information regarding the best strategy to attack the cancer at the molecular level (8,9).

Molecular imaging offers a non-invasive means of complementing com-monly used prognostic and diagnostic methods such as IHC following biop-sies of the primary tumor. Molecular imaging of cancer, primarily via FDG-

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PET, is currently a broadly applicable method that is used in most industrial-ized territories (10). Although useful and broadly applicable, imaging tech-niques can be imprecise and can produce false-positive or false-negative re-sults while offering limited information about treatment strategies (11,12). In comparison, targeted molecular imaging aimed at specific, cancer-associated markers, can assist the selection of the therapeutic strategy to be pursued (8,13,14).

The need for precision For decades, cytostatic chemotherapeutic compounds have been a workhorse and gold standard of cancer therapy accompanied by radiotherapy and surgery (15). Unfortunately, the nonspecific nature of these compounds and the asso-ciated toxicities can wreak havoc on healthy tissues without eradicating the cancer (16,17). Additionally, tumor cell resistance, both intrinsic and ac-quired, present a challenge that is hard to overcome with the broadly applica-ble, commonly used treatments (18,19). The need for more precise compounds along with fast-paced technological advances has resulted in an increase in the number of targeted cancer therapies available, with many more underway in preclinical or clinical pipelines (20–23). Essentially, targeted therapies may turn the tables on cancer and utilize the specific characteristics of each tumor to eradicate the cancer.

The role of precision medicine in radiodiagnostics and radiotherapy One test may tell all The gold standard in molecular imaging of cancer remains FDG-PET. FDG-PET is an established method and a common step on the path to diagnosis and prognosis (10). Though highly useful, FDG-PET has certain limitations (24). This method relies on the increased glucose uptake of rapidly dividing cancer cells (i.e., the Warburg effect (25)), which is less relevant for slow-growing cancers (26). False-positives from either active immune cells during inflam-mation or adipose tissue further limit the accuracy of the assessments made based on FDG-PET imaging (11). FDG-PET is likely to visualize the primary tumor as well as metastases but offers no additional information regarding possible therapeutic strategies (27). Targeted radiotracers, on the other hand, offer more precise and perhaps crucial information for diagnosis and progno-sis and for selecting a therapeutic course of action (13,14,28–30).

Several novel targeted radiotracers have entered clinical trials. One exam-ple is the affibody ABY-025, which targets the human epidermal growth fac-tor receptor 2 (HER2) in breast cancer and is labeled with 68Ga, a short-lived

13

positron-emitting radiometal (31). Compared to commonly used diagnostic methods (i.e., FDG-PET and IHC), [68Ga]-ABY-025 provides more precise information on the expression of HER2 throughout both primary tumors and metastases (Figure 1). FDG-PET will likely detect both primary tumors and metastatic burden, while IHC of primary tumors may detect an elevated HER2 expression. However, the heterogeneity of both primary tumors and metasta-ses can result in false-negative assessments of IHC following biopsies. As HER2 expression in breast cancer is crucial for the selection of a therapeutic strategy, it is imperative that the expression level be accurately assessed (32). [68Ga]-ABY025 provides the precise information needed, ultimately increas-ing the success rates of therapy.

Figure 1. FDG-PET versus [68Ga]ABY-025 images of Patient A (HER2-negative): (A1) FDG-PET image, (A2) [68Ga]ABY-025 image. Patient B (HER2-positive): (B1) FDG-PET image, (B2) [68Ga]ABY-025 image. From Sörensen et al. “Measuring HER2-Receptor Expression in Metastatic Breast Cancer Using [68Ga]ABY-025 Af-fibody PET/CT”. Theranostics, 2016.

Additionally, targeted molecular imaging can be used for monitoring the re-sponse to therapy, another element that is gaining importance in precision medicine (30). The ability to estimate whether a patient is responding to the chosen therapy at an early stage is a crucial part of increasing the success rates of therapies. Targeted radiotracers can improve this assessment in a minimally invasive manner (33).

An age-old but ever-rising star Radio-iodinetherapy (RAI), peptide receptor radionuclide therapy (PRRT), and radio-immunotherapy (RIT) are all examples of targeted radionuclide therapies (TRNT). RAI ([131I]-NaI) has been in use for more than half a cen-

14

tury against thyroid cancer and utilizes an innate mechanism (the sodium-io-dide symporter) that ensures uptake and accumulation of the radionuclide in thyroid cells (34). Similarly, the newly approved Xofigo® (223RaCl2) utilizes another innate process, namely, the uptake of Ca2+ ions during bone formation in bone metastases (35,36). 223Ra, an alpha-emitting radionuclide, replaces the Ca2+ ions needed for bone formation, thus irradiating the newly formed bone-metastases of disseminated prostate cancers from within, with limited expo-sure to adjacent, healthy tissues (37).

Table 1. Examples of current targeted radionuclide therapies, either approved or un-dergoing clinical trials. PSMA, prostate-specific membrane antigen; CEA, carci-noembryonic antigen; SSTR, somatostatin receptor; HLA-DR10, peptide anchor motif of human leukocyte antigen; MUC1, mucin 1; PEM, polymorphic epithelial mucin (identical target to MUC1); GBM, gliobastoma; CLL, chronic lymphocytic leukemia; NSCLC, non-small cell lung cancer; H&N, head and neck (44).

Compound Target Nuclide Cancer(s) Status

Cotara DNA 131I GBM, anaplastic astrocytoma Phase III

J591 PSMA 177Lu Castrate-resistant prostate cancer with bone metastases

Phase II

Labetuzumab CEA 90Y/131I Breast, lung, pancreatic, stomach, colo-rectal carcinoma

Phase III

Licartin HAb18G/CD147 131I Hepatocellular carcinoma Phase II Lutathera® SSTR 177Lu Metastatic GEP NETs Approved Lymphocide CD22 90Y Non-Hodgkin’s lymphoma/CLL Phase III MIBG Norepinephrine trans-

porter

131I Neuroblastoma, pheochromocytoma, paraganglioma

Phase III

Oncolym HLA-DR10 131I Non-Hodgkin’s lymphoma/CLL Phase III PAM4 MUC1 90Y Pancreatic adenocarcinoma Phase III Radretumab Fibronectin 131I Refractory Hodgkin’s lymphoma,

NSCLC, melanoma, H&N carcinoma Phase II

Theragin PEM 90Y Ovarian, gastric carcinoma Phase III Xofigo® None 223Ra Metastatic castration-resistant prostate

cancer Approved

Zevalin® CD20 90Y Non-Hodgkin’s lymphoma Approved

In contrast to RAI and Xofigo®, PRRT and RIT utilize specific cancer-asso-ciated molecular targets in order to deliver cytotoxic radiation (38,39). For these methods to be effective, the targeted antigens must be exclusively or predominantly expressed in the cancerous tissues (39). In 2017 and early 2018, the European Medicines Agency and the US Food and Drug Admin-istration approved the radiolabeled somatostatin analogue Lutathera® (177Lu-DOTA-Tyr3-octreotate) for treatment of gastro-enteropancreatic neuroendo-crine tumors (GEP NETs) (40,41). Clinical trials with Lutathera® gathered promising results in terms of increased progression free survival and improved the quality of life of otherwise terminal patients (42). The remarkable results of the Lutathera® trials further emphasize the potency of TRNT.

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As mentioned earlier, RIT exploits the overexpression of cancer-associated antigens on the cell surface. The most established RIT is Zevalin® (ibri-tumomab tiuxetan), an anti-CD20 antibody radiolabeled with 90Y, a beta-emit-ting radiometal. Zevalin® was approved for therapy of primarily non-Hodg-kin’s lymphoma more than a decade ago and continues to provide positive outcomes (43). Recent successes within the field of TRNT in combination with historical successes such as Zevalin® and RAI have rapidly advanced the field of TRNT. As a result, several new TRNTs are undergoing various stages of clinical trials (Table 1) (44).

The established workhorse External beam radiotherapy (EBRT) is a non-invasive therapy and remains the most commonly used form of radiotherapy worldwide. Given that more than 50% of cancer patients receive EBRT, this method is not only the most com-mon form of radiotherapy worldwide but also among the most common cancer therapies (45). Despite being widely used across the world, EBRT has its lim-itations. First, EBRT has little effect other than palliative on disseminated dis-ease (46,47). Second, EBRT has limited applicability in cases of tumor-infil-tration into vital tissues such as lungs, esophagus or trachea (48). Third, EBRT can result in both short- and long-term side effects such as acute toxicity and secondary malignancies, which are of great concern (49–51).

Despite being a broadly applicable, commonly used therapy, EBRT can be adapted for the field of precision medicine. To avoid adverse effects, there exist guidelines for radiation absorbed doses and doses tolerated by dose-lim-iting organs. Although such guidelines are based on empirical evidence, they do not account for the fact that each patient can respond differently to therapy (52). The use of a precision medicine-based approach by monitoring and as-sessing individual responses to ongoing therapy to adjust therapeutic doses may increase success rates or limit side effects (53).

The strategic targets An overexpressed cell-surface receptor One of the primary challenges of RIT is the identification of a suitable target. To avoid unnecessary toxicity in healthy tissues, the targeted antigen should be expressed primarily in the cancerous tissues. In an ideal “magic bullet” scenario, the targeting moiety would course through the body and seek out even the smallest of metastasis, while ensuring that the dose of cytotoxic ra-diation is delivered solely to the cancer.

Several targets have been investigated for use in RIT in past decades. One of these targets, CD44v6, was successfully utilized in a Phase I trial using

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186Re against head and neck squamous cell carcinoma, yielding promising re-sults for future RIT applications using this strategy (54). The antigen itself is a splice variant of the glycoprotein CD44 (cluster of differentiation 44) cell surface receptor. While CD44 is commonly found in most tissues, the splice variants (CD44v) are much more tissue specific (55). The splice variants are generated as a result of post-translational modifications and alternative splic-ing (56). Proteins of the CD44 family are predominantly involved in facilitat-ing cell-cell and cell-extracellular-matrix interactions via their main ligand, hyaluronic acid (57). Several of the splice variants have been the focus of can-cer research as possible cancer stem cell-like markers and are often associated with a more aggressive disease. CD44v6, in particular, is associated with a more aggressive, invasive, and radio- and chemoresistant disease, which nat-urally translates to a poor prognosis (58,59). An overexpression of CD44v6 can be detected in several cancers, and when present, is likely expressed at an extreme level (60,61). The expression of CD44v6 in normal tissues is limited primarily to the basal membrane and suprabasal stratum spinosum epithelial layers (62). The combination of the scarce distribution of CD44v6 in healthy tissues and extreme expression in cancerous tissues is what makes CD44v6 an exciting and promising target for RIT.

The guardian of the genome At the very center of the innate defense against cancer, lie tumor suppressors (63). These guardians help maintain cellular integrity by for example initiating cell cycle arrest, allowing damaged cells time to repair, or inducing pro-grammed cell death, such as apoptosis (64). Perhaps the most well-known tu-mor suppressor is the transcription factor TP53, sometimes referred to as the guardian of the genome (65). While p53 affects a variety of cellular mecha-nisms, this protein is most commonly associated with cycle arrest and apop-tosis following DNA damage or cellular stress (66). The importance of p53 as a tumor suppressor is evident in the mutational rate of the gene in malignan-cies. It is estimated that TP53 is mutated in approximately 50% of all human cancers (64). In addition to neutralizing the native tumor-suppressive role of a functioning wild-type p53 protein (wt p53), the mutated p53 protein can ac-quire gain-of-function properties, which essentially transform the protein into a malicious oncogene (67). In cancers where wt p53 is retained, dysfunctional activation pathways or overexpression of negative regulators of p53 such as murine double-minute 2 (MDM2) limit the innate function of wt p53 as a tu-mor suppressor and genomic watchdog (68–70).

p53 and cell cycle arrest Once DNA damage is detected, cell cycle arrest is initiated by wt p53. Once activated, wt p53 induces the expression of p21cip1/waf1, which inhibits cyclin dependent kinases (CDKs) that are essential for cell cycle progression (Figure

17

2) (71). By inhibiting CDKs, p21 inhibits both the G1-to-S-phase transition and the G2-to-M-phase transition. Furthermore, wt p53 downregulates the ex-pression of cyclin A, which can result in a secondary pause in cell cycle pro-gression into and through the S-phase (66,71).

Figure 2. Simplified schematic depiction of p53-induced cell cycle arrest via induc-tion of p21cip1/waf1.

Apoptosis The role of wt p53 in apoptosis is complex. However, a simplified explanation can be found in the relationship between wt p53 and members of the B-cell lymphoma 2 (Bcl-2) family (Figure 3) (72). The Bcl-2 family can be classified into three subgroups: a group with anti-apoptotic function and two groups with pro-apoptotic function (73). The pro-apoptotic proteins are sectioned into ef-fectors and sensitizers of apoptosis: Bim, Bid and PUMA (p53 upregulated modulator of apoptosis) are examples of effector proteins, which bind with high affinity to anti-apoptotic Bcl-2 proteins such as Bcl-2 and Bcl-xL (72,73). Sensitizer proteins include Noxa (phorbol-12-myristate-13-acetate-induced protein 1) and Bad. Upon wt p53-induced transcription, Noxa localizes to the mitochondria and assists in liberating pro-apoptotic BH3-only (Bcl-2 homol-ogy 3) proteins, thereby inducing further inhibition of anti-apoptotic Bcl-2 proteins (72). Inhibition of anti-apoptotic proteins and activation and stabili-zation of pro-apoptotic proteins culminates in Bax/Bak activation, resulting in mitochondrial outer membrane permeabilization (MOMP) and initiation of caspase-cascades essential for apoptosis (73,74). While these interactions are far more complex than stated, wt p53 remains at the very center, as Bax, Noxa, Puma, Bid, etc. are all downstream targets of wt p53 (73).

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Figure 3. Simplified depiction of p53-mediated apoptosis via interactions with pro- and anti-apoptotic members of the Bcl-2 family following p53 activation.

The highly conserved feedback loop balancing the scales p53 expression in normal cells is nearly non-existent due to the short half-life of the protein, which is between 5-30 minutes depending on the cell type (75). The degradation of p53 in the proteasome lies at the center of this short half-life, a degradation which is facilitated by the main negative regulator of p53; MDM2 (66,76). Regulation of p53 by MDM2 and its structural homolog MDMX (commonly referred to as MDM4) is a highly conserved autoregula-tory feedback loop found in all jawed vertebrates and even in some inverte-brate species (77). MDM2 acts in a heteromeric complex with MDMX to neg-atively regulate p53. MDM2/X-heterodimers are more efficient as well as more stable than MDM2-homodimers in terms of p53-inhibition (68,78). The MDM2/X-complex has potent ubiquitin E3 ligase activity, which marks p53 for degradation. Once the complex binds to p53, it facilitates the relocation of the protein from the nucleus to the cytoplasm and ultimately to the 26S pro-teasome for degradation (79). The conserved autoregulatory relationship be-tween p53 and MDM2/X maintains the cause-and-effect balance of p53 in response to cellular stress (76).

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Table 2. Selection of antagonists of the p53-MDM2 protein-protein interaction un-dergoing clinical trials. AML; acute myeloid leukemia; ALL, acute lymphocytic leu-kemia; CML, chronic myeloid leukemia; SCLL, small cell lymphocytic lymphoma; R/R, relapsed or refractory (80,81).

Drug Type Target cancer(s) Combination Phase

RG7112 Cis-imidazoline MDM2 antagonist

Solid tumors, AML, ALL, CML, refractory CLL/SCLL, liposarcoma

I

Soft-tissue sarcoma Doxorubicin I AML Cytarabine I

RG7388 Cis-imidazoline MDM2 antagonist

Multiple myeloma Ixazomib citrate, dexamethasone

I/II

R/R AML Cytarabine III R/R Follicular lymphoma Obinutuxumab I/II Diffuse large B-cell lym-phoma

Rituximab I/II

R/R AML Venetoclax I/II Prostate cancer Idasanutlin with

abiraterone or en-zalutamide

I/II

SAR-405838

Spiro-oxindole MDM2 antagonist

Advanced cancer I Advanced solid tumors Pimasertib I

MK-8242 Piperidines MDM2 an-tagonist

AML Cytarabine I

AMG232 Piperidinone MDM2 antagonist

R/R AML Trametinib Ib R/R multiple myeoloma Carfilzomib, le-

nalidomide, dexa-methasone

I

Advanced solid tumors and multiple myeloma

I

Metastatic cutaneous mela-noma

Trametinib and dabrafenib

Ib/IIa

DS-303b Imidazothiazole MDM2 antagonist

AML, ALL, CML, R/R mul-tiple myeoloma, advanced solid tumors and lymphomas

I

HDM201 Dihydroisoquinolinone MDM2 antagonist

Liposarcoma LEE011 Ib/II Advanced solid and hemato-logical tumors

I

CGM097 Dihydroisoquinolinone MDM2 antagonist

Advanced solid tumors I

ALRN-6924

Stapled peptide MDM2/X inhibitor

AML or advanced myelo-dysplastic syndrome

I/Ib

AML or advanced myelo-dysplastic syndrome

Cytarabine I/Ib

Advanced solid tumors or lymphomas

I/IIa

As the amount of p53 protein present in cells is defined by the rate of degra-dation as opposed to the rate of production, the regulatory factors involved are crucial for a functional p53 response (66). In tumors that retain wt p53, irreg-ularities in the autoregulatory feedback loop with the negative regulators MDM2/X are a common occurrence. However, dysfunctional activation path-ways, for example epigenetic silencing of p14ARF expression, can disrupt the native function of wt p53. p14ARF binds to MDM2/MDMX and inhibits their activity, leading to increased levels of p53 expression (66).

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In recent years, several wt p53-targeted therapies have entered clinical tri-als (Table 2). Small-molecule antagonists of the p53-MDM2 protein-protein interaction inhibit the binding of MDM2 with wt p53. These MDM2 inhibitors all have tumor-regressive effects; however, to date, the observed effects have been meager and primarily in non-solid tumors (81). A majority of the inhib-itors are in clinical trials in combination with either well-known chemothera-peutic agents or new cytostatic compounds (Table 2) (80,81). As wt p53 is central to both DNA repair and radiation response mechanisms, it seems odd that none of these inhibitors are involved in clinical trials with radiotherapy. Utilizing MDM2 inhibitors as radiosensitizers could potentially increase the therapeutic effect of radiotherapy or perhaps lower the radiation doses other-wise required for tumor regression.

Our entities An anti-CD44v6 mini-antibody construct functionally equivalent F(ab’)2

AbD19384, a fully human recombinant, bivalent mini-antibody construct, that is functionally equivalent to a F(ab’)2, is comprised of two Fab AbD15179 peptides linked non-covalently via a spontaneous dimerization domain (82,83). Fab AbD15179 was developed by our group and binds specifically and with high affinity to the CD44v6-receptor (84,85). The mini-antibody construct (AbD19384) was produced with a potential dual-purpose function. First, the smaller size (113 kDa) of this construct compared to a full-length antibody (150 kDa) should result in a more rapid elimination from the blood-stream, albeit slower than that of a Fab (50 kDa) (86). Second, the bivalent nature of the dimer could result in higher affinity and slower dissociation rate than that of Fab AbD15179. These characteristics indicate a possible dual-function of AbD19384 in precision medicine as both an imaging tracer and a possible targeting moiety for RIT (82,83).

A fully human, full-length anti-CD44v6 antibody The number of antibody-based TRNT is rapidly increasing, as is the number of antibody-based targeted therapies utilized in precision medicine. Previous studies on anti-CD44v6 targeting moieties (i.e., U36, an U36-derived Fab and F(ab’)2) demonstrated how tumor uptake of the full-length antibody was su-perior to that of both the Fab and F(ab’)2 (87). The greater tumor uptake of U36 compared to the smaller moieties illustrates the potential benefit of uti-lizing a full-length antibody for RIT (87). Therefore, following the evaluation of AbD19384, a full-length anti-CD44v6 antibody (here referred to as AbN44v6) was developed for use in RIT in the hopes of increasing tumor

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uptake. Identically to AbD19384, the full-length, fully human recombinant monoclonal antibody originated from the original Fab construct, AbD15179. Thus, the binding sequence was identical throughout all three moieties (Fab AbD15179, AbD19384, and AbN44v6).

A dual MDM2/X-inhibitor The current generation of MDM2 inhibitors (Table 2) primarily targets MDM2 and not MDMX (78,80,81). The development of resistance following continuous treatment with the inhibitors has been observed and only a meager apoptotic response to MDM2 inhibition-based therapies have so far been re-ported (78). Relatively short biological half-lives and hematological toxicities are challenges that need to be overcome as this generation of inhibitors pro-ceeds through clinical trials.

PM2 (SMTide02), is a novel stapled peptide targeting the p53-MDM2 pro-tein-protein interaction (70). ‘Stapling’ occurs via a covalent, hydrocarbon linker between two non-adjacent amino acids. The hydrocarbon linker thus connects the turns of the α-helix of the peptide, resulting in greater stability (88,89). However, in addition to binding and inhibiting MDM2, PM2 also binds to and blocks the binding of MDMX to wt p53. This dual inhibition results in a more comprehensive network of interactions with not only MDM2 but also MDMX, which exceeds the interaction network of similar, small mol-ecule MDM2 inhibitors in terms of complexity (90). In fact, recent studies have shown that PM2 can bind to and antagonize otherwise inhibition-re-sistant MDM2 (90). The increased stability of the secondary structure of PM2, in addition to increasing the affinity for MDM2/X by reducing the entropic cost of binding, also results in an increase in the in vivo half-life of PM2 (89). These factors are all greatly important for the future utilization of this the pep-tide as an anti-cancer therapeutic.

The importance of radionuclides Criteria for the suitability of radionuclides for molecular imaging or therapy differ greatly. While some radionuclides possess properties that are important for both applications, most radionuclides are limited in their fields of use. For molecular imaging, gamma- or positron-emitting nuclides are adept; however, additional characteristics such as decay half-life, energy spectra, abundance and availability are essential for the suitability and applicability of these nu-clides (91).

As for nuclides suitable for therapeutic use, among the most important properties are range of the therapeutic particle in tissue, decay half-life, and abundance of unwanted radiation (92). Beta and alpha particle-emitting radi-onuclides are highly valued for radiotherapy. Alpha particles are by far the most powerful, causing devastating DNA damage as they pass through the cell

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(93). However, the short ranges of alpha particles, which are restricted to a few cell diameters, limit their use, particularly in larger tumors and metastases (94). Beta particles on the other hand, have a greater range in tissues and ben-efit from the so-called cross-fire effect, although the damage of each particle may be less devastating than that of an alpha particle (93,95). The range of beta particles in tissue results in a cross-fire effect via irradiation from nearby cells. The cross-fire effect is thus beneficial for the targeted treatment since not every cancerous cell must express the targeted antigen in order to receive a dose of cytotoxic radiation (96). However, the greater range of beta particles also potentially increases the absorbed dose received by healthy tissues, once again emphasizing the importance of precision, specificity, and stability of the targeting moieties.

This thesis has focused on several radio-iodine isotopes (124I (paper I), 125I (papers I and II) and 131I (paper II)) and one radiometal, 177Lu (papers II and V). All radiohalogenations were a result of direct labeling by electrophilic substitution using chloramine-T (125I and 131I) or [1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril] (iodogen) (124I). 177Lu was conjugated to AbN44v6 by chelation with CHX-A’’-DTPA ([(R)-2-Amin-3-(4-isothiocyanatophenyl)-propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaacetic acid).

Residualizing and non-residualizing radionuclides An element of importance in TRNT is the biological nature of the molecular target. The accessibility, whether it shreds into circulation or the expression is retained in metastases and if the target is affected as a result of therapy are important for therapeutic success. For therapeutic use, cell-surface receptors remain the most common target. Activation of the receptor by ligand binding and consequent internalization of the receptor is crucial to the choice of radi-onuclide. Radiohalogens are considered non-residualizing labels as they are subject to diffusion following lysosomal degradation due to their lipophilicity (97). Radiometals, on the other hand, are residualizing and are thus retained within the cell (98). Therefore, it is crucial to understand the characteristics of the chosen molecular target, in order to select an appropriate radionuclide. Two of the most commonly used beta-emitting therapeutic radionuclides are 177Lu and 131I. These radionuclides share similar characteristics in terms of decay half-lives (6.7 days and 8.0 days, respectively) and mean range of the beta particle in tissue (0.25 mm and 0.4 mm, respectively) (95). The half-lives of these radionuclides make them both highly suited for conjugation with full-length antibodies for therapeutic use in precision medicine.

The promise of combination therapies Two of the major risks involved with radiotherapy are long-term side effects (i.e., secondary malignancies) and immediate adverse events (i.e., toxicities)

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in healthy tissues and cells, such as those of the hematopoietic system (51). While the risks differ among the various forms of radiotherapy, they share a common denominator: radiation-induced toxicities in healthy tissues. Similar issues apply to both existing chemotherapeutic compounds and novel anti-cancer therapies (99).

Table 3. Examples of approved targeted radionuclide therapeutic compounds partak-ing in combination studies in clinical trials. CVP, cyclophosphamide, vincristine sul-fate, prednisone; CHOP, cyclophosphamide, doxorubicin hydrochloride, vincristine sulfate, prednisone (101).

Compound Target Nu-clide

Combination Cancer(s) Phase

DOTATATE or octreotide

SSTR 177Lu or 111In

Fluorouracil NETs

DOTATATE SSTR 177Lu Capecitabine with/without te-mozolomide

Advanced NETs I/II

DOTATATE SSTR 177Lu 90Y-DOTATATE Metastatic NETs II MIBG Norepi-

nephrine transporter

131I Irinotecan and vincristine R/R neuroblastoma I/II

Xofigo® None 223Ra Abiraterone, enzalutamide or both Castration-resistant prostate cancer with bone metastases

IIIb

Zevalin® CD20 90Y RIT before high-dose BEAM chem-otherapy and autologous stem-cell transplantation

Relapsed, diffuse large B-cell lym-phoma

III

Zevalin® CD20 90Y Consolidation after first-line CVP, CHOP, CHOP-like regimen, fludar-abine or rituximab combination

Stage III/IV follicu-lar non-Hodgkin’s lymphoma

III

For increased success rate and improved quality of life for the patient, it is critical to limit the toxicities of cancer therapies. One way of limiting thera-peutic toxicity and possibly avoiding adverse events is via combination ther-apies, which offer a number of distinct advantages. First, decreasing the inci-dence of toxicity while retaining the therapeutic outcome by combining ther-apies that have different toxicity profiles. Second, additive or synergistic ef-fects of combination therapies may reduce the toxicity profiles upon reducing the doses of one or both therapies. Third, the promise of increased success rates without having to increase the doses of either therapy or risking increased toxicity rates (100).

Potentiating radiotherapy As more than half of all cancer patients undergo radiotherapy, an advanta-geous strategy involves combining radiotherapy with radiosensitizing com-pounds (102). Radiosensitization involves potentiating the effects of or sensi-tizing the cancer cells to the effects of radiotherapy prior to, during or post radiotherapy (103). EBRT is routinely used in combination with approved

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chemotherapeutic compounds such as etoposide and fluorouracil in order to enhance therapeutic success (104). However, despite the increase in the num-ber of approved TRNT compounds, few are being assessed as combination therapies with chemotherapeutic compounds (Table 3) (101). Those that are, are tested in combination with chemotherapeutic compounds that have already been approved. Similarly, only a few trials involving approved chemothera-peutic compounds are undergoing clinical assessment for possible combina-tion with experimental (i.e., not yet approved) TRNT (Table 4) (101).

Table 4. Examples of experimental targeted radionuclide therapeutic compounds cur-rently undergoing clinical trials as combination therapies. GI, gastrointestinal (101).

Compound Target Nuclide Combination Cancer(s) Phase

A5B7 CEA 131I Combretastatin-A4-phosphate

Advanced GI carcino-mas

Phase I

HEDP Bone 186Re Docetaxel Prostate cancer with bone metastases

Phase I

J591 PSMA 177Lu Docetaxel or prednisone

Castrate-resistant prostate cancer with bone metastases

Phase II

L19SIP Fibronectin 131I Whole-brain EBRT

Brain metastases Phase II

Lintuzumab CD33 213Bi Cytarabine AML Phase I/II

mAb425 EGFR 125I EBRT and te-mozolomide

GBM Phase II

However, the current climate appears to be changing and paints an encourag-ing picture. Current studies focus on novel therapies that influence pathways involved in cell cycle arrest, DNA damage repair, and apoptosis (102–104). While each strategy is promising, we believe that targeting upstream influenc-ers or effectors is an interesting and promising concept. Upstream of cell cycle arrest, DNA repair and radiation response mechanisms as well as apoptotic pathways, lies wt p53. The potential of targeting all of the abovementioned mechanisms simultaneously via wt p53 activation is a tempting radiosensiti-zation strategy.

PM2 as a radiosensitizer At the preclinical level, early research into MDM2 inhibitors delved into the correlation with radiation. Upon irradiating cancer cells, a p53 induction oc-curs, thus resulting in upregulation of the therapeutic target, MDM2. As a re-sult, the term ‘radiosensitization’ was frequently used in early studies of these inhibitors. Once the inhibitors entered clinical trials, their functions as radio-sensitizers appeared to have been completely abandoned, leading to combina-

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tion therapies with cytostatic compounds (Table 2) (80,81). To date, the ther-apeutic response to MDM2 inhibition has been lower than anticipated, primar-ily due to a meager level of apoptotic response (81).

PM2 belongs to a new generation of MDM2/X-inhibitors and has yet to be tested in a clinical setting. Studies have shown that PM2 is able to bind to and inhibit otherwise resistant MDM2, which offers promise for the future appli-cation of PM2 or new generations of the peptide. This ability has been at-tributed to the dual inhibition of both MDM2 and MDMX by PM2 as well as to the increased in vivo stability of stapled peptides. Dual inhibition could lead to increased accumulation of wt p53, resulting in a greater apoptotic response to MDM2/X therapy (88,90).

A suitable combination therapy for PM2 has yet to be determined. The pre-liminary results from preclinical studies involving PM2 and radiotherapy pre-sented in this thesis indicate the best way the drug could be used in a combi-nation therapy trial. Induction of p53 activation by radiotherapy could poten-tially not only increase the therapeutic response to MDM2/X inhibition but also potentiate the effects of radiotherapy. This hypothesis covers both the promise and the premise of combining PM2 with radiotherapy to achieve greater precision in selection of the most effective and least invasive individ-ualized treatments for cancer patients.

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Aim

The overall aim of the work included in this thesis comprises three parts:

First, to evaluate the novel CD44v6 mini-antibody construct, AbD19384, for potential application as a diagnostic tool for the detection of CD44v6-positive xenografts and for monitoring the response to treatment.

Second, to evaluate the novel, full-length anti-CD44v6 antibody, AbN44v6, for potential use in radio-immunotherapy of CD44v6-positive cancers.

Third, to evaluate PM2-based treatment of wt p53 cancers and establish whether PM2-based therapy in combination with radiotherapy, both external beam radiotherapy and radio-immunotherapy, would potentiate the effects of both therapies.

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The present study

Treatment response studied via molecular imaging of two overexpressed antigens (paper I) The epidermal growth factor receptor (EGFR) and CD44v6 are two commonly overexpressed cell surface antigens in several cancers including squamous cell carcinomas (SCC). Therefore, these receptors are potential targets for preci-sion medicine for diagnosis and response monitoring using targeted molecular imaging modalities. In addition, these receptors are potential targets for tar-geted therapies. By utilizing targeted imaging modalities, as opposed to gold standard tracers such as FDG-PET, useful information regarding diagnosis and response to therapy can be extracted in a non-invasive manner.

EGFR and CD44v6 expression levels were evaluated to test the response to Heat shock protein 90 (HSP90) inhibition via treatment with the novel HSP90 inhibitor AT13387 (onalespib), a promising therapeutic compound and potential radiosensitizer. The expression levels were evaluated in vitro before and after AT13387 treatment using radiolabeled cetuximab (an anti-EGFR antibody) and AbD19384 (an anti-CD44v6 mini-antibody construct functionally equivalent to a F(ab’)2). An in vivo dual-xenograft study, which included a small animal PET comparison of targeted molecular imaging mo-dalities 124I-cetuximab and 124I-AbD19384, with the gold standard imaging probe FDG-PET, was conducted.

All experiments, both in vitro and in vivo, were carried out on a high EGFR and high CD44v6-expressing SCC-cell line (A431) and a low EGFR and low CD44v6-expressing SCC-cell line (UM-SCC-74B) and corresponding xeno-grafts. Both radio-iodinated cetuximab and AbD19384 were stable following labeling and retained their specificity for their respective target antigens. AT13387 treatment downregulated EGFR expression levels by 58% on the A431 samples and 64% on the UM-SCC-74B samples (Figure 4). The expres-sion levels of CD44v6 were unaffected by HSP90 inhibition regardless of con-centration on both cell lines. The in vitro results correlated with the in vivo xenograft studies. 124I-cetuximab clearly distinguished between the high and low EGFR-expressing tumors in control animals. Additionally, 124I-AbD19384 clearly visualized the high CD44v6-expressing tumor (A431) in both the control and AT13387 treatment groups. FDG-PET was unable to dis-criminate between the control and treatment groups. While AT13387 had no

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effect on tumor growth, the uptake of 124I-cetuximab was significantly dimin-ished in the drug-treated animals, with a 67% and 40% reduction in the A431 and the UM-SCC-74B xenografts, respectively. The uptake of neither 124I-AbD19384 nor FDG-PET was altered following treatment with AT13387 in the A431 or the UM-SCC-74B tumors. Ex vivo IHC confirmed the imaging results, i.e., downregulation of EGFR and unchanged CD44v6 expression in both tumor models.

Figure 4. EGFR and CD44v6 expression and binding specificity in A) A431 and B) UM-SCC-74B cells. Cells were incubated with increasing concentrations of 124I or 125I-labeled cetuximab or AbD19384 (0.01-60 nM). Non-radiolabeled antibody (100-fold molar excess compared to radiolabeled) was added to the highest concentrations to correct for nonspecific binding. n=3, error bars presented as SD.

Discussion As the arsenal of targeted cancer therapies grows, so do the therapeutic op-tions. The ability to assess the initial response to a chosen course of therapy via non-invasive methods is not only an important tool but perhaps a growing necessity. Precision medicine may offer an increased success rate, but even when based on the individual specifications of the patient, precision medicine does not secure a positive outcome. Therefore, monitoring the specific re-sponse to a chosen therapy increases the chances of success by evaluating the

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treatment at an early stage. Thus, clinicians gain the opportunity to change the chosen course of action.

The targets of paper I offer valuable information on two levels. First, the EGFR expression is clearly affected by the AT13387 treatment, indicating that HSP90 inhibition has an effect on the tumor. Second, the unchanged expres-sion levels of CD44v6, as exemplified by the uptake of radiolabeled AbD19384 following AT13387 treatment, indicates that this particular recep-tor could also be utilized in a different capacity.

Figure 5. Representative images of small-animal PET/CT of a) 124I-cetuximab, b) 124I-AbD19384, and c) FDG-PET. The left posterior flank (T1) represents the high EGFR and CD44v6-expressing xenografts (A431) and the right posterior flank (T2) repre-sents the low EGFR and CD44v6-expressing xenografts (UM-SCC-74B). Cross sec-tions of the xenografts and planar maximum intensity projection images are presented on the upper and lower rows, respectively.

Downregulation of EGFR is a positive clinical outcome (105,106). Targeted molecular imaging for monitoring treatment response could assess the early therapeutic response in real-time in a precise, non-invasive manner that sur-passes solely measuring tumor size. Although the tumor size was unaffected by the five AT13387 treatments prior to PET/CT-imaging, this result need not indicate a lack of efficiency of the novel drug. Rather, this result was obtained due to the experimental setup, which was designed to evaluate the expression levels of the receptors directly after the initial treatment with AT13387. In comparison, the lack of downregulation of CD44v6 following AT13387 ther-apy makes it a possible target for RIT in combination with HSP90 inhibition. However, proceeding with AbD19384 for RIT is unlikely. The bivalent AbD19384 construct is formed through spontaneous dimerization through a non-covalent bond with the monomer and dimer in a state of equilibrium (82). Therefore, the injected 124I-AbD19384 was comprised of both 124I-labeled monomers and 124I-labeled dimers, which may raise future questions concern-ing in vivo stability. Furthermore, studies on U36, its Fab and F(ab’)2, have shown that tumor uptake of a full-length antibody is >20-fold compared to that of either smaller targeting moieties (87). As such, a full-length antibody

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is more likely to achieve superior tumor-to-organ ratios and absorbed doses to the tumors and, ultimately, be the more suitable RIT candidate (86).

In conclusion, the application of targeted molecular imaging at an early stage in order to monitor the therapeutic response would greatly benefit the selection of therapeutic strategies. In precision medicine, this strategy is of increasing importance to ensure the best clinical outcome.

In the present thesis perspective, paper I evaluated and characterized AbD19384, functionally equivalent to a F(ab’)2 (based on the original Fab AbD15179 construct), for potential use in radio-immunodiagnostics. Radio-labeled AbD19384 bound specifically to CD44v6 in vitro and in vivo, as demonstrated by e.g., 124I-AbD19384 PET/CT xenograft imaging, where 124I-AbD19384 imaging revealed a clear difference between high and low CD44v6-expressing xenografts. Lastly, the lack of downregulation of CD44v6 following HSP90 inhibition indicated a potential future RIT applica-tion for CD44v6-binders in combination with radiosensitizers. Furthermore, this may be a promising construct to further develop into a full size antibody for potential RIT applications.

Preclinical assessment of an anti-CD44v6 antibody for RIT (paper II) CD44v6, with limited expression in normal tissues and high expression in sev-eral cancers, is an excellent candidate for RIT. A novel, fully human, full-length anti-CD44v6 antibody, AbN44v6, was developed from the same Fab AbD15179 construct as AbD19384 in paper I.

AbN44v6 was evaluated in vitro and in vivo for suitability of use in RIT with two therapeutic radionuclides, 177Lu and 131I. Specificity of the antibody following radiolabeling was assessed prior to in vitro RIT using 3D multicel-lular tumor spheroids of a moderate CD44v6-expressing cell line (HCT116) and a low CD44v6-expressing cell line (UM-SCC-74B). A dual-nuclide in vivo investigation of normal tissue biodistribution and dosimetry concluded the study.

Stability and specificity were unaffected following all radiolabeling proce-dures. A clear activity-dependent growth inhibition was measured in the sphe-roids for both cell lines tested, although the greater response was observed in the moderate CD44v6-expressing spheroids (HCT116). Growth inhibition was mainly caused by non-targeted radiation, i.e., the presence of a therapeutic beta-emitting nuclide in the cell medium for the UM-SCC-74B spheroids, as exemplified in the radiolabeled rituximab samples. HCT116 spheroids pre-sented with a greater response to targeted RIT, which at the higher activities did not regain normal spheroid growth rates even after removal of the activity. In the in vivo study, both radioconjugates presented similar biodistribution

31

profiles, with rapid clearance from most organs. The 177Lu-labeled conjugate demonstrated higher uptake in liver and spleen than the 125I-labeled conjugate, making these organs the dose-limiting organs for 177Lu-AbN44v6. 125I-AbN44v6 was not cleared quite as rapidly from the circulation, resulting in a higher absorbed dose to the red marrow, consequently making the red marrow the dose-limiting organ for this conjugate. The total effective dose for both radioconjugates was estimated to be approximately 0.1 mSv/MBq.

Figure 6. 3D in vitro RIT of either 177Lu-AbN44v6 or 131I-AbN44v6. A) Growth-re-sponse of UM-SCC-74B, normalized to % of untreated controls at each time point. B) Representative images of UM-SCC-74B spheroids shown in A, as well as 100 kBq of radiolabeled rituximab. C) Growth-response of HCT116 spheroids normalized to % size of untreated controls at each time point. D) Representative images of HCT116 spheroids shown in C, as well as 100 kBq of radiolabeled rituximab. n≥5, error bars represent 95% confidence intervals.

Discussion The use of RIT is usually restricted to high antigen-expressing cancers. Natu-rally, the cumulative absorbed dose to the tumor increases with increasing an-tigen density. However, HCT116, a moderate CD44v6-expressing cell line, demonstrated a clear activity-dependent response to RIT in an in vitro 3D set-ting. The highest activity treatment, 100 kBq of either 177Lu- or 131I-AbN44v6, resulted in a decreased size by approximately 50%. Additionally, the majority of the growth inhibition was a direct result of targeted as opposed to non-targeted radiation. In contrast, the majority of the growth inhibition detected in the UM-SCC-74B spheroids was caused by the mere presence of therapeu-tic nuclides in close proximity to the spheroid. These results verify that the

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cytotoxic effect of RIT was greater in the cell line with a greater antigen-den-sity while also indicating that this therapeutic strategy may be feasible for both high and moderate antigen-expressing cancers.

The dual-nuclide normal tissue biodistribution results were similar for both radioconjugates. However, the 177Lu-labeled conjugate demonstrated a ten-dency to accumulate more within organs (e.g., liver and spleen) but was cleared faster from circulation. This finding indicates possible tendencies to-ward aggregation in vivo that would result in elimination from circulation via a size-dependent mechanism, transchelation to metal-binding proteins or a lowered stability of the conjugate in vivo (107). The 125I-conjugate was more rapidly cleared from all organs, but remained in the bloodstream for an ex-tended period compared to the 177Lu-conjugate. This increased the absorbed dose to the red marrow, making the red marrow the primary dose-limiting or-gan of the iodine-conjugate. The differences in biodistribution profiles of each radioconjugate offer valuable information. One of the limiting factors of any radiopharmaceutical, is the absorbed dose to healthy tissues, usually accumu-lated during elimination (108,109). Previous studies on the radiolabeled Fab AbD15179, which contains an identical binding sequence to the full-length antibody, highlighted the liver and spleen (111In-AbD15179), and kidneys (125I-AbD15179) as possible dose-limiting organs.

The biodistribution of the radiolabeled AbN44v6 conjugates demonstrated an increase in uptake in the liver, spleen and kidneys compared to the biodis-tribution of the smaller AbD15179 and AbD19384. Additionally, the full-length antibody demonstrated an increased time in circulation compared to AbD15179 and AbD19384. This was expected, as studies have shown that full-length antibodies are present in the circulation for an extended period compared to smaller moieties (86,91). The extended time in the blood of both radiolabeled AbN44v6 conjugates is likely the reason for the increased uptake in % ID/g in high-perfusion organs (e.g., the liver, spleen and kidneys).

The relatively low uptake of 125I-AbN44v6 by organs of elimination such as the liver indicates that the full-length antibody is possibly suitable as a tar-geting moiety. Furthermore, the differences in dose-limiting organs, depend-ing on radiolabel, make it possible to customize therapy for individual patients or patient groups, depending on their medical profile to avoid toxicity issues. The indication of decreased in vivo stability or possible aggregation of the 177Lu-labeled conjugate, as demonstrated in the rapid elimination from the blood, is of concern and should be further addressed. Moreover, other poten-tial chelators and labeling techniques for radiometal labeling should be as-sessed.

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Figure 7. Biodistribution presented as % ID/g of 177Lu-AbN44v6. n=5, error bars rep-resent SD.

Notably, AbN44v6 does not cross-react with the murine CD44v6-receptor, and the results of the xenograft study are not completely extrapolatable to hu-mans without further studies. However, previous studies using anti-CD44v6 antibodies in humans have demonstrated very limited uptake in normal tis-sues, which is promising for future studies with AbN44v6. However, size, ag-gregation, charge, lipophilicity and choice of radiolabel are crucial to deter-mine both the clearance mechanisms and potential off-target effects. There-fore, this study still offers important information about the future prospects of utilizing AbN44v6 as a radio-immunotherapeutic agent. However, as tumors were not included in the biodistribution and dosimetry studies, the tumor-to-organ ratios as well as absorbed dose to the tumors have yet to be defined. As both AbD15179 and AbD19384 demonstrated excellent specific binding in vivo, we expect greater uptake by the full-length antibody in future xenograft studies.

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Figure 8. Biodistribution presented as % ID/g of 125I-AbN44v6. n=5, error bars rep-resent SD.

In the present thesis perspective, paper II further evaluated AbD15179-based CD44v6-constructs by moving from the F(ab’)2-construct to a full-length an-tibody for potential use in RIT applications. The full-length antibody was suc-cessfully radiolabeled with both a radiometal and radio-iodine and retained antigen-binding. RIT using radiolabeled AbN44v6 resulted in activity-de-pendent growth inhibition in vitro and demonstrated promising normal tissue clearance and dosimetry in vivo, indicating that AbN44v6 could be a promis-ing future candidate for RIT in CD44v6-expressing cancers.

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The potency of PM2 in combination with external beam radiotherapy (papers III & IV) Due to its role as a crucial tumor suppressor and high mutation rate in cancer, p53 remains an interesting target for precision medicine. Small-molecule an-tagonists of the p53-MDM2 protein-protein interaction are generating prom-ising results in clinical trials; however, so far without exploring the combina-tion with radiotherapy despite the central role played by wt p53 in radiation response and DNA damage mechanisms. The aim of papers III and IV was therefore to investigate the potential of PM2-based therapy and evaluate the combination of PM2 treatment and EBRT in a proof-of-principle setting.

Paper III determined the potency of PM2 monotherapy and the combination therapy in vitro. The paper sheds light on the mechanisms behind the observed effects via e.g., Western blotting and flow cytometry. Paper IV further estab-lished the potency of PM2 treatment in combination with EBRT in an in vivo setting using wt p53 (HCT116) and p53-knockout (HCT116 -/-) xenografts.

While PM2 treatment demonstrated anti-tumorigenic effects on wt p53, Human papillomavirus (HPV)-negative cell lines, the combination of PM2 and EBRT proved a more potent cocktail. Upregulated expression levels of wt p53, cleaved caspase-3, and Noxa were measured following PM2 exposure (Figure 9). The expression levels were further elevated in the combination-treated samples (i.e., PM2 with EBRT), exemplifying the potential of merging the two therapies. The chosen in vitro therapy setting, 3D multicellular tumor spheroids, confirmed dose-dependent growth inhibition for both monothera-pies. Five repeated doses of PM2 or EBRT at 48 h intervals resulted in signif-icantly decreased spheroid sizes compared to untreated controls (Figure 10A, 10B, 10D, 10E). The effects of the combination therapies were however su-perior to each monotherapy. Three repeated combination doses resulted in near-complete stagnation in growth and five repeated combination doses led to complete spheroid disintegration and cell death (Figure 10C and 10F).The selectivity of PM2 towards wt p53 cancers was evident following the evalua-tion of the effects of the peptide on a p53-knockout cell line (HCT116 -/-). The peptide had no detectable anti-tumorigenic effects on the HCT116 -/- samples in either 2D or 3D cultures, regardless of combination with radiother-apy (Figure 2, paper IV). This was confirmed in vivo, where no significant effects on the growth of HCT116 -/- xenografts were observed following PM2-based therapy (Figure 11G-11I). In contrast, the wt p53 xenografts were greatly affected by both monotherapies and the combination therapy, ulti-mately resulting in a prolonged survival of 50% in the combination-treated group compared to the controls (Figure 11A-11F). Lastly, 125I-labeled PM2 biodistribution demonstrated clear tumor uptake and retention with rapid elim-ination from normal tissues and even distribution of the compound throughout the tumor, as visualized by autoradiography (Figure 3, paper IV).

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Figure 9. Western blot analyses of (A) p53 expression at 24 h and (B) cleaved caspase-3 expression at 72 h and 96 h of UM-SCC-74B and HCT116 samples post treatment with either 2 Gy, PM2 (20 µM), or the combination. Samples (n≥3) were normalized to controls, represented as a dotted line at y=1. Error bars presented as SD. (C) Representative Western blot images of p53 and cleaved caspase-3 expres-sion of UM-SCC-74B samples at 24 h (p53) and 72 h (cleaved caspase-3) post treat-ment, and HCT116 samples (D) at 24 h (p53) and 96 h (cleaved caspase-3) post treatment. Flow cytometric analyses of UM-SCC-74B and HCT116 samples at 24 h, 48 h, 72 h, and 96 h post treatment of (E) cleaved caspase-3 expression and (F) Noxa expression. n≥3, error bars presented as SD. Significance was determined us-ing one-way ANOVA: p≤0.05 (*), p≤0.01 (**), p≤0.001 (***), p≤0.0001 (****).

Discussion Two of the main cellular responses to p53 activation are cell cycle arrest and apoptosis. While cell cycle arrest is relevant for cancer therapy, apoptosis is of far greater interest as a therapeutic response to p53 activation. It has been suggested that the abovementioned responses are dependent on the level of p53 protein accumulation, with lower levels resulting in cell cycle arrest and higher levels in p53-mediated apoptosis (110). The findings presented in paper III support this hypothesis, as PM2 monotherapy indicated p53-mediated apoptosis, as exemplified by the upregulation of both cleaved caspase-3 and Noxa (Figure 9). The detected apoptosis in the PM2 monotherapy samples is an example of the potency of a therapeutic strategy that targets the negative regulators of the p53 protein. By inhibiting MDM2 and MDMX and thus re-storing expression levels, wt p53 is able to assert its innate capacity as a tumor suppressor.

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Figure 10. Growth of UM-SCC-74B spheroids over time following treatment in 48 h intervals with (A) EBRT (2 Gy), (B) PM2 treatment (20 µM), and (C) combination therapy of EBRT and PM2 treament. Representative images during EBRT (D), PM2 treatment (E), and treatment combinations (F). n≥4, error bars represent 95% confi-dence intervals.

However, in order to potentiate the p53-mediated tumor suppressive mecha-nisms, a stimulant of p53 activation pathways, such as ionizing radiation, could be of great beneficial use. Induction of a wt p53 response via exposure to ionizing radiation should potentiate the effects of both radiation and p53-based therapy. In paper III, this hypothesis was supported as the levels of pro-apoptotic proteins (cleaved caspase-3 and Noxa) were amplified in all wt p53 combination-treated samples compared to monotherapy samples (Figure 9). In the 3D in vitro therapeutic setting, this result was even more evident. Re-peated monotherapies resulted in growth inhibition, whereas repeated combi-nation treatments resulted in complete sample disintegration of the UM-SCC-74B spheroids (Figure 10).

Modest clinical successes of current anti-MDM2 therapies have been at-tributed to overexpression of MDMX as well as mutations affecting the bind-ing properties of MDM2 as a consequence of MDM2-targeted therapy (81). As both MDM2 and MDMX are in an autoregulatory loop with p53, induction of p53 accumulation will naturally result in a similar upregulation of the neg-ative regulators. Unlike most antagonists of the p53-MDM2 protein-protein interaction, PM2 targets and inhibits both MDM2 and MDMX. Therefore, PM2 could potentially negate these limitations by blocking the interaction be-tween wt p53 and both MDM2 and MDMX. Additionally, directly inducing a p53 response via EBRT in combination with a potent MDM2/X inhibitor could further increase the therapeutic success rates. In paper IV, this strategy was assessed in vivo. Using HCT116 in parallel with HCT116 -/- xenografts, combination therapy using PM2 and EBRT was tested. The results obtained

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in vitro in an identical experimental setting correlated well with the outcome of the in vivo xenograft trial. Both monotherapies resulted in growth inhibition of the wt p53 xenografts, whereas only EBRT had an inhibitory effect on the growth of HCT116 -/- xenografts (Figure 11).

Figure 11. HCT116 wt p53 and p53 -/- xenografts treated with PM2 and EBRT. A) Growth of wt p53 tumors until time of first termination of animals from each group (controls, 10xPM2 (daily injections), 3xEBRT (48 h intervals) or the combination of the two monotherapies. n≥6, error bars presented as 95% confidence intervals. B) Tu-mor size of wt p53 xenografts shown in (A) at day 10 post start of treatment. n≥6, error bars presented as SD. p<0.05 defined as *. C) Survival proportions of wt p53 xenografts. D) Growth of wt p53 tumor size until time of first termination of animals from each group (controls, 3xPM2 (48 h intervals), 3xEBRT (48 h intervals) or the combination. n=6, error bars presented as 95% confidence intervals. E) Tumor size of wt p53 xenografts shown in (D) at day 10 post start of treatment. n≥6, error bars pre-sented as SD. p<0.05 defined as *. F) Combination index plot of synergistic effects of 3xPM2 and 10xPM2 and 3xEBRT. G) Growth of p53 -/- tumors until time of first termination of animals from each group with identical treatment regimens as animals presented in (A). n=5, error bars presented as 95% confidence intervals. H) Tumor size of -/- p53 xenografts shown in (G) at day 10 post start of treatment. n=5, error bars presented as SD. I) Survival proportions of -/- p53 xenografts.

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Additionally, combination treatments yielded synergistic results, and an in-creased median survival by 50% between combination-treated and control groups in HCT116 xenografts was observed. No such effects were detected in the p53-knockout samples, in vitro or in vivo as only EBRT resulted in growth inhibition. These results confirm the specificity of PM2 toward wt p53 cancers as well as the potency of combining PM2-based therapy with EBRT in wt p53, HPV-negative cancers. Furthermore, radiolabeled PM2 (125I-PM2), injected subcutaneously above the tumor, demonstrated excellent tumor penetration with an even distribution throughout the tumor, which was retained for at least 48 h post injection.

A rapid elimination rate and lack of accumulation of PM2 in normal tissues could possibly circumvent the familiar toxicities observed with current MDM2 inhibitors. However, as PM2 was injected subcutaneously, conclu-sions from the biodistribution results are merely indicative of the distribution of 125I-PM2 following localized subcutaneous injection. Additional studies are needed to assess the toxicity profile of PM2, preferably in the intended means of future clinical administration. The potency of PM2, despite the subcutane-ous injections at low doses, is exemplified in the success of the proof-of-prin-ciple in vivo combination study presented in paper IV. Granted, the need for further optimization concerning for example delivery, doses, and treatment-intervals as well as toxicity studies of PM2-based therapy exists. However, there is no denying the anti-tumorigenic effects of PM2 and its potential as a future cancer therapy of wt p53 cancers. Furthermore, the greatly increased therapeutic response to combination therapy with EBRT illustrates the prom-ise of utilizing PM2 as a future radiosensitizer.

An indication of the benefit of combining two precision medicines (paper V) Merging a radiosensitizing compound such as PM2 with a novel RIT agent (AbN44v6) is a unique strategy. The combination of two precision therapeu-tics is a promising concept that could exceed the success rate of traditional therapies, particularly in cases where EBRT is less likely to have a therapeutic effect (e.g., disseminated disease or infiltrating tumors). Earlier studies in this thesis focused on potentiating EBRT with PM2-based therapy on wt p53 can-cer cell lines. The final study (paper V) combines the novel combination of wt p53-targeted therapy with RIT via radiolabeled AbN44v6 targeting CD44v6.

The evaluation was conducted in the same in vitro setting as Paper II, using a moderate CD44v6-expressing cell line (HCT116) and a low CD44v6-ex-pressing cell line (UM-SCC-74B), both wt p53 cell lines. The therapy was assessed in a 3D multicellular tumor spheroid setting using previously deter-mined doses of PM2 and activities of 177Lu-AbN44v6 (papers II-IV).

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Figure 12. A) HCT116 3D in vitro RIT treated with 30 kBq and 100 kBq of 177Lu-AbN44v6 and 100 kBq of 177Lu-rituximab. Also shown are representative images of the same treatments, B) HCT116 3D in vitro therapy of spheroids treated with 20 µM of PM2 and the combination of PM2 and RIT Also shown are representative images of the same treatments. n≥4, error bars presented as 95% confidence intervals.

Repeated treatments with PM2 (20 µM) at 48 h intervals failed to increase the level of growth inhibition in both HCT116 and UM-SCC-74B spheroids com-pared to a single dose incubated for six-to-seven days (Figure 2E and 2F, paper V). RIT using 177Lu-AbN44v6 resulted in activity-dependent growth inhibi-tion of the HCT116 spheroids, which differed significantly from the growth inhibition observed using identical activities of the negative control 177Lu-rituximab (Figure 12). In contrast, RIT using 177Lu-AbN44v6 did not differ significantly from the effects of 177Lu-rituximab on the UM-SCC-74B sphe-roids (Figure 13). The combination of RIT and PM2-based therapy greatly improved the growth inhibition of both cell lines. The HCT116 spheroids treated with 100 kBq of 177Lu-AbN44v6 and a single dose of PM2 diminished two-fold in size compared to spheroids treated with RIT alone, and three-fold compared to PM2 monotherapy. Similar effects were observed in the UM-SCC-74B spheroids, where the highest activity treatment (100 kBq) had little effect on spheroid growth in itself, and yet, the addition of PM2 resulted in spheroid regression.

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Discussion Previous work presented in this thesis on the effects of PM2 treatment in a 3D in vitro model system (paper III), has illustrated the potency of combining radiotherapy with PM2-based therapy. However, as EBRT is rarely applicable in cases of disseminated disease, the combination therapy of PM2-based ther-apy and EBRT has certain limitations. Changing the nature of the applied ra-diotherapy by substituting EBRT with RIT may help overcome these limita-tions.

As seen in paper III, repeated treatments with PM2 resulted in partial growth inhibition, whereas the combination with repeated doses of radiother-apy resulted in complete growth inhibition and even in spheroid regression and disintegration (Figure 4, paper III). However, the repeated treatments with PM2, at 48 h intervals, only resulted in momentary growth inhibition, which ended nearly directly after the peptide was removed from the cell medium. A direct comparison between three repeated PM2 treatments at 48 h intervals and one PM2 treatment incubated for the same period was executed in paper V (Figure 2E and 2F, paper V). The two different treatment schedules resulted in near identical growth inhibition of both HCT116 and UM-SCC-74B sphe-roids, establishing that PM2 remains active in cell culture medium for at least six days. Additionally, as repeated treatments failed to improve the therapeutic response in the in vitro setting further, there was little interest in assessing repeated treatments in combination with RIT in the in vitro spheroids. Exclud-ing the need for repeated PM2 treatments prolonged the incubation time of 177Lu-AbN44v6 to six days. The six-day incubation of both therapies resulted in remarkable amplifications in terms of growth inhibition on both cell lines. The combination of a single PM2 treatment with ≥30 kBq of 177Lu-AbN44v6 induced a significant size reduction of both HCT116 and UM-SCC-74B sphe-roids compared to monotherapies as well as repeated PM2 treatments (Figures 12 and 13). Furthermore, combination therapy of PM2 with 100 kBq of 177Lu-AbN44v6 resulted in complete growth inhibition of the UM-SCC-74B sphe-roids, which toward the end of the assay demonstrated signs of disintegration (Figure 13).

While the potency of the combination cocktail is by far more striking in the UM-SCC-74B samples, it is imperative to remember that the primary radia-tion damage in UM-SCC-74B was caused by non-targeted radiation. In all studies presented in this thesis, UM-SCC-74B has proven highly sensitive to the combination of PM2 with radiotherapy. Therefore, although impressive, the spheroid results of combined PM2 treatment and RIT are slightly mislead-ing when using this cell line as these results would likely not be translatable to an in vivo setting. Additionally, attempting RIT on a low antigen-expressing cell line is likely not a plausible strategy in a clinical setting. Furthermore, although all wt p53, HPV-negative cell lines responded to PM2-based therapy (Table 1, paper III), there was a difference the sensitivity of each cell line to

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the treatment, particularly in combination with radiotherapy. The difference in sensitivity should be investigated in future studies in order to ensure the applicability for wide-scope use of PM2-based therapy of wt p53 cancers. The effects of combined RIT and PM2-based therapy on HCT116 spheroids were significant and indicate that this may be a feasible strategy for CD44v6-ex-pressing cancers. This finding further elucidates the promise of combining two targeted compounds and could translate well to an in vivo setting using high or moderate CD44v6-expressing xenografts.

Figure 13. A) UM-SCC-74B 3D in vitro RIT treated with 30 kBq and 100 kBq of 177Lu-AbN44v6 and 100 kBq of 177Lu-rituximab. Also shown are representative im-ages of the same treatments, B) UM-SCC-74B 3D in vitro therapy of spheroids treated with 20 µM of PM2 and the combination of PM2 and RIT. Also shown are repre-sentative images of the same treatments. n≥4, error bars presented as 95% confidence intervals.

The radiosensitizing or potentiating effects of PM2 were greater when the pep-tide was administered after irradiation (data not shown). This observation is attributed in part to the rapid elimination of the peptide and to the peak ex-pression levels of MDM2/X following irradiation. Barring an excretion pro-cess, PM2 is active in vitro for an extended period and remains active for at least six days in cell culture medium, as demonstrated by the spheroid data

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presented in paper V. The therapeutic window of PM2 is thus wider in the in vitro assays, and the timing and method of drug administration is of lesser importance. However, the in vitro therapeutic effects measured as a result of the widened therapeutic window can be falsely amplified and should be taken into consideration when translating the in vitro data into an in vivo setting.

The findings presented in paper V illustrate the potency of combining PM2-based therapy with RIT using radiolabeled AbN44v6. Through combining PM2-based therapy with a “magic bullet” (RIT), this potent cocktail will seek out and interact with tumor tissues. Furthermore, barring no notable expres-sion of the molecular target in healthy tissues, the combination therapy will likely only affect organs involved in the excretion pathways. Overall, these factors highlight the potency of this combination therapy and offer a promis-ing and exciting future for radiosensitization strategies.

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Conclusions

Due to rapid technological advances, a deeper understanding of the molecular characteristics of cancer has warranted a revolution within the field of preci-sion medicine. Targeted compounds for both diagnosis and therapy have in-creased the success rates of cancer therapies and improved the means of early detection. However, the field is complex and far from complete and new tar-gets, tracers and therapies are essential for ultimately defeating cancer.

The work presented here is comprised of two main components, namely, targeting the CD44v6-receptor and combining MDM2/X inhibition with radi-otherapy. In particular, this work has focused on combination therapies, pri-marily of PM2 and radiotherapy, which we believe is a potent and promising strategy for treatment of wt p53 cancers. The main findings are listed below:

AbD19384 labeled with 124I can detect CD44v6-positive xenografts and can selectively distinguish between high- and low-expressing tumors.

CD44v6 was not downregulated following HSP90 inhibition and can be used as a target for radio-immunotherapy in combination with HSP90 inhibition.

In vitro 3D RIT using radiolabeled AbN44v6 resulted in activity-dependent targeted growth inhibition of a moderate CD44v6-ex-pressing cell line.

Normal tissue biodistribution and dosimetry of radiolabeled AbN44v6 resulted in a total effective dose of approximately 0.1 mSv/MBq for both radioconjugates.

PM2-based therapy indicated increased p53-mediated apoptotic ac-tivity in wt p53, HPV-negative cancer cell lines in vitro.

The effects of PM2-based therapy are potentiated by external beam radiotherapy, resulting in increased levels of pro-apoptotic proteins in vitro.

Repeated PM2 treatment and external beam radiotherapy results in wt p53 spheroid disintegration in vitro and growth inhibition of wt p53 xenografts in vivo.

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The combination of radio-immunotherapy using 177Lu-AbN44v6 and PM2-based therapy is a potent strategy for future therapeutic treatment of wt p53, HPV-negative CD44v6-expressing cancers.

The work presented here has illuminated the possibility of utilizing CD44v6 as a target for both radiodiagnostics and therapy. Furthermore, we have estab-lished the potency of combining external beam radiotherapy with MDM2/MDMX inhibition using PM2 both in vitro and in vivo. Lastly, a proof-of-principle in vitro study confirmed the promising and unique concept of combining radio-immunotherapy targeting CD44v6 with PM2-based ther-apy. It remains our hope that utilizing not one but several of the molecular characteristics of a specific cancer will help turn the tables on cancer and im-prove patient outcomes in the future.

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The future at a glance

Efficacy of PM2 Although the effects of PM2-based therapy and combination therapy with EBRT were evaluated in vivo, the doses and particularly the delivery of PM2, (i.e., testing intravenous or intraperitoneal injections) would benefit from fur-ther investigation. Furthermore, while PM2-based therapy had a growth sup-pressive effect on all wt p53, HPV-negative cell lines, the responses varied in strength. This finding is exemplified by the differences in growth inhibition and spheroid disintegration between HCT116 and UM-SCC-74B. The reasons behind the differences in efficacy observed between cell lines have not yet been investigated. However, HCT116 has a KRAS mutation, which is up-stream of p53, and could potentially influence the level of p53 activation fol-lowing irradiation and explain the lack of potency of PM2-based therapy. A thorough investigation into the mechanisms behind these differences in effi-cacy could pinpoint new and interesting combination therapies using PM2.

RIT and PM2 in vivo The original goal of the work presented in this thesis was to assess the combi-nation of RIT and PM2-based therapy. However, for proof-of-principle, EBRT replaced RIT for simplicity as well as practicality. Paper V evaluated the potency of combining RIT targeting CD44v6 with PM2-based therapy in vitro and paper II assessed the biodistribution and dosimetry of AbN44v6. The next logical step of evaluation is to not only examine RIT in vivo but also test the combination of RIT with PM2. The use of a high CD44v6-expressing, wt p53, HPV-negative xenograft would be ideal in this experimental setup. Ad-ditionally, for future therapy with radiometals (i.e., 177Lu), a different chelator should be considered. Although the results from chelating AbN44v6 with CHX-A’’-DTPA were promising, concerns of aggregation or stability issues in vivo were observed in the biodistribution study in paper II. Moreover, the specific activities of 177Lu-AbN44v6 were generally lower than those follow-ing direct radio-iodination. These issues could potentially be overcome by us-ing a different chelator.

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Targeted drug delivery of PM2 and newer generations of the peptide Studies have shown that PM2 enters the cytoplasm and further localizes to the nucleus, but the mechanism by which the stapled peptide makes its way both into the cell and the nucleus remains unclear. Furthermore, PM2 does not tar-get cancer cells specifically. To ensure delivery of PM2 or newer generations of the peptide specifically to cancer cells, targeted drug delivery of PM2 could be highly beneficial. The use of targeted lipo-discs or nanoparticles loaded with the stapled peptide could ensure a higher delivery rate to cancer cells while lowering uptake in normal tissues. This is an interesting concept, which in the future could lower the toxicity following therapy with MDM2/X inhib-itors. Furthermore, by labeling PM2 with 125I, the need for combining PM2-based therapy with EBRT or RIT as well as the need for lipo-discs may be-come redundant. The Auger-electrons emitted by 125I would cause extensive damage to the DNA of cancer cells when in close proximity to the nucleus.

Combining RIT with HSP90 inhibition Although featured and discussed in paper I, the combination of RIT and HSP90 inhibition was not the main focus of this thesis. A substantial decrease in EGFR expression was observed following treatment with AT13387, indi-cating that EGFR would be a poor target for combination therapy. However, the unchanged expression levels of CD44v6 following AT13387 therapy were highly promising for future combination studies. In vivo therapy with AT13387 in combination with EBRT has been assessed by our group and it would be easy to repeat the study, albeit this time in combination with RIT targeting CD44v6.

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Acknowledgements

The journey thus far has truly been a wonderful experience, albeit with long days and even a few long nights. It was never a lonely journey and along the path, more than a few people deserve my deepest thanks and sincerest appre-ciation. I would like to thank all of my colleagues, collaborators and former students for their contributions to this thesis. The following people certainly deserve my most heartfelt gratitude:

Marika Nestor, my supervisor and role model both at work and as a parent. Thank you for your continuing faith in me and for having patience with me when I struggle to see the positive side of things. I will always admire your ability to find a thousand solutions in a single second, your creativity and ap-proach to both life and science. We set out to solve the cancer enigma and I hope we will get there one day.

My co-supervisor, Vladimir Tolmachev, whose encouragement, support and kindness has meant the world to me. Your knowledge of radiochemistry is unparalleled, but I think what I have cherished the most are our talks on his-tory.

TBRGE-members, both past and present. From summer kick-offs to late nights at the lab, you have all been wonderful, helpful and supportive:

Diana, I have learned so much from you over the years that it is far too much to list! You have an innate ability to sense when I am having a tough day and I know I have not thanked you enough for both that and your endless help and support.

Sara, from student to teacher - of statistics and software (of which you will need to teach me continuously!). Thank you for sharing my passion for mak-ing lists, schedules and plans, but mostly for being my outlet and friend when I needed it the most.

Sina, you have taught me more about receptors and interactions than you probably know. I will miss taking care of your herbs in the summer (all of that delicious basil!).

João, who understands my appreciation for football and music. I will never forget when Portugal played the EUROs in 2016 and you were too nervous to watch without a little support from afar.

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Last but not least, Anna-Karin, for the dissection tips and important life-lessons (not choosing a hotel with a beachfront in Darwin due to the extreme crocodilian infestation was a lifesaver - literally).

Andris, for all of our passionate discussions, silly Friday afternoon humor after too much fika and lending a helping hand whenever I have needed it.

Bo Stenerlöw, for always having great advice at hand, the helpful support and for being a fantastic role model.

Kicki, for your continuous support and insightful observations about life and work. Your door is always open for me (probably since I know the passcode), regardless of the issue or your own lack of time.

The rest of old “BMS” and “PPP”, particularly Javad and Anzhelika for al-ways trying to help, Anna Orlova, for all of the advice and help you have offered me, Hans, for taking the time from a busy retirement to assist in do-simetry studies and trying to teach me physics even though I am a lost cause.

All of Ridgeview, particularly Jonas, whose infectious positivity I dearly miss as part of TBRGE, Hanna, Jos, Kalle for always being available and helpful with whatever silly question I might have about LigandTracer.

I would also like to thank the IGP PhD student council members for teaching me that there may be more to life at Uppsala University than the bottom floor at Rudbeck!

Björn & Lis-Marie, for always believing in me and appreciating me for who I am. Thank you for always supporting me, whether it meant living in your basement for four months or showing me love whenever I needed it. Henrik & Elsa, for always being positive and welcoming (and allowing me to steal your son whenever we visit and lending us muscle-power during hectic mov-ing days). Bertil, for being inquisitive and listening to me talk about the silly bits of knowledge no one else has the patience for.

Last but definitely not least, my family: Mor, for reading nearly every academic paper I have ever written (including this thesis) and teaching me more about writing than any teacher ever did. You have consistently encouraged me to do whatever makes me happy in life and both you and Gert deserve a medal for always being there for us (especially for the cat-sitting recently!).

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Far, for putting up with me and supporting me through thick and thin through-out my life (and for passing on your love of history). You & Helle have con-tinuously showed me love, support and understanding and kept your doors open for Malin and me, making it so incredibly welcoming to come home.

Christina, the perhaps proudest big sis in the world. I cannot remember a time in my life when you were not there to support me. To me, you will always be the smartest and strongest in the world.

Johan, for being the most encouraging and protective not-so-little-anymore brormand in existence. You were always my sunshine growing up and in my eyes; you continue to outshine the rest of the world.

Malin, the love of my life. You mean and are the world to me. Thank you for having the illusion of me being perfect.

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References

1. Hanahan D, Weinberg RA. The Hallmarks of Cancer. Cell. 2000 Jan 7;100(1):57–70.

2. Shin SH, Bode AM, Dong Z. Precision medicine: the foundation of future can-cer therapeutics. Npj Precis Oncol. 2017 Apr 24;1(1):12.

3. Fisher R, Pusztai L, Swanton C. Cancer heterogeneity: implications for tar-geted therapeutics. Br J Cancer. 2013 Feb 19;108(3):479–85.

4. Sun X, Yu Q. Intra-tumor heterogeneity of cancer cells and its implications for cancer treatment. Acta Pharmacol Sin. 2015 Oct;36(10):1219–27.

5. Senft D, Leiserson MDM, Ruppin E, Ronai ZA. Precision Oncology: The Road Ahead. Trends Mol Med. 2017 Oct 1;23(10):874–98.

6. Schmidt KT, Chau CH, Price DK, Figg WD. Precision Oncology Medicine: The Clinical Relevance of Patient Specific Biomarkers Used to Optimize Can-cer Treatment. J Clin Pharmacol. 2016 Dec;56(12):1484-1499.

7. Klement GL, Arkun K, Valik D, Roffidal T, Hashemi A, Klement C, et al. Future paradigms for precision oncology. Oncotarget. 2016 Jul 19;7(29):46813-46831.

8. Dunphy MPS, Lewis JS. Radiopharmaceuticals in preclinical and clinical de-velopment for monitoring of therapy with PET. J Nucl Med. 2009 May;50 Suppl 1:106S–21S.

9. Dhingra VK, Mahajan A, Basu S. Emerging clinical applications of PET based molecular imaging in oncology: the promising future potential for evolving personalized cancer care. Indian J Radiol Imaging. 2015 Dec;25(4):332–41.

10. Kostakoglu L, Agress H, Goldsmith SJ. Clinical Role of FDG PET in Evalua-tion of Cancer Patients. RadioGraphics. 2003 Mar 1;23(2):315–40.

11. Ibrahim N, Cho S, Hall L, Perlman S. Is it cancer? Potential pitfalls of FDG PET/CT imaging. J Nucl Med. 2016 Jan 5;57(supplement 2):1315–1315.

12. Long NM, Smith CS. Causes and imaging features of false positives and false negatives on 18F-PET/CT in oncologic imaging. Insights Imaging. 2011 Dec;2(6):679-698.

13. Szyszko TA, Yip C, Szlosarek P, Goh V, Cook GJR. The role of new PET tracers for lung cancer. Lung Cancer. 2016 Apr 1;94:7–14.

14. Sharma P, Mukherjee A. Newer positron emission tomography radiopharma-ceuticals for radiotherapy planning: an overview. Ann Transl Med. 2016 Feb;4(3):53.

15. Ke X, Shen L. Molecular targeted therapy of cancer: The progress and future prospect. Front Lab Med. 2017 Jun 1;1(2):69–75.

16. Maranhão RC, Vital CG, Tavoni TM, Graziani SR. Clinical experience with drug delivery systems as tools to decrease the toxicity of anticancer chemo-therapeutic agents. Expert Opin Drug Deliv. 2017 Oct 3;14(10):1217–26.

17. Angsutararux P, Luanpitpong S, Issaragrisil S. Chemotherapy-Induced Cardi-otoxicity: Overview of the Roles of Oxidative Stress. Oxidative Medicine and Cellular Longevity. 2015;2015:795692.

52

18. Lippert TH, Ruoff H-J, Volm M. Intrinsic and Acquired Drug Resistance in Malignant Tumors. Arzneimittelforschung. 2008;58(06):261–4.

19. Housman G, Byler S, Heerboth S, Lapinska K, Longacre M, Snyder N, et al. Drug Resistance in Cancer: An Overview. Cancers. 2014 Sep 5;6(3):1769–92.

20. Lheureux S, Denoyelle C, Ohashi PS, De Bono JS, Mottaghy FM. Molecularly targeted therapies in cancer: a guide for the nuclear medicine physician. Eur J Nucl Med Mol Imaging. 2017;44(Suppl 1):41–54.

21. Padma VV. An overview of targeted cancer therapy. BioMedicine. 2015 Nov;5(4):19.

22. Buffery D. The 2015 Oncology Drug Pipeline: Innovation Drives the Race to Cure Cancer. Am Health Drug Benefits. 2015 Jun;8(4):216–22.

23. Talwar V, Pradeep Babu KV, Raina S. An overall review of targeted therapy in solid cancers. Curr Med Res Pract. 2017 May 1;7(3):99–105.

24. Ahmad SS, Duke S, Jena R, Williams MV, Burnet NG. Advances in radiother-apy. BMJ. 2012 Dec 4;345:e7765.

25. Liberti MV, Locasale JW. The Warburg Effect: How Does it Benefit Cancer Cells? Trends Biochem Sci. 2016 Mar;41(3):211–8.

26. Masui T, Doi R, Ito T, Kami K, Ogawa K, Harada D, et al. Diagnostic value of 18F-fluorodeoxyglucose positron emission tomography for pancreatic neu-roendocrine tumors with reference to the World Health Organization classifi-cation. Oncol Lett. 2010 Jan;1(1):155–9.

27. Almuhaideb A, Papathanasiou N, Bomanji J. 18F-FDG PET/CT Imaging In Oncology. Ann Saudi Med. 2011;31(1):3–13.

28. Virgolini I, Decristoforo C, Haug A, Fanti S, Uprimny C. Current status of theranostics in prostate cancer. Eur J Nucl Med Mol Imaging. 2018 Mar;45(3):471-495. Epub 2017 Dec 28.

29. Jalilian AR. An overview on Ga-68 radiopharmaceuticals for positron emis-sion tomography applications. Iran J Nucl Med. 2016 Jan 1;24(1):1–10.

30. Weissleder R. Molecular Imaging in Cancer. Science. 2006 May 26;312(5777):1168–71.

31. Sörensen J, Velikyan I, Sandberg D, Wennborg A, Feldwisch J, Tolmachev V, et al. Measuring HER2-Receptor Expression In Metastatic Breast Cancer Us-ing [68Ga]ABY-025 Affibody PET/CT. Theranostics. 2016;6(2):262–71.

32. Zhang B, Hurvitz S. Long-term outcomes of neoadjuvant treatment of HER2-positive breast cancer. Clin Adv Hematol Oncol HO. 2016 Jul;14(7):520–30.

33. Mankoff DA. Molecular imaging to select cancer therapy and evaluate treat-ment response. Q J Nucl Med Mol Imaging. 2009 Apr;53(2):181–92.

34. Zukotynski K, Jadvar H, Capala J, Fahey F. Targeted Radionuclide Therapy: Practical Applications and Future Prospects. Biomark Cancer. 2016 May 18;8(Suppl 2):35–8.

35. European Medicines Agency - Find medicine - Xofigo [Internet]. [cited 2018 Feb 13].

36. Xofigo (radium Ra 223 dichloride) FDA Approval History - Drugs.com [In-ternet]. [cited 2018 Feb 13].

37. Jadvar H, Challa S, Quinn DI, Conti PS. One-Year Postapproval Clinical Ex-perience with Radium-223 Dichloride in Patients with Metastatic Castrate-Resistant Prostate Cancer. Cancer Biother Radiopharm. 2015 Jun 1;30(5):195–9.

38. Bison SM, Konijnenberg MW, Melis M, Pool SE, Bernsen MR, Teunissen JJM, et al. Peptide receptor radionuclide therapy using radiolabeled somato-statin analogs: focus on future developments. Clin Transl Imaging. 2014;2(1):55–66.

53

39. Larson SM, Carrasquillo JA, Cheung N-KV, Press OW. Radioimmunotherapy of human tumours. Nat Rev Cancer. 2015 Jun;15(6):347–60.

40. European Medicines Agency - Find medicine - Lutathera [Internet]. [cited 2018 Feb 13].

41. Lutathera (lutetium Lu 177 dotatate) FDA Approval History [Internet]. Drugs.com. [cited 2018 Feb 13].

42. Strosberg J, El-Haddad G, Wolin E, Hendifar A, Yao J, Chasen B, et al. Phase 3 Trial of (177)Lu-Dotatate for Midgut Neuroendocrine Tumors. N Engl J Med. 2017 Jan 12;376(2):125–35.

43. Tomblyn M. Radioimmunotherapy for B-cell non-hodgkin lymphomas. Cancer Control. 2012 Jul;19(3):196–203.

44. Gudkov SV, Shilyagina NY, Vodeneev VA, Zvyagin AV. Targeted Radionu-clide Therapy of Human Tumors. Int J Mol Sci. 2015 Dec 28;17(1).

45. Borras JM, Lievens Y, Dunscombe P, Coffey M, Malicki J, Corral J, et al. The optimal utilization proportion of external beam radiotherapy in European countries: An ESTRO-HERO analysis. Radiother Oncol. 2015 Jul 1;116(1):38–44.

46. Fotouhi Ghiam A, Dawson LA, Abuzeid W, Rauth S, Jang RW, Horlick E, et al. Role of palliative radiotherapy in the management of mural cardiac metas-tases: who, when and how to treat? A case series of 10 patients. Cancer Med. 2016 Feb 16;5(6):989–96.

47. Tharmalingham H, Hoskin PJ. The changing role of radiation therapy in the management of oligometastatic disease. Tech Innov Patient Support Radiat Oncol. 2017 Mar 1;1:13–5.

48. Dornfeld K, Simmons JR, Karnell L, Karnell M, Funk G, Yao M, et al. Radi-ation Doses to Structures Within and Adjacent to the Larynx are Correlated With Long-Term Diet- and Speech-Related Quality of Life. Int J Radiat Oncol Biol Phys. 2007 Jul 1;68(3):750–7.

49. Dieckmann K, Widder J, Pötter R. Long-term side effects of radiotherapy in survivors of childhood cancer. Front Radiat Ther Oncol. 2002;37:57–68.

50. Fridriksson JÖ, Folkvaljon Y, Nilsson P, Robinson D, Franck-Lissbrant I, Ehdaie B, et al. Long-term adverse effects after curative radiotherapy and rad-ical prostatectomy: population-based nationwide register study. Scand J Urol. 2016 Jun 22;1–8.

51. Hall S, Rudrawar S, Zunk M, Bernaitis N, Arora D, McDermott CM, et al. Protection against Radiotherapy-Induced Toxicity. Antioxid Basel Switz. 2016;5(3).

52. Peeters STH, Heemsbergen WD, Koper PCM, van Putten WLJ, Slot A, Diel-wart MFH, et al. Dose-Response in Radiotherapy for Localized Prostate Can-cer: Results of the Dutch Multicenter Randomized Phase III Trial Comparing 68 Gy of Radiotherapy With 78 Gy. J Clin Oncol. 2006 May 1;24(13):1990–6.

53. Zietman AL, Bae K, Slater JD, Shipley WU, Efstathiou JA, Coen JJ, et al. Randomized Trial Comparing Conventional-Dose With High-Dose Conformal Radiation Therapy in Early-Stage Adenocarcinoma of the Prostate: Long-Term Results From Proton Radiation Oncology Group/American College of Radiology 95-09. J Clin Oncol. 2010 Mar 1;28(7):1106–11.

54. Börjesson PKE, Postema EJ, Roos JC, Colnot DR, Marres HAM, van Schie MH, et al. Phase I therapy study with (186)Re-labeled humanized monoclonal antibody BIWA 4 (bivatuzumab) in patients with head and neck squamous cell carcinoma. Clin Cancer Res Off J Am Assoc Cancer Res. 2003 Sep 1;9(10 Pt 2):3961S–72S.

54

55. Bellerby R, Smith C, Kyme S, Gee J, Günthert U, Green A, et al. Overexpres-sion of Specific CD44 Isoforms Is Associated with Aggressive Cell Features in Acquired Endocrine Resistance. 2016 Jun 20;6:145.

56. Wu X-J, Li X-D, Zhang H, Zhang X, Ning Z-H, Yin Y-M, et al. Clinical sig-nificance of CD44s, CD44v3 and CD44v6 in breast cancer. J Int Med Res. 2015 Apr;43(2):173–9.

57. Sneath RJ, Mangham DC. The normal structure and function of CD44 and its role in neoplasia. Mol Pathol. 1998 Aug;51(4):191–200.

58. Shi J, Zhou Z, Di W, Li N. Correlation of CD44v6 expression with ovarian cancer progression and recurrence. BMC Cancer. 2013;13:182.

59. Tjhay F, Motohara T, Tayama S, Narantuya D, Fujimoto K, Guo J, et al. CD44 variant 6 is correlated with peritoneal dissemination and poor prognosis in patients with advanced epithelial ovarian cancer. Cancer Sci. 2015 Oct;106(10):1421–8.

60. Lv L, Liu H-G, Dong S-Y, Yang F, Wang Q-X, Guo G-L, et al. Upregulation of CD44v6 contributes to acquired chemoresistance via the modulation of au-tophagy in colon cancer SW480 cells. Tumour Biol. 2016 Jul;37(7):8811-24.

61. Afify A, Durbin-Johnson B, Virdi A, Jess H. The expression of CD44v6 in colon: from normal to malignant. Ann Diagn Pathol. 2016 Feb;20:19–23.

62. Mack B, Gires O. CD44s and CD44v6 expression in head and neck epithelia. PloS One. 2008;3(10):e3360.

63. Guo XE, Ngo B, Modrek AS, Lee W-H. Targeting Tumor Suppressor Net-works for Cancer Therapeutics. Curr Drug Targets. 2014 Jan;15(1):2–16.

64. Sherr CJ, McCormick F. The RB and p53 pathways in cancer. Cancer Cell. 2002 Aug 1;2(2):103–12.

65. Merkel O, Taylor N, Prutsch N, Staber PB, Moriggl R, Turner SD, et al. When the guardian sleeps: Reactivation of the p53 pathway in cancer. Mutat Res. 2017 Jul 1;773:1–13.

66. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature. 2000 Nov 16;408(6810):307-10.

67. Liu J, Zhang C, Feng Z. Tumor suppressor p53 and its gain-of-function mu-tants in cancer. Acta Biochim Biophys Sin. 2014 Mar;46(3):170–9.

68. Mancini F, Di Conza G, Moretti F. MDM4 (MDMX) and its Transcript Vari-ants. Curr Genomics. 2009 Mar;10(1):42–50.

69. Lane DP, Cheok CF, Lain S. p53-based cancer therapy. Cold Spring Harb Perspect Biol. 2010 Sep;2(9):a001222.

70. Hoe KK, Verma CS, Lane DP. Drugging the p53 pathway: understanding the route to clinical efficacy. Nat Rev Drug Discov. 2014 Feb 28;13(3):217–36.

71. Shaw PH. The role of p53 in cell cycle regulation. Pathol Res Pract. 1996 Jul;192(7):669–75.

72. Shamas-Din A, Brahmbhatt H, Leber B, Andrews DW. BH3-only proteins: Orchestrators of apoptosis. Biochim Biophys Acta. 2011 Apr 1;1813(4):508–20.

73. Pistritto G, Trisciuoglio D, Ceci C, Garufi A, D’Orazi G. Apoptosis as anti-cancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies. Aging. 2016 Mar 27;8(4):603–19.

74. Elmore S. Apoptosis: A Review of Programmed Cell Death. Toxicol Pathol. 2007;35(4):495–516.

75. Cai X, Yuan Z-M. Stochastic Modeling and Simulation of the p53-MDM2/MDMX Loop. J Comput Biol. 2009 Jul;16(7):917–33.

76. Shi D, Gu W. Dual Roles of MDM2 in the Regulation of p53. Genes Cancer. 2012 Mar;3(3–4):240–8.

55

77. Coffill CR, Lee AP, Siau JW, Chee SM, Joseph TL, Tan YS, et al. The p53-Mdm2 interaction and the E3 ligase activity of Mdm2/Mdm4 are conserved from lampreys to humans. Genes Dev. 2016 Feb 1;30(3):281–92.

78. Pellegrino M, Mancini F, Lucà R, Coletti A, Giacchè N, Manni I, et al. Tar-geting the MDM2/MDM4 Interaction Interface as a Promising Approach for p53 Reactivation Therapy. Cancer Res. 2015 Nov 1;75(21):4560–72.

79. Mendoza M, Mandani G, Momand J. The MDM2 gene family. Biomol Con-cepts. 2014 Mar 1;5(1):9–19.

80. Burgess A, Chia KM, Haupt S, Thomas D, Haupt Y, Lim E. Clinical Overview of MDM2/X-Targeted Therapies. Front Oncol. 2016 Jan 27;6:7.

81. Tisato V, Voltan R, Gonelli A, Secchiero P, Zauli G. MDM2/X inhibitors under clinical evaluation: perspectives for the management of hematological malig-nancies and pediatric cancer. J Hematol Oncol. 2017 Jul 3;10(1):133.

82. Haylock A-K, Spiegelberg D, Mortensen AC, Selvaraju RK, Nilvebrant J, Eriksson O, et al. Evaluation of a novel type of imaging probe based on a re-combinant bivalent mini-antibody construct for detection of CD44v6-express-ing squamous cell carcinoma. Int J Oncol. 2016 Feb;48(2):461–70.

83. Spiegelberg D, Mortensen AC, Selvaraju RK, Eriksson O, Stenerlöw B, Nestor M. Molecular imaging of EGFR and CD44v6 for prediction and response monitoring of HSP90 inhibition in an in vivo squamous cell carcinoma model. Eur J Nucl Med Mol Imaging. 2016 May;43(5):974–82.

84. Haylock A-K, Spiegelberg D, Nilvebrant J, Sandström K, Nestor M. In vivo characterization of the novel CD44v6-targeting Fab fragment AbD15179 for molecular imaging of squamous cell carcinoma: a dual-isotope study. EJNMMI Res. 2014;4(1):11.

85. Nilvebrant J, Kuku G, Björkelund H, Nestor M. Selection and in vitro charac-terization of human CD44v6-binding antibody fragments. Biotechnol Appl Bi-ochem. 2012 Oct;59(5):367–80.

86. Nestor MV. Targeted radionuclide therapy in head and neck cancer. Head Neck. 2010 May;32(5):666–78.

87. Sandström K, Haylock AK, Spiegelberg D, Qvarnström F, Wester K, Nestor M. A novel CD44v6 targeting antibody fragment with improved tumor-to-blood ratio. Int J Oncol. 2012 May 1;40(5):1525–32.

88. Wei SJ, Chee S, Yurlova L, Lane D, Verma C, Brown C, et al. Avoiding drug resistance through extended drug target interfaces: a case for stapled peptides. Oncotarget. 2016 Apr 4;7(22):32232–46.

89. Brown CJ, Quah ST, Jong J, Goh AM, Chiam PC, Khoo KH, et al. Stapled peptides with improved potency and specificity that activate p53. ACS Chem Biol. 2013 Mar 15;8(3):506–12.

90. Wei SJ, Joseph T, Chee S, Li L, Yurlova L, Zolghadr K, et al. Inhibition of nutlin-resistant HDM2 mutants by stapled peptides. PloS One. 2013;8(11):e81068.

91. Boswell CA, Brechbiel MW. Development of radioimmunotherapeutic and di-agnostic antibodies: an inside-out view. Nucl Med Biol. 2007 Oct 1;34(7):757–78.

92. Yeong C-H, Cheng M, Ng K-H. Therapeutic radionuclides in nuclear medi-cine: current and future prospects. J Zhejiang Univ Sci B. 2014 Oct;15(10):845–63.

93. Graf F, Fahrer J, Maus S, Morgenstern A, Bruchertseifer F, Venkatachalam S, et al. DNA Double Strand Breaks as Predictor of Efficacy of the Alpha-Particle Emitter Ac-225 and the Electron Emitter Lu-177 for Somatostatin Receptor Targeted Radiotherapy. PLoS One. 2014 Feb;9(2):e88239.

56

94. Behr TM, Sgouros G, Stabin MG, Béhé M, Angerstein C, Blumenthal RD, et al. Studies on the red marrow dosimetry in radioimmunotherapy: an experi-mental investigation of factors influencing the radiation-induced myelotoxicity in therapy with beta-, Auger/conversion electron-, or alpha-emitters. Clin Can-cer Res Off J Am Assoc Cancer Res. 1999 Oct;5(10 Suppl):3031s–3043s.

95. Jødal L. Beta emitters and radiation protection. Acta Oncol Stockh Swed. 2009;48(2):308–13.

96. Carlsson J, Forssell Aronsson E, Hietala SO, Stigbrand T, Tennvall J. Tumour therapy with radionuclides: assessment of progress and problems. Radiother Oncol. 2003 Feb;66(2):107–17.

97. Tolmachev V, Orlova A, Lundqvist H. Approaches to Improve Cellular Reten-tion of Radiohalogen Labels Delivered by Internalising Tumour-Targeting Proteins and Peptides.Curr Med Chem. 2003 Nov;10(22):2447-60.

98. Zhou Y, Baidoo KE, Brechbiel MW. Mapping Biological Behaviors by Appli-cation of Longer-Lived Positron Emitting Radionuclides. Adv Drug Deliv Rev. 2013 Jul;65(8):1098–111.

99. Torrisi JM, Schwartz LH, Gollub MJ, Ginsberg MS, Bosl GJ, Hricak H. CT Findings of Chemotherapy-induced Toxicity: What Radiologists Need to Know about the Clinical and Radiologic Manifestations of Chemotherapy Toxicity. Radiology. 2011 Jan 1;258(1):41–56.

100. Bayat Mokhtari R, Homayouni TS, Baluch N, Morgatskaya E, Kumar S, Das B, et al. Combination therapy in combating cancer. Oncotarget. 2017 Jun 6;8(23):38022–43.

101. Gill MR, Falzone N, Du Y, Vallis KA. Targeted radionuclide therapy in com-bined-modality regimens. Lancet Oncol. 2017 Jul 1;18(7):e414–23.

102. Ding M, Zhang E, He R, Wang X. Newly developed strategies for improving sensitivity to radiation by targeting signal pathways in cancer therapy. Cancer Sci. 2013 Nov 1;104(11):1401–10.

103. Maier P, Hartmann L, Wenz F, Herskind C. Cellular Pathways in Response to Ionizing Radiation and Their Targetability for Tumor Radiosensitization. Int J Mol Sci. 2016 Jan 14;17(1):102.

104. Wang H, Mu X, He H, Zhang X-D. Cancer Radiosensitizers. Trends Pharma-col Sci. 2018 Jan 1;39(1):24–48.

105. Baselga J. The EGFR as a target for anticancer therapy—focus on cetuximab. Eur J Cancer. 2001 Sep 1;37:16–22.

106. Okada Y, Kimura T, Nakagawa T, Okamoto K, Fukuya A, Goji T, et al. EGFR Downregulation after Anti-EGFR Therapy Predicts the Antitumor Effect in Colorectal Cancer. Mol Cancer Res. 2017 Oct 1;15(10):1445–54.

107. Fricker SP. Metal Compounds in Cancer Therapy. Springer Science & Busi-ness Media. 2012, pp 225-269.

108. Loke KSH, Padhy AK, Ng DCE, Goh ASW, Divgi C. Dosimetric Considera-tions in Radioimmunotherapy and Systemic Radionuclide Therapies: A Re-view. World J Nucl Med. 2011;10(2):122–38.

109. Hobbs RF, Wahl RL, Frey EC, Kasamon Y, Song H, Huang P, et al. Radiobi-ological Optimization of Combination Radiopharmaceutical Therapy Applied to Myeloablative Treatment of Non-Hodgkin’s Lymphoma. J Nucl Med. 2013 Sep;54(9):1535–42.

110. Goh AM, Lane DP. How p53 wields the scales of fate: arrest or death? Tran-scription. 2012 Oct;3(5):240–4.

Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1430

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