The Clinical Role of Nuclear Imaging in Immunotherapeutic Drug Development

Journal of Central Pharmacology & Clinical Toxicology Special Issue on Pharmacokinetics and Pharmacodynamics

Review Article *Corresponding author Berlinda J. de Wit – van der Veen, Department of Nuclear Medicine, NKI-AVL, Plesmanlaan 121, 1066 CX The clinical role of nuclear Amsterdam, the Netherlands; Tel: 31-20 512 2287; Fax: 31-20 512 2290; Email: Submitted: 04 May 2015 imaging in immunotherapeutic Accepted: 17 August 2015 Published: 20 August 2015 drug development Copyright © 2015 van der Veen et al. BerlindaJ. de Wit – van der Veen*, Marcel P.M. Stokkel Department of Nuclear Medicine, NKI-AVL, Netherlands OPEN ACCESS

Keywords Abstract • Positron emission tomography Advances in the oncological sciences have led to the development of agents • Biodistribution that are designed to affect specific molecular pathways. However, many of these • Immunotherapeutic agents immunotherapeutic agents do not successfully pass phase 2 trials due to concerns with • Imaging biomarker efficacy, toxicity or side effects. The generalized endpoints that traditionally have been used in these trials, such as maximum tolerated dose and overall response, may be less optimal for immunotherapeutic agents. The addition of in vivo nuclear imaging in these early trials can aid in patient stratification, evaluation of drug bio distribution, pharmacodynamic effects and tumor heterogeneity. This review discusses the advantages of nuclear imaging, and outlines the first application of these imaging biomarkers in clinical trials.

ABBREVIATIONS the tumor microenvironment, transmembrane or intracellular

SPECT: Single Photon Emission Computer Tomography, PET: tumor growth and limiting metastatic progression (cytostatic), Positron Emission Tomography, CT: Computer Tomography, ratherprocesses. than Thedirectly triggered kill tumor effects cells are (cytotoxic) often aimed [1]. at inhibiting MRI: Magnetic Resonance Imaging, HER2: Human Epidermal Growth Factor 2, VEGF: Vascular Endothelial Growth Factor, EGFR: Epidermal Growth Factor Receptor, GCPII or PSMA: approval were compounds like Rituximab (Rituxan, Biogen Glutamate Carboxypeptidase II, CAIX: Carbonic Anhydrase 9, RIT: Idec,Among 1997), the Trastuzumab first immunotherapeutic (Herceptin, Genentech, agents to receive 1998), FDA and Radioimmunotherapy Cetuximab (Erbitux, Bristol-Myers Squibb, 2003). Still, many INTRODUCTION target-based therapies do not successfully pass phase 2 trials due Anticancer drugs have traditionally been discovered either the question whether traditional pharmacological studies (e.g., to concerns with efficacy, toxicity or side effects. This has raised by chance or by widespread screening programs. However, the progress in molecular and genetic oncological sciences has [2,3]. Traditionally, phase 1 trials seek to determine the optimal preclinical, phase 1, phase 2, etc.) are optimal for these drugs led to the identification of new drug targets. This resulted in a dose,dose biological scheme and dose general range and efficacy tumor of response. a new drug However, in humans. these paradigm shift towards the development of anticancer agents The main endpoints consist of aspects like maximum tolerated that are specifically designed to affect one or multiple molecular pathways. Unlike conventional cytotoxic drugs, most of which generalized endpoints do not reflect the true complexity and disrupt processes in the nucleus, these compounds can affect individualization of targeted therapeutic agents. Cite this article: van der Veen BJW, Stokkel MPM (2015) The clinical role of nuclear imaging in immunotherapeutic drug development. J Pharmacol Clin Toxicol 3(4):1057. van der Veen et al. (2015) Email: Central

pharmaceutical research. Although, newer techniques like mass

Identification of key molecular oncogenic pathways or drivers in a set population is the foundation for targeted- usedspectrometry today due are its highly available reproducible to quantify nature specific [12,13]. compounds Typically, drug development. Consequently, molecular stratification without the need forex radioactive vivo autoradiography labeling, autoradiography involves the designis still of patients is vital for successful clinical implementation of designimmunotherapeutic and preclinical agents. evaluation. Biomarkers In early thatclinical provide trials, specificsimaging isotopesthe first like step Hydrogen of (3H), Carbon (14C) or Phosphorus (32P). biomarkerson the drug-target are increasingly are preferably used as identified they can attribute during compound in patient and production of a drug labelled with long-lived beta-emitting selection, tumor characterization and early response assessment is quickly cryosectioned (typically 20-50 micrometer). The [4,5]. sectionsAfter administration are placed between of the radiolabelled detector layers, drug, such the specimen as X-ray Imaging in drug development

Anatomical imaging modalities, such as computer isfilm desired, or phosphorous radioactive detector calibration plates, and standards are left are for periodplaced tomography (CT) and magnetic resonance imaging (MRI) are alongsideof time (typically the specimens. hours-days). The resulting When quantitative digital images evaluation provide generally used to localize lesions and evaluate tumor shrinkage. Micro-autoradiography can be applied to localize radiolabelled information on tissue concentrations and biodistribution. ‘ResponseRadiological Evaluation assessment Criteria of therapy in Solid response Tumors’- or RECIST- disease progression is performed according to the highly standardized imagescompounds can beat combineda cellular levelwith whenimmunohistochemical there is a specific staining organ orto tissue of special interest. After image development, the resulting criteria. Additionally, image-guided biopsies are performed to evaluate the local pharmacodynamic effects in specific target response have however some important drawbacks; 1) they only assess radioactivity accumulation in relation to specific molecular lesions. These conventional strategies to identify tumor load and on the detector material, isotope (e.g., high energy beta-emitters providetargets. The blurred achieved images) resolution and distance of autoradiography between detector is dependent plates tumor heterogeneity into account, and, 3) they do not provide characteristicsprovide a generalized on the drug-target estimate of withinresponse, the tumor2) they lesions. do not take Scintigraphyand specimen, but can range from 10-100 μm [12,14,15]. has gained popularity as a method to characterize tumors in vivoIn the era of targeted therapies, nuclear molecular imaging gamma-rays emitted by isotopes using one or multiple large In vivo nuclear detectors,Scintigraphic known imagingas a gamma relies camera. on the This detection imaging technique of single . In itself this approach is not new, and has been used coupledfor decades to a in biological cell cultures compound and tissue (e.g., slices. radiopharmaceutical), imaging relies on the administration of a radioactive isotope neurology,has been around orthopedics, for almost oncology, 60 years, and etc.). is routinely A gamma used camera in the clinical practice for a broad range of indications (e.g., cardiology, and on the detection of radiation emitted by the isotope. The than detailed anatomical structures. The radiopharmaceutical area, also known as planar acquisitions. Single photon emission resulting images represent specific biological processes, rather concentration that can be detected with nuclear imaging computerproduces tomography two-dimensional (SPECT) images is similar of a to relatively conventional large planar body imaging, but uses a gamma camera that rotates around the patient based anatomical imaging modalities such as CT and MRI, these modalities is in the range of picomolar, whereas for contrast- and acquires images from different angles. Three-dimensional biologicalconcentrations active are compound in the order to aid of inmillimolar. early clinical This studiesensures [6,7]. that representations of the isotope distribution are produced after a nuclear imaging can be performed using micro dosages of the set of mathematical procedures known as image reconstruction therapeutic drug development to visualize biodistribution, target [16]. After proper calibration of the camera, SPECT has the ability This strength explains the wide spread use of nuclear imaging in to both visualize and quantify radioactivity distributions in structures larger then roughly 1.5cm in size. Depending on the occupancy, pharmacodynamic effects, tumor heterogeneity, or regarding subject preparation, administration, image acquisition radiopharmaceutical and clinical indication, specific protocols trials,just to optimizes localize tumor treatment targets schemes before and or during provides treatment background [8-10]. on This approach holds great benefit as it increases the power of pharmacodynamic measures. andPositron quantification emission are tomographyapplied. Translational nuclear imaging techniques Positron emission tomography like SPECT, positron emission tomography (PET) is also a three-dimensional nuclear imaging in vivo or The fore most advantage of radiochemical tracing is the ex vivo depending on the techniques used. The most widely modality, but has roughly a 10-fold higher sensitivity, increased ability to quantify biodistribution and metabolism Unlike the gamma camera, which has multiple rotating detectors, used imaging techniques in translational drug development accuracy, and hence, superior quantitative analysis of images. include autoradiography, scintigraphy, and positron emission the PET scanner consists of a continues ring with numerous sections [11]. tomography (PET), which will be briefly introduced in the next isstationary converted detector into two elements. photons Whenthat are a emitted PET-isotope under decays, a 180 Autoradiography degreea positron angle is (e.g., emitted the process that, after is called collision annihilation). with an Allelectron, these Ever since the mid-1900s quantitative whole-body photon-pairs are detected by the opposing detector elements autoradiography has been a valuable tool in preclinical in the PET-ring. After image reconstruction, three-dimensional J Pharmacol Clin Toxicol 3(4): 1057 (2015) 2/10 van der Veen et al. (2015) Email: Central

representations of the isotope imaging distribution in oncology are produced is Fluor-18 in hours) should be used. Whereas for intact antibodies that have (which18 structures of ~5mm in size can be evaluated. The principal a half-life of several days, isotopes with a matching physical isotope used for traditional PET half-life are used. Depending on the radioisotope and molecular F) labeled to the glucose-analog fluorodeoxyglucose [17]. Both withstructure respect of to the the drug, biodistribution different radiolabeling and pharmacokinetics strategies canshould be arePET increasingly and SPECT incorporated are regularly into combined PET systems with [18]. low-dose CT for applied. Still, the biological behavior of the radiopharmaceutical anatomical localization of specific findings. Also, MRI scanners drugbe similar should to be the minimal, unlabeled to drug. reduce Additionally, the radiation targeting dose to of the drug should be good, and off-target binding of the radiolabelled imaging.The differences Because SPECT-isotopes between SPECT emit and low-energyPET regarding radiation image quality and sensitivity are far less profound for small animal patient. The final consideration for isotope selection is related to radiation is absorbed within tissue. Small animals have less decay characteristics and the type of imaging modality that will volumecompared and to mass PET-isotopes, compared to a relativelyhumans, so larger radiation portion will ofbetter the be used. Isotopes that emit gamma rays are used for scintigraphic penetrate through the subject, and hence, relatively more translationalimaging, whereas pharmacological positron emitters research. are used for PET. Table 1 radiation will be detected by the SPECT scanner. Additionally, provides an overview of the most important isotopes used in great technological improvements have been made in the last Focus on antibody PET in clinical trials decade. The newest generation commercially available animal Several antibodies have been radiolabelled in the SPECT systems are able to provide ultra-high-resolution images In vivo developmental phase or shortly after clinical introduction to multi-isotopefor identification imaging of structures are other important below 0.5mm developments. in size [19]. These characterize lesions, quantify response or to select suitable quantification of whole body biodistribution over time and withpatients the for key immunotherapy. PET isotopes 89 TableZr, 64 Cu 2 providesand 124 an extended development, and accordingly, its use in clinical trials. overview of all the listed clinical trials that have been performed advances enhanced the role of preclinical nuclear imaging in drug I.The following Clinical radiopharmaceuticals paragraphs will focus on targets that have been used in a clinical For in vivo setting, including CD20, CD44, human epidermal growth factor requirements have to be met; 1) appropriate decay characteristics, (GCPII,2 (HER2), also vascular known endothelialas PSMA), and growth carbonic factor anhydrase (VEGF), epidermal 9 (CAIX) imaging specific biological and physics-related (markedgrowth factor with a receptor star in Table (EGFR), 2). glutamate carboxypeptidase II 2) the labeling should not influence pharmacokinetics and CD20-targeting (B-cell lymphoma) biodistribution of the drug, and, 3) possibility of easy and stable labeling. The choice of an isotope depends not only on the chemistry of the drug, but also on its biological half-life. If the monotherapyRetuximab and and in ibritumomabtiuxetan combination with other belong chemotherapies. to the first drug is for instance a small molecules or antibody fragments and tumor-targeted therapies approved by the FDA, both as a has fast kinetics, isotopes with a short half-life (i.e., minutes to Table 1: Important radionuclides used in translation clinical studies.

Half-life Main emitter, Emax (yield %) Autoradiography & ADME studies Carbon-14 14C >5700 y ß-, 0.156 MeV (100) Hydrogen-3 3H 12.3 y ß-, 0.019 MeV (100) Phosphorus-32 32P 14.3 d ß-, 1.709 MeV (100) SPECT Technetium-99m 99mTc 6.0 h

111 Indium-111 In 2.81 d Ƴ, 142 keV (90) 131 Iodine-131 I 8.0 d Ƴ, 171 keV (90.2) and 245 keV (94.0) PET Ƴ, 364 keV (82) Oxygen-15 15O 2 min. ß+, 1.73 MeV (99,9) Carbon-11 11C 20 min. ß+, 0.98 MeV (99,8) Gallium-68 68Ga 68 min. ß+, 1.89 MeV (89.1) Fluor-18 18F 109 min. ß+, 0.633 MeV (96.7) Copper-64 64Cu 12.7 h ß+, 0.653 MeV (17.4) Zirconium-89 89Zr 78.4 h ß+, 0.897 MeV (22.7) Iodine-124 124I 100.3 h ß+, 1.54 MeV (11.8) and 2.14 MeV (10.9)

Abbreviations: Emax Maximal energy, y years, d days, h hours, min. minutes, keV kilo electron Volt, MeV Mega electron Volt

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Table 2: A. # of trials Radiopharmaceutical Overview of clinical studies withTarget 89Zr-, 64Cu- or 124I-labelled antibodiesTumor or fragments types (# completed) 89Zr-Bevacizumab VEGF * 8 (6)

89 Zr-Trastuzumab HER2 * MultipleBreastcancer|Esophagogastriccancer Myeloma|Breastcancer|RCC|NET 8 (1) 89Zr-Cetuximab EGFR * Colorectal cancer 3 (2) 89Zr-Panitumumab EGFR * 1 (0)

89 Zr-huJ591 PSMA * NSCLC|Urothelialcancer|SarcomasProstatecancer|Glioblastoma 2 (0) 89 PSMA * Prostate cancer 3 (0)

89 Zr-GirentuximabZr-Df-IAB2M CA-IX * RCC 1 (0) 89Zr-RO5429083 1 (1)

89 Zr-MSTP2109A STEAP1CD44 * CD44-expressingProstate cancer solid tumors 1 (0) 89 EphA2 EphA2-expressing solid tumors 1 (0)

89 Zr-MMOT0530AZr-DS-8895a Pancreaticcancer|Ovariancancer 1 (0) 89 Zr-GSK2849330 MLSNHER3 HER3-expressing solid tumors 1 (0) 89Zr-GC1008 Gliomas 1 (0)

64 Cu-Trastuzumab HER2TGF-β * Breastcancer|Esophagogastriccancer 4 (1) 64Cu-Rituximab Lymphoma 1 (0)

64 Cu-M5A IV CD20M5A * CEA-expressing solid tumors 1 (0) 64Cu-U3-1287 HER3 HER3-expressing solid tumors 1 (1) 64 CXCR4 Solid tumors 1 (0)

64 Cu-PlerixaforCu-AE105 uPAR Prostate cancer|Breastcancer|Urothelial cancer 1 (1) 124I-cG250 CAIX * RCC 6 (4) 124I-hu3F8 1 (0)

124 I-anti8H9 GD28H9 Melanoma|Neuroblastoma|Sarcoma 2 (0) 124 I-huA33 A33 Peritonealcancer|Gliomas|NeuroblastomaColorectal cancer 1 (0) 124 PS Solid tumors 1 (0) A This overview only includes completed and active trials registered at the ClinicalTrails.gov database. I-PGN650 Abbreviations:

RCC: Renal Cell Carcinoma; NET: Neuroendocrine Tumors; NSCLC: Non-Small Cell Lung Cancer Lymphoma’s are typically very radiosensitive, so it did not the othe clinical setting. r CD20 tracers similar results were achieved in the (pre-) take long before these antibodies were radiolabeled with α- or CD44-targeting (head/neck carcinoma, solid tumors) β-emitting isotopes to provide targeted radiotherapy (also known as radioimmunotherapy) or with ƴ-emitting isotopes for imaging.90 ibritumomabtiuxetanAt this moment, two (Zevalin) FDA-approved and 131Itositumomab radiopharmaceuticals (Bexxar), Many types of solid tumors, among which squamous cell are available for the treatment of lymphoma, Yttrium-90 ( Y) carcinoma of the head and neck (HNSCC), express moderate to Currently, well over 60 completed or ongoing clinical trials with high levels of CD44. All efforts of radiolabeling CD44-targeting both of which are directed against different epitopes of CD20 [20]. radiolabelled retuximab or ibritumomabtiuxetan are registered. theantibodies 89 were initially focused on RIT, however, application Prior to radioimmunotherapy (RIT), proper patient selection was with variable success. Borjesson et al. were the first to use is crucial, thus tumor load, biodistribution and dosimetry are Zr-labeled anti-CD44v6 chimeric monoclonal antibody evaluated with either 111In, 89Zr or 64 cU36 in 20 patients to localize and characterize HNSCC lesions 89Zr to ibritumomabtiuxetan to predict [27]. All primary tumors were visualized and 18 of the 25 90 Cu anti-CD20 imaging [21- dosimetric evaluation indicated that the highest observed dosage tumor-containing lymph node levels were correctly identified. A 25] for example coupled the biodistribution of Y-ibritumomabtiuxetan and identify the thyroid, lungs and spleen [28]. The average radiation dose to was found in the liver (±1.3 mSv/MBq), followed by the kidneys, dose limiting organs in seven patients with CD20-positive B-cell 89 90lymphoma [26]. Rituxan was given one week before injection the novel of ±70MBq 89Zr-ibritumomab tiuxetan, as is done in standard thethat wholebinds to body another was 0.6epitope, mSv/MBq. shows A promising preclinical results evaluation in mice of Y-ibritumomab tiuxetan treatments. Most intense accumulation and monkeysZr-labelled [29]. Recently, RO5429083, a clinical a trial humanized with this anti-CD44 drug was was89 seen in the liver and spleen, and the whole90 body effective dose completed in which the tissue distribution and pharmacokinetics wasthat ±0.87PET can mSv/MBq. be used Theto predict correlation the therapy between biodistribution. the pre-treatment For were assessed using PET. Zr-PET and the actual distribution of Y was high, suggesting

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HER2-targeting (breast & gastric carcinoma) 89Zr-Trastuzumab, a highly heterogeneous accumulation pattern was visualized within and among patients. In primary breast cancer, Gaykema et al. used 89 emerged using 111In-Trastuzumab SPECT to evaluate HER2- Approximately a decade ago the first preclinical studies111 In- Zr-Bevacizumab (37MBq/5mg) Trastuzumab was highly stable in vivo and the accumulation successfully to localize lesions with a diameter larger then 10mm expression in xenograft tumor models. Though, was related to HER2-expression, translation to humans proved (96.1%EGFR targeting of lesions visualized) (Colorectal [45]. & head/neck carcinoma) Though several EGFR-directed therapies have been developed, the approved antibodies cetuximab and panitumumab clinicalnot feasible grade due 89Zr-Trastuzumab to poor imaging (37MBq). characteristics The results [30]. indicated Dijkers are the main targets used in imaging studies. In preclinical studies thatet al. unlabeled-Trastuzumab published the first in-human should biodistributionbe co-administered study with with the a mixed results are achieved with these 64Cu- and 89Zr-labelled 89Zr-Trastuzumab in both Trastuzumab-naïve (50mg) and on- antibodies [46-51]. Aerts et al. demonstrated a disparity between radioactivity accumulation in three positive tumor cell lines and appropriate antibody dosages, tumor lesions could be visualized treatment (10mg) patients to limit off-target accumulation. At the ex vivo

64 al. showed that EGFR expression [52]. The group of Chen et al. also 4-5 days after injection [31,32]. Mortimer et al. and Tamura et HER2-positive tumor load in metastatic breast cancer, despite showed this disparity, which was especially profound in HNSCC Cu-DOTA-Trastuzumab64 is also able89 to visualizes Cu compared to Zr [33,34]. The tumor models [48-50]. A number of factors could explain these 64 89 imaging time points and reduced tumor vascularity or vascular Cu over conflicting results, like suboptimal antibody concentration or the patients.much shorter Still, bothhalf-life tracers of showed substantial accumulation main advantage of Zr, is alowered radiation dose for trials.permeability. Only recently, Nevertheless, Makris et these al. reported early findings on the biodistribution probably have and bladder. restricted the use 89of EGDR-PET as imaging biomarker in clinical in the blood pool (only in the first 1-2 days), liver, kidney, spleen Zr-labeled cetuximab in seven patients with Other groups have used this imaging approach to assess the colorectal cancer [53]. The 89Zr-Cetuximab administration and dosimetry of 2 andpharmacodynamics HER2 tyrosine of kinase novel inhibitor)targeted therapies in small like animals PU-H71 [35,36]. (i.e., (37MBq) was preceded by infusion of unlabeled cetuximab Alsoheat shock in patients protein with 90 (HSP90) metastatic inhibitor) HER2-positivebreast and afatinib (i.e., cancer, EGFR (500mg/mobserved in). the The liver, average kidneys, effectivespleen and whole-body lungs. At this dose moment was 89 there0.61mSv/MBq, are two ongoingand highest clinical radioactivity trials in patients accumulation with colon was carcinoma to include 89 Zr-Trastuzumab PET has successfully been used to evaluate guided treatment optimization. scalepharmacodynamics and ongoing, bothof the studies novel suggestHSP90 inhibitor,that an early NVP-AUY922, response Zr cetuximab PET as a method for image toand treatment the novel could anti-HER2 be visualized. drug, T-DM1 A substantial [37,38]. heterogeneous Though small PSMA-targeting (prostate carcinoma) response throughout the lesions within and among patients a possible treatment strategy in androgen resistant metastatic heterogeneous HER2-related accumulation in proven HER2- This target for prostate cancer was already investigated as was observed in both studies. The clinical implications of this positive breast cancer are still under investigation. Additionally, an 111In-labelled anti-PSMA antibody known as ProstaScintTM 89 disease(111 in the early 1990’s. The first approved imaging antibody,

Zr-Trastuzumab imaging (37MBq/50mg)was used in specific patient has a complex medical history with HER-positive and In capromabpendetide,ProstaScintTM EUSA Pharma), was introduced for clinical dilemmas when biopsies cannot be performed or the HER-negative tumors [39]. suboptimalthe detection targeting of soft tissuecharacteristics and bone resulting metastasis in a [54]. low sensitivity However, routine use of was found to be restricted due to VEGF-targeting (solid tumors) ligands that target the PSMA receptor have been evaluated ever for detecting metastases, especially in smaller lesions. Other angiogenesis in solid tumors and metastatic lesions, VEGF is among the extracellular domain and are internalized upon binding the Thoughmost prominent. various The factors anti-VEGF are mAbBevacizumab known to contribute (Avastin) to since. Initial attempts focused on intact antibodies89Zr-J591 thatPET targetin 11 was labeled with 111In and 123 men with localized PCa showed promising results. First 20mg [55]. The first results of a pilot study with 64 I for SPECT imaging with limited 89 121 Zr-J591, and acquisitions were made 5-7 days post injection. A success. Cai et al. for the first time demonstrated PET imaging of unlabeled J591 was infused before injection89Zr-J591 of 185MBq/2mgPET to detect of Cu-DOTA-VEGF (i.e.,89Zr-labeled antibody Bevacizumab fragment of Bevacizumab)as an imaging in a glioblastoma mouse model [40]. Whereas, van der Bilt et subsequent study to assess the ability of al. were the first to use lesions and pharmacokinetics of this tracer are still ongoing [56]. biomarker to study the antiangiogenic effect of everolimus in an Additionally, a number of small molecule inhibitors and antibody antiangiogenicovarian xenograft treatment model [41].with serialBoth Van89Zr-labeled Asselt et Bevacizumab al and Oosting in [57,58].fragments, which have considerably faster kinetics but still target et al. used this same approach in humans to evaluate the effect of the extracellular domain of PSMA, are also used in clinical trials these studies, a typical antibody distribution was seen in normal CAIX-targeting (RCC) organsneuroendocrine with highest (NET) accumulation and renal cell incarcinoma the circulation, (RCC) [42-44]. kidneys, In indicated as a promising biomarker in renal cell carcinoma (RCC) Since its discovery in the early 1990’s, G250/CAIX was liver and spleen four days after injection (37MBq/5mg). Like with J Pharmacol Clin Toxicol 3(4): 1057 (2015) 5/10 van der Veen et al. (2015) Email: Central

Figure 1

Schematic overview of the traditional pharmacological study design and the role of nuclear imaging within (pre)clinical studies.

Figure 2 89Zr-Cetuximab (60 MBq) was administered [66].

(red arrows). Cetuximab On the PET-imagingearly 89 in a patient with HPV-negative HNSCC. As part of a clinical trial 89Zr-PET On the FDG-PET a large primary tumor with high glucose metabolism is visualized in the oral cavity, together with one positive node metastasis Zr-PET extensive accumulation is seen both in the circulation and at the site89Zr of and the 18 primaryF PET. tumor. On the late accumulation is still visible at the site of the tumor, while the activity in the circulation has greatly reduced. At later time-points accumulation in the bone marrow can be visible. Furthermore, there is a marked difference in image quality between the as it has good targeting characteristics and limited expression by PET and digital autoradiography was directly related to CAIX expression by immunohistochemistry [61]. Based on these results, a large Phase 3 trials was initiated to validate 124I-cG250 as in normal tissues [59]. Radiolabeling of G250 (also known as Girentuximab) has been performed by various groups to a 124 differentiate between benign versus malignant124 renal masses, an imaging biomarker in RCC [62]. A total of 195 patients received anddemonstrated to study its in 26biodistribution patients with as renal preliminary masses that work a forpositive RIT. I-PET/CT scan 2-6days post-injection (185MBq/13.7mg) and The first clinical study in humans with I-cG250 by Divgi et al. underwent surgery. This study confirmed the initial results124I-cG250 of the pilot study, an average sensitivity and specificity of respectively PET scan was highly predictive of clear-cell RCC (sensitivity 94%; 86.2% and 85.9% were found. It was concluded that specificity 100%) [60]. Radioactivity accumulation as visualized PET/CT is able to characterize a malignant phenotype, which J Pharmacol Clin Toxicol 3(4): 1057 (2015) 6/10 van der Veen et al. (2015) Email: Central

68 89Zr-labelled cG250 suggest thatcould this have tracer a major might impact have betteron patient binding management. and internalization The first bothal. successfully radiopharmaceuticals demonstrated are PET small with molecules a Ga-HBEDCC-PSMA that target characteristicsresults of preclinical compared studies to with124I-cG250 [63]. Accordingly, a themolecule PSMA-receptor, in 37 men only their 1 and distribution 3 hours after pattern injection on PET[65]. isThough quite

RCC with a less aggressive tumor type using 89Zr-cG250 PET in clinical trial has been initiated to identify patients suspected of ligands.different. In Illustrating drug development, that the agreementpharmacokinetic between and the targeting study- properties of antibody-fragments can differ significantly among whichFuture watchful directions waiting of can antibody be considered. PET biodistribution and targeting studies. However, when imaging isdrug primarily and radiopharmaceuticalused as a biomarker to is select crucial patients, when characterize performing lesions or assess treatment response, absolute accordance in The great potential of nuclear imaging during89Zr-based the imaging clinical biodistribution between study-drug and radiopharmaceutical is introduction of immunotherapeutic agents has been presented in lesscrucial [66]. this overview. The growing use of especially The complete picture quantitativein clinical multicenter imaging biomarkersstudies has led to to describe a need for pharmacokinetics harmonization of imaging protocols among the different centers. The use of and pharmacodynamics is only valuable if the exchange of those The fact that cancer is an extremely complex molecular findings among different centers is possible. Recently, the EATRIS disease that is dependent on local tissue for oncogenesis and (European Infrastructure for Translational Medicine) and the growth is conceptualized by Hanahan and Weinberg through EANM (European Association of Nuclear Medicine)18F imaging. collaborated seeminglytheir ‘Hallmarks ‘normal’ of cancer’biological [67]. processes The tumor and microenvironment cells that support to facilitate a standardization and cross-calibration program for is defined as the surrounding of the cancer cells including PET-scanners,Up to now, most as has clinical already imaging been trialsinitiated with for immunotherapeutic tumor growth such as blood vessels, immune inflammatory cells, cancer-associated fibroblasts, endothelial cells, extracellular agents have been using intact radiolabeled124I and antibodies. 89Zr are Due the to most the domatrix not recon and signalingwith the microenvironment. molecules. As briefly Standard mentioned biopsies before, and longsuitable biological isotopes. half-life Though of radiolabeling these compounds with imagingIodine is is common carried thestandard ex vivo tumor assessments stratification such asor histology,evaluation proteomics, of response genomics, generally outpractice up to in 9 many days afterlabs, injection,looking at so imaging characteristics 89Zr has better properties. Several centers are currently involved in 89 in time. Still, even in a biopsy sample heterogeneity can already Zr-labeling procedures to etc. only focus on a small area of the tumor at one specific moment the development of new and easier be appreciated through the varying degrees of cell proliferation, manufacture stable compounds. Still, both isotopes share common invasiveness,Molecular inflammation,imaging can provide receptor a expression,more complete vascularity, systematic etc. problems, 1) a limited availability due to the dependency of a body.cyclotron As a forresults, their the production, administered and, dose 2) a ishigh typically radiation between burden 37 andto the 100MBq, patients which due to in long term residence results in times less ofoptimal the isotope images in andthe thatpicture enable of all in these vivo aspects in the living tumor, as is stated microenvironmentby Weissleder and include Mahmood multi-parametric [68]. Well-known MRI, technologies nuclear and imaging of tumor physiology and the long acquisition times (i.e., >45 minutes for typical whole body evenacquisition). are entirely Despite missed these on extended 89Zr and acquisition124 times, lesions optical imaging (e.g., relies on fluorescence, bioluminescence, radiationsmaller than dose 1cm per in injection,diameter imagingshould not with be quantified, these isotopes and can reflectance or absorbance of light). Specialized algorithms are I PET. Due to the high then applied to extract quantitative measures for signal intensity, patient population. To put this in perspective, a conventional shape and texture of the cancerous tissues, a novel methodology only be performed a limited number of times and in a specific known as ‘Radiomics’. This approach of transferring imaging an 89 data into phenotypic tumor information has been shown by FDG-PETantibodies scan is unlikely is ±5 mSv, to develop a diagnostic into a abdominal generally usedCT ±8mSv, diagnostic and ‘virtualAerts et molecular al. in over imaging 1000 biopsy’ patients has with been lung suggested and head/neck to study modality.Zr-immunoPET ±20-40mSv. Consequently, PET with intact thecancer cancerous [69]. With tissues this broad as an range alternative of technologies, to standard even the biopsies term [68]. Although medical imaging has indeed gained a central role in personalized medicine, standard biopsies still remain To optimize imaging characteristics of these radiopharmaceuticals focus is shifting towards smaller antibody- ex vivo a crucial part of tumor identification. Combining the image- radiolabelingfragments (i.e., with affibodies, alternative diabodies, positron nanobodies, emitters like etc)68Ga, and18F tobased identity phenotypic the heterogeneous information tumorwith data and gathered measure from response to smalland 64 Cu molecules. becomes These possible. compounds The shorter have residence faster kinetics, time in thus the targetedassessments therapies. of specific areas results in a synergistic approach CONCLUDING REMARKS isbody the and recently physical published half-life studiesof these with isotopes 18F-labelled results in and a lower68Ga- radiationlabelled PSMA-targeting dose to the patient. small Amolecules. good example Cho etof al. this showed approach the 18 Advances in oncological sciences led to the development of feasibility of F-DCFBC PET only 2 hours after injection in five novel anticancer agents that are designed to affect key molecular men with proven prostate carcinoma [64]. Afshar-Oromieh et pathways or drivers. Nuclear molecular imaging techniques hold J Pharmacol Clin Toxicol 3(4): 1057 (2015) 7/10 van der Veen et al. (2015) Email: Central

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Cite this article van der Veen BJW, Stokkel MPM (2015) The clinical role of nuclear imaging in immunotherapeutic drug development. J Pharmacol Clin Toxicol 3(4):1057.

J Pharmacol Clin Toxicol 3(4): 1057 (2015) 10/10