Zirconium-89 Labeled Antibodies: A New Tool for Molecular Imaging in Cancer Patients

Antibody based positron emission tomography (immuno-PET) imaging is of increasing importance to visualize and characterize tumor lesions. Additionally, it can be used to identify patients who may benefit from a particular therapy and monitor the therapy outcome. In recent years the field is focused on 89Zr, a radiometal with near ideal physical and chemical properties for immuno-PET. In this review we will discuss the production of  89Zr, the bioconjugation strategies, and applications in (pre-)clinical studies of  89Zr-based immuno-PET in oncology. To date, 89Zr-based PET imaging has been investigated in a wide variety of cancer-related targets. Moreover, clinical studies have shown the feasibility for 89Zr-based immuno-PET to predict and monitor treatment, which could be used to tailor treatment for the individual patient. Further research should be directed towards the development of standardized and robust conjugation methods and improved chelators to minimize the amount of released Zr4+ from the antibodies. Additionally, further validation of the imaging method is required. The ongoing development of new 89Zr-labeled antibodies directed against novel tumor targets is expected to expand applications of  89Zr-labeled immuno-PET to a valuable method in the medical imaging.


Introduction
Molecular biomarkers can be used to monitor, image, and measure biological processes at molecular or cellular level. Different types of biomarkers are known, including diagnostic, prognostic, and predictive biomarkers, or a combination of these [1]. Extensive research has been done on the development of molecular imaging biomarkers in the field of cancer. This has led to tools that can be used to visualize and characterize tumor lesions. An advantage of using molecular imaging agents is the noninvasive nature of these procedures, whereas in conventional methods a more invasive procedure is used (e.g., blood sample or biopsy). Various imaging modalities can be used for tumor visualization such as fluorescent imaging, magnetic resonance imaging (MRI) or radionuclide imaging with positron emission tomography (PET), or single photon emission computed tomography (SPECT). In most cases, the use of PET is preferred over SPECT since higher spatial resolution images can be obtained and images can be analyzed quantitatively more accurately with PET. Specific uptake of molecular biomarkers can be achieved using radiolabeled targeting agents such as antibodies, directed against tumor-associated antigens like epidermal growth factor receptor (EGFR) [2], human epidermal growth factor receptor 2 (HER2), and many others. The high specificity and affinity of radiolabeled antibodies make them attractive candidates as an imaging agent. For example, 89 Zr-labeled anti-HER2 antibodies can be used to differentiate between HER2 + and HER2 − tumors [3], also appreciating intra-and intertumoral heterogeneity. An additional application of radiolabeled antibodies is to identify patients who may benefit from a particular therapy and monitor therapy outcome based on the level of tumor-associated antigen expression [4]. However, the relative slow pharmacokinetics of intact antibodies ( 1/2 = 3-4 days) requires the use of radionuclides with long half-lives (e.g., 111 In (2.8 days) for SPECT or 89 Zr (3.3 days) and 124 I (4.2 days) for PET [5]). For antibody based PET imaging (immune-PET) 89 Zr has several advantages: 89 Zr has a half-life of 78.4 h which matches the pharmacokinetics of antibodies and it has a relative low average positron energy of 395 keV, making it an ideal candidate for high resolution PET imaging of slow-accumulating biomolecules. In addition, 89 Zr-based agents are safer to handle and more stable in vivo making them better candidates than 124 I-based agents for clinical applications. Due to the numerous advantages of 89 Zr-based immuno-PET, the field is progressing at a rapid and exciting pace. In this review, the potential of 89 Zr-based immuno-PET in oncology will be reviewed. The production of 89 Zr, the bioconjugation strategies, and applications in (pre-)clinical studies are discussed.  89 Zr provides high resolution PET images. In addition, the energy disparity between the photons (511 keV) and the gamma rays (909 keV) prevents the latter from interfering with the detection of 511 keV photons. In contrast, its halogen competitor, 124 I, produces high energy photons of different energies (603 keV (63.0%), 1691 keV (10.9%), and 723 keV (10.4%) [6]) which may result in random and scatter coincidences and therefore in more background noise as compared to 89 Zr. Hence, reconstruction of 89 Zr-based PET scans is more straightforward to attain good image quality compared to 124 I. Although 89 Zr has many advantages over other PET radionuclides, some essential shielding requirements during transport and handling of 89 Zr are needed (half-value layer of 89 Zr in lead is roughly 10 mm). High energy and highly penetrating photons (909 keV) are emitted during 89 Zr decay in high abundance.

Production of 89 Zr
The first production of 89 Zr was done by Link et al. [7] by a (p,n) nuclear reaction by bombarding 89 Y on Y foil with 13 MeV protons [5]. The produced 89 Zr needed several purification steps and was obtained in 80% yield with radionuclidic purity exceeding 99%. Nowadays, many medical centers are able to produce medical isotopes using low-energy cyclotrons that are capable of bombarding targets with protons of low energy (<20 MeV). Therefore, the most common route to produce 89 Zr is via the 89 Y (p, n) 89 Zr reaction on commercially available 89 Y target foils. The above route will in general result in high yields (94-95%) and high radionuclidic purities (>99%). Competing nuclear reaction, like (p, 2n) reactions, can result in small amounts radionuclidic byproducts, such as 88 Zr and 88 Y [8]. Several separation and purification techniques with variable outcomes are used including anion exchange, cation exchange, and solvent extraction [9][10][11]. For synthesizing such radiopharmaceuticals for patients, automated units for a clean, fast, safe, and reproducible radionuclide synthesis according to good manufacturing practice (GMP) are necessary. Several groups have designed and built automated systems for 89 Zr [12,13]. For example Wooten et al. [14] reported a custom-made system to safely and routinely produce 89 Zr with high radionuclidic purity (>99.99%) and satisfactory effective specific activity (5-353 mCi⋅ mol −1 (0.01%-0.88% of theoretical specific activity)) based on previous developments in separation and purification techniques [9][10][11]15].

The Need for Efficient Chelators
The release of 89 Zr 4+ from the antibodies needs to be prevented, because the free radionuclide can accumulate in the mineral bone and can associate with plasma proteins. This leads to depositing significant doses to the bone marrow [16]. Therefore, an appropriate chelator system is necessary to minimize the disassociation of 89 Zr from the antibodies. Over the years, several chelators have been used with different success, such as diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraacetic acid (DOTA), and desferoxamine (DFO) [17]. The stability of Zr-DOTA, Zr-DTPA, and Zr-EDTA was found to be limited. The thermodynamic stability of Zr-DTPA is slightly higher than that of Zr-EDTA, most likely because DTPA coordinatively saturates the Zr 4+ , while EDTA requires exogenous water molecules [18]. DFO is the most prominent chelator of Zr 4+ . DFO is a hexadentate siderophore containing three hydroxamate groups for chelating metals and a primary amine tail for conjugation to a biomolecule ( Figure 1). Besides a zirconium chelator, it is a chelating agent for several other metal ions [19]. It demonstrated good stability, releasing less than 0.2% of Zr 4+ after 24 h in serum [20] and after seven days in serum still less than 2% demetallation occurs [21]. Several proof-of-principle preclinical studies have been conducted using DFO to label antibodies with 89 Zr; however, the in vivo stability of this complex remains an issue, because free 89 Zr is observed in the bone dependent on the in vivo behavior of the antibody [22]. Several studies have attempted to improve the linkage between DFO and the antibody [9,23], whereas others have focused on improving the chelate itself [24]. Eventually, a ligand that is both octadentate and oxygen-rich is believed to be the most stable Zr 4+ chelator, since it would be able to incorporate all eight coordination sites of zirconium [22]. This novel high stability Zr 4+ ligand would in theory minimize the uptake of liberated Zr 4+ in the bone and other nontargeted tissues. To date, the design, synthesis, and the evaluation of such a Zr 4+ chelate requires further research.

Conjugation of Antibodies with DFO.
As DFO is currently the most promising chelator for 89 Zr 4+ , conjugation of antibodies with DFO will be discussed here in detail ( Figure 1). Several methods are available to conjugate DFO based on the reaction of an activated bifunctional chelator with a lysine or cysteine residue of the antibody. The different conjugation techniques do not only have different conjugation efficiencies, but also affect the biodistribution of the radiolabeled antibody [22]. The earliest reports on conjugation of DFO to bioactive molecules is based on the addition of thiols to the amino group of DFO [20]. In this approach DFO was modified by Nsuccinimidyl-S-acetylthioacetate (SATA), resulting in an Sacetyl-protected thiol derivatized form of chelator. In parallel, maleimide moieties were introduced in the antibody by the reaction with 4-(N-maleimidomethyl) cyclohexane carboxylic acid N-hydroxysuccinimide ester (SMCC). Next, the two formed compounds were combined and in the presence of hydroxylamine at physiological pH the DFO-antibody conjugate was formed. Following this early work, Verel et al. introduced a novel conjugation approach, which was based on an activated 2,3,5,6-tetrafluorophenol (TFP) chelate ester which can form a stable amide bond with the -amino-groups of the lysine residues of the monoclonal antibody (mAb) [9]. This laborious approach consisted of 5 steps which involve (i) the extension of DFO with succinyl anhydride, (ii) protection of side-reactions of the hydroxamate groups of the ligand by complexation with Fe 3+ , (iii) formation of activated TFP ester, (iv) conjugation of activated DFO-ester to the unmodified antibody, and (v) removal of Fe 3+ from the chelator. Nowadays the most widely used method in preclinical 89 Zrbased immuno-PET uses the conjugation strategy with Nsuccinimidyl-DFO, which addresses -amino groups of lysine side chains [22,[25][26][27][28][29][30]. Since the Zr 4+ field is rapidly growing and becoming more mainstream in the clinical setting, simple methods for DFO conjugation preferably using commercial available starting materials are essential. Perk et al. introduced a simplified method using a commercially available p-isothiocyanatobenzyl-DFO (DFO-Bz-NCS) chelate, which can be directly attached to the -aminogroups of the lysine residues of an antibody by forming a stable thiourea linkage [23]. Despite the fact that this method is simpler than the N-succinimidyl-DFO chemistry, it requires more expertise mainly because of the limited water solubility of the chelator precursor.
A limitation of the conjugation of DFO to antibodies is compromised immunoreactivity, because the chelator may interfere with the antigen-binding domain of the antibody, especially if there are lysines in or close to the complementarity determining regions of the antibody. To overcome these limitations site-specific strategies using engineered cysteine residues can be used in combination with thiol-reactive DFO derivatives such as bromoacetamido-desferrioxamine (DFO-Bac), iodoacetamido-desferrioxamine (DFO-Iac), and maleimidocyclohexyl-desferrioxamine (DFO-CHX-Mal) [31]. The radiolabeled antibodies using these thiol-reactive DFO derivatives were stable and showed similar characteristics as the lysine-linked complexes. Remarkably, no significant difference was observed between the immunoreactivity of the site-specific complex and the lysine-linked complexes.
Another novel conjugation approach for effective labeling of 89 Zr to antibodies is the use of click chemistry between an acetylene group and an azide. This approach might not significantly improve the targeting of tumors compared with DFObased conjugation strategies; however, with this approach it is possible to fully tailor the constructs. Furthermore, the modular system can be used for direct comparison of bioconjugates with different radiometals as the the chelatormodified antibodies are synthesized using identical ligation conditions resulting in similar immunoreactivity and chelator/antibody ratios [32]. Several studies have been reported on bioorthogonal click chemistry [32], Staudinger ligation [33], or catalyst-free click chemistry [34]. The click chemistry as specialized conjugation method is expected to expand the scope of 89 Zr-based PET.

Preclinical Studies with 89 Zr
Over the last years several 89 Zr-labeled antibodies directed against different tumor types have been evaluated in preclinical studies (e.g., [9,18,22,35,36]; see Table 1). Here these developments of 89 Zr-labeled antibodies in preclinical studies will be discussed based on their tumor target.

5.
1. Targeting CD20. The glycosylated phosphoprotein, CD20, is expressed on the surface of B-cell lymphomas, hairy leukemia, B-cell chronic lymphocytic leukemia, and melanoma cells. 89 Zr-labeled antibodies directed against CD20 might be useful to measure and monitor the therapeutic effect of non-Hodgkin's lymphoma (NHL) therapy [18,37]. The 89 Zr-Desferrioxamine-rituximab, an antibody directed against CD20, specifically targeted the human CD20 antigen in a humanized CD20-expressing transgenic mouse model (huCD20TM). 90 Y-labeled anti-CD20 mAb ibritumomab tiuxetan (Zevalin) is approved for treatment of patients with relapsed and refractory NHL. In a pilot study, 89 Zr-labeled ibritumomab tiuxetan was shown to have a nearly identical biodistribution compared to 90 Y-labeled counterpart [18]. This indicated that a scout scan with 89 Zr-ibritumomab immuno-PET can be used to assess, predict, and quantify the biodistribution of 90 Y-ibritumomab tiuxetan.

Targeting CD44.
The cell-surface glycoprotein, CD44, is involved in many biological processes including adhesion of cells to extracellular matrix proteins, lymphocyte-endothelial cell interactions, metastasis formation, migration of cells, and T cell activation/adherence [38]. The v6 splice variant of CD44 is involved in tumorigenesis, tumor cell invasion, and metastasis and is expressed preferentially in squamous cell carcinomas [39]. Preclinical studies using 89 Zr-labeled anti-CD44v6 chimeric monoclonal antibody cU36 demonstrated that the tracer was able to detect small tumors in nude mice with HNSCC xenografts [9,40]. In addition, it was reported that 89 Zr-cU36 PET imaging was a suitable candidate for scouting of therapeutic doses of 90 Y-cU36 [40,41].
Recently, evaluation of 89 Zr-RG7356, an antibody directed against the constant part of CD44, was performed in mice bearing tumor xenografts with different levels of CD44 expression and RG7356 responsiveness, namely, MDA-MB-231 (CD44+, responsive), PL45 (CD44+, nonresponsive), and HepG2 (CD44−, nonresponsive) [42]. 89 Zr-RG7356 selectively targeted CD44+ responsive and nonresponsive tumors in mice. 89 Zr-RG7356 whole body immuno-PET in healthy cynomolgus monkeys revealed antibody uptake in spleen, salivary gland, and bone marrow, which might be related to the expression of CD44 in these organs. The 89 Zr-RG7356 uptake in the normal organs decreased with increasing dose of unlabeled RG7356, indicating saturable targeting of CD44 in these animals.

Targeting EGFR.
The epidermal growth factor receptor (EGFR) is a member of the ErbB family. It plays a crucial role in differentiation, proliferation, and survival of many different tumor types, including breast, lung bladder, and colon carcinoma [2]. The overexpression of EGFR is associated with more aggressive tumors and poor prognosis due to the resistance of treatment [43,44]. Many mAbs have been developed to inhibit the EGFR activation [2]. A well-known example is cetuximab (Erbitux), a chimeric IgG, which upon binding to the ligand-binding domain induces internalization of EGFR and thereby blocking downstream signalling [45,46]. Several studies showed tumor regression upon treatment with cetuximab [47][48][49][50]. 89 Zr-labeled cetuximab was evaluated for scouting the biodistribution of 90 Y-and 177 Lu-cetuximab in tumor bearing mouse and thus potentially allowing the estimation of the radiation dose delivered to tumors and normal tissues during radioimmunotherapy with 90 Y-and 177 Lu-cetuximab [51]. It was reported that the 89 Zr-immuno-PET could be used for in vivo scouting of 90 Y-and 177 Lu-labeled mAbs. However, an increased bone uptake of 89 Zr-cetuximab, compared with 90 Y-and 177 Lu labeled cetuximab, was observed indicating that 89 Zr is more efficiently incorporated in the bone compared to the other radiometals ( 90 Y-and 177 Lu). Therefore estimating bone marrow doses based on 89 Zr-bone uptake is not straightforward. Another study investigated the relation between the in vivo expression of EGFR and the tumor uptake of 89 Zr-cetuximab [52]. In this study no clear-cut relationship was found, suggesting that apart from antigen Another approved mAb to inhibit the EGFR signalling is panitumumab. It was the first recombinant human monoclonal antibody (IgG2) approved by the FDA for the treatment of patients with EGFR-expressing metastatic colorectal cancer (mCRC) [53]. In several studies the use of panitumumab for noninvasive, in vivo imaging of HER1 expression in tumors is reported [54][55][56][57][58]. The use of 89 Zr-panitumumab for immuno-PET of HER1 expression was recently evaluated in a direct comparison with 111 In-panitumumab. The organ biodistribution between 111 In-and 89 Zr-panitumumab was almost identical [55]. In addition, the targeting of 89 Zrpanitumumab correlated well with the HER1 expression. Recently, a standardized and straightforward stepwise ∼5 h production method was reported for the production of clinical-grade 89 Zr-panitumumab [59]. In this method clinical-grade panitumumab is conjugated with DFO chelate and subsequently radiolabeled with 89 Zr resulting in high yields (>70%) and high radiochemical purity (>98%).

Targeting HER2.
Human epidermal growth factor receptor 2 (HER2) is another member of the ErbB family. It is involved in angiogenesis, differentiation, metastasis, proliferation, and cell survival upon heterodimerization with other members of the EGF receptor family [60]. HER2 overexpression is found in many types of tumors including breast and ovarian cancer. The FDA approved anti-HER2 mAb trastuzumab (Herceptin, Genentech, CA, USA) to be used for the treatment of HER2 positive breast tumors, since it blocks the HER2 activation [60]. The efficacy of the treatment is dependent on the HER2 expression level. The HER2 expression level in a tumor is not static and may vary over time [60]. In addition, the HER2 expression is found to be different between the primary lesion and the distant metastatic lesions in the same patient. Noninvasive in vivo imaging to visualize HER2 expressing using radiolabeled trastuzumab has been extensively investigated [29,30,61]. PET imaging using 89 Zr-trastuzumab has been performed in different murine tumor models and accumulation of the tracer was found to be HER2 specific [29,30,61]. For example, the tumor uptake of 89 Zr-trastuzumab in nude mice with a subcutaneous human ovarian cancer xenografts (SK-OV-3) was high (∼30% ID/g) and the biodistribution was similar to that of 111 In-trastuzumab [29]. Recently, the specificity of 89 Zrtrastuzumab, 18 F-FDG, and 18 F-FLT PET for HER2-positive gastric cancer was evaluated ([3]; Figure 2). The study revealed a high specific uptake of 89 Zr-trastuzumab in HER2positive tumors, whereas 18 F-FDG and 18 F-FLT PET were unable to differentiate between HER2-positive and HER2negative tumors. In addition, 89 Zr-trastuzumab was used to quantitatively determine the HER2 expression level after treatment. For example, after treatment with a heat shock protein 90 (hsp90) inhibitor a significant decrease in HER2 expression could be measured based on the 89 Zr-trastuzumab tumor targeting [30,62]. A combination treatment of hsp90 inhibitor 17AAG and the EGFR/HER2 tyrosine kinase inhibitor, lapatinib, revealed an even stronger reduction of the HER2 expression levels using 89 Zr-Trastuzumab-F(ab ) 2 fragment as probe [63]. Additionally, the biological effect of afatinib, an EGFR/HER2/HER4 inhibitor, in a HER2-positive gastric xenograft models was evaluated [3]. In this model the uptake of 18 F-FDG did not change after afatinib therapy, whereas a decrease in 89 Zr-trastuzumab uptake was observed upon treatment. The lower uptake of the 89 Zr-trastuzumab correlated with the decreased HER2 expression as determined by immunoblots and immunohistochemistry. Thus, 89 Zr-trastuzumab PET might be useful for the characterization, treatment planning, and treatment monitoring of HER-2 positive cancers.

Targeting VEGF.
Vascular endothelial growth factor (VEGF) is a proangiogenic factor in both normal tissues and in tumors. The overexpression of VEGF and its receptors (VEGFR) are associated with poor prognosis [64]. The humanized anti-VEGF mAb, bevacizumab (Avastin, Genentech/Hoffmann-La Roche), is capable of blocking angiogenesis by depleting VEGF and thereby preventing its binding to the VEGFR. This neutralizes VEGF actions (see, e.g., [65][66][67][68][69][70][71][72]). A direct comparison between 89 Zrbevacizumab and an irrelevant 89 Zr-labeled IgG revealed a significantly higher tumor uptake of 89 Zr-bevacizumab in nude mice with human ovarian SK-OV-3 tumors [73]. Besides using 89 Zr-bevacizumab as PET tracer for noninvasive in vivo imaging of VEGF expression in the tumor microenvironment, potentially it can also be used to predict or monitor an antiangiogenic response. For example, hsp90 is crucial player in VEGF transcription and can be used to treat ovarian tumors. In nude mice with a subcutaneous human ovarian cancer xenografts (A2780), uptake of 89 Zrbevacizumab in the tumors correlated with the therapeutic effect of the hsp90 inhibitor, NVP-AUY922, [74]. In another study the effect of the mTOR inhibitor, everolimus, on the VEGF production was evaluated [75]. Everolimus treatment caused decreased 89 Zr-bevacizumab uptake in subcutaneous A2780 human ovarian tumor. The results were in line with the lower VEGF-A protein levels in tumor lysates of treated versus untreated tumors. These results indicate 89 Zrbevacizumab can be used to monitor tumor VEGF-A levels as an early biomarker of the antiangiogenic effect of mTOR inhibitor treatment. 89 Zr-labeled ranibizumab, a monoclonal antibody fragment (Fab) derivative of bevacizumab, was used to detect and monitor the early antiangiogenic response to treatment with sunitinib, a VEGFR tyrosine kinase inhibitor, in nude mice bearing a subcutaneous A2780 human ovarian tumor or Colo205 human colon cancer xenografts. 89 Zr-ranibizumab PET matched better with the observed results obtained by histology, immunohistochemistry, and tumor proliferation and vascularization assays, than 18 F-FDG PET and 15 O-water PET. Since ranibizumab has a serum half-life of only 2 to 6 hours, rapid and sequential follow-up PET scans are feasible with 89 Zr-ranibizumab [76]. Therefore, in contrast to 89 Zrbevacizumab, 89 Zr-ranibizumab can be used for imaging of rapid dynamic alterations in VEGF response in tumors.

Targeting PIGF.
The clinical benefits of angiogenesis inhibitors can be compromised by the upregulation of proangiogenic factors such as the placental growth factor (PIGF). PIGF, a VEGF homolog, is expressed in low levels in normal tissue and can be overexpressed in tumor cells. PIGF contributes to angiogenesis in pregnancy, wound healing, ischemic conditions, and tumor growth [77,78]. PIGF inhibitors are able to reduce the angiogenesis and tumor cell motility. The antitumor activity of a humanized mAb directed against PIGF-1 and PIGF-2, RO5323441, in human tumor xenograft models has been reported [79]. To further explore and validate the use of RO5323441, the tumor and normal tissue uptake of 89 Zr-RO5323441 at different time points was evaluated in mice bearing human PlGF-expressing Huh7 hepatocellular cancer xenografts. Tumor accumulation of 89 Zr-RO5323441 was specific and time-and dose-dependent.

Targeting PSMA.
Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein which is associated with increased tumor progression, development of castration resistance, and/or resistance to hormone-based treatments [80][81][82]. PMSA is expressed in a limited range of normal tissues including benign prostatic epithelium, renal proximal tubule, small bowel, and the brain; however, the expression level is 2 to 3 times lower than in prostate cancer specimens [83]. 89 Zr-labeled anti-PSMA mAb, J591, was able to differentiate between subcutaneous PSMA positive and negative tumors in athymic nude mice [21], making it a potential target for clinical noninvasive identification and quantification of PSMA-positive tumors.
5.8. Targeting CD147. CD147, a member of the immunoglobulin superfamily, is involved in many physiological functions including embryo implantation, early stage neural network formation, and spermatogenesis [85]. Overexpression of CD147 is found in many types of cancer including pancreatic cancer and induces expression of matrix metalloproteinases (MMPs) and VEGF [86,87]. Several (pre-)clinical studies have been performed using anti-CD147 antibodies to inhibit the actions of CD147 and revealed a reduction in proliferation, invasion and metastasis of tumors [88][89][90]. Almost 90% of the pancreatic cancers have high CD147 expression levels [86]. Sugyo et al. evaluated the CD147 expression in four pancreatic cancer cell lines (MIA Paca-2, PANC-1, BxPC-3, and AsPC-1) using the human 125 I-, 67 Ga-, or 89 Zr-labeled anti-CD147 mAb (059-053) [84]. Additionally, the in vivo CD147 expression was evaluated using 125 I-or 89 Zr-labeled 059-053 in mice with s.c. and orthotopic MIA Paca-2 and A4 (non-CD147-expressing) tumors. The biodistribution data revealed significantly higher tumor uptake of 89 Zr-059-053 in MIA Paca-2 tumors than in the A4 tumors ( Figure 3). PET/CT imaging demonstrated that orthotopic MIA Paca-2 tumors could be visualized with 89 Zr-059-053 PET. High expression of CD147 is not only restricted to pancreatic cancer, but is also found in other types of cancer including bladder, breast, colorectal, cervical, liver, and ovarian cancer [84][85][86]. Therefore, 89 Zr-059-053 might also be applied in patients with these cancer types.

Targeting CAIX.
Hypoxia in tumors is associated with a poor prognosis in many tumor types since it is associated with resistance to radiotherapy and chemotherapy. In many tumor types carbonic anhydrase IX (CAIX) has been validated as an intrinsic hypoxia-related cell marker [91]. Using antibodies directed against CAIX it is possible to select patients for hypoxia-targeting or -modifying treatment combined with radiotherapy. For example, it is possible to visualize tumor hypoxia in mice bearing s.c. SCCNij3 head and neck squamous cell carcinomas using 89 Zr-cG250-F(ab ) 2 , an anti-CAIX antibody fragment [92]. In a direct comparison, the tumor uptake of mAb 89 Zr-cG250 in mice with CAIXexpressing clear cell renal cell carcinoma (ccRCC) xenografts (NU-12) was significantly higher compared to that of 124 I-cG250 [93]. This indicates that PET imaging of ccRCC tumors with 89 Zr-cG250 could be more sensitive than 124 I-cG250-PET. CAIX targeted 89 Zr-PET imaging is a candidate for imaging hypoxia in different types of tumors and deserves further exploration.

5.10.
Targeting IGF-1R. The insulin like growth factor 1 receptor (IGF-1R) is a transmembrane receptor expressed in many human cancers, including in ∼35% of all triple-negative breast carcinomas. It is involved in the proliferation, apoptosis, angiogenesis, and tumor invasion. Heskamp et al. reported excellent tracer uptake of 111 In-R1507 and 89 Zr-R1507, a human mAb directed against IGF-1R, in mice with s.c. SUM149 triple-negative breast cancer xenografts [94]. This suggests that the use of 89 Zr-R1507 in patient selection of IGF-1R-targeted therapy is possible.

Targeting
Met. The expression of hepatocyte growth factor receptor tyrosine kinase (Met) was measured by PET using 76 Br or 89 Zr-labeled-onartuzumab, a mAb against Met [95]. Both tracers specifically targeted Met; however, at later time points a higher tumor uptake was observed with 89 Zr-Onartuzumab. This suggests that 89 Zr-onartuzumab is the preferred tracer to identify Met expression in cancer patients and possibly to predict and monitor the treatment with Met-targeted therapeutics. In another study, the potential of immune-PET using 89 Zr (residualising radionuclide) or 124 I-labeled (non-residualising radionuclide) anti-Met mAb DN30 was evaluated in mice with s.c. GLT-16 (high Met expression) and FaDu (low Met expression) tumors [96]. The biodistribution data revealed significantly higher tumor uptake of 89 Zr-DN30 than 124 I-DN30 in GTL-16 tumorbearing mice. Similar blood levels were found indicating that DN30 is internalized. 89 Zr-DN30 immuno-PET imaging was able to visualize small tumor lesions with a higher 89 Zr tumor uptake in GTL-16 than FaDu tumor-bearing mice. Additionally, the correlation was high for PET-image-derived 89 Zr tumor uptake and the ex vivo-assessed 89 Zr tumor uptake. This indicates that 89 Zr-labeled immuno-PET is an attractive method to evaluate Met-targeted therapeutics.

5.
12. Targeting GPC3. The glypican-3 (GPC3) is a hepatocellular-specific cell surface proteoglycan overexpressed in most hepatocellular carcinomas (HCC). Sham et al. reported excellent tracer uptake of 89 Zr-GPC3, a mAb directed against GPC3, in mice with GPC3-expressing HepG2 liver tumors [97]. This suggests that the use of 89 Zr-GPC3 to image HCC in the liver is possible.

Clinical Translation of 89 Zr Immuno-PET
The 89 Zr-labeled antibodies against the targets mentioned above all show promising results for clinical translation. To date, several clinical investigations using 89 Zr-labeled antibody constructs have been reported [1,22,98]. Here these recent clinical studies will be discussed.
6.1. 89 Zr-Labeled cU36. The first clinical trial using the 89 Zr-cU36 PET to target CD44 expressing tumors showed that the tracer was able to detect primary tumors as well as metastases in the neck region with similar sensitivity as computed tomography (CT) and magnetic resonance imaging (MRI) [99]. The results are promising, although several issues remain to be addressed. In the clinical study micrometastases were missed with 89 Zr-cU36 PET, so immuno-PET may be less suited as a staging tool, but more suitable to characterize tumors. Moreover, 2 out of the 20 patients developed antibodies against the chimeric cU36 antibody (HACA), which may hinder repetitive imaging procedures.  [102]. After binding of 89 Zr-trastuzumab to these extracellular domains, the immune complex was cleared by the liver and excreted in the intestines. In patients treated with trastuzumab at the time of injection, higher doses of 89 Zr-trastuzumab did not improve imaging since complex formation was minimal.
Overall, the uptake of 89 Zr-trastuzumab in the tumor lesions was high. The best time to assess tumor uptake was 4 to 5 days after injection of 89 Zr-trastuzumab ( Figure 4). All the known and even some unknown lesions were detected with PET. Of interest, metastatic brain lesions were detected in several patients, despite the fact that trastuzumab cannot penetrate the blood-brain barrier. This is probably because the blood-brain barrier in patients with brain metastasis is disrupted allowing 89 Zr-trastuzumab to pass. In this study HER2 overexpressing lesions could be distinguished from non-HER2 expressing lesions. These data indicate the potential use of 89 Zr-trastuzumab to improve the diagnosis of patients with HER2-positive breast cancer especially when lesions are inaccessible for biopsy.  Figure 5(a)). 25 of the 26 breast cancer nodules (96.1%) were detected using 89 Zrbevacizumab. Also, a correlation between the VEGF-A protein level in the tumors observed as measured by VEGF-A ELISA and the tumor uptake 89 Zr-bevacizumab was found ( Figure 5(b)). This study provides evidence that 89 Zrbevacizumab might be a potential candidate for the classification of breast tumors and to predict and monitor the effect of VEGF-A targeted therapies.

Conclusions
Clinical studies revealed that the use of 89 Zr-based immuno-PET results in high spatial resolution images with high tumor uptake and a good signal to noise ratio. Therefore, the use of 89 Zr-labeled antibodies is very promising for noninvasive visualization of tumor-associated antigens before, during, and after therapy. This makes 89 Zr-based immuno-PET an excellent imaging modality to predict and monitor treatment and to tailor treatment for individual patients. However, to fully integrate 89 Zr-based immuno-PET in the clinic several hurdles still need to be overcome. For example, standardized and robust methods for stable conjugation of DFO to antibodies should become available to obtain clinical-grade conjugates. In addition, research should focus on the development of improved chelators to minimize the amount of liberated Zr 4+ . Although some direct comparison studies between 89 Zr-based immuno-PET and immuno-PET using other PET isotopes have been performed, and supplementary quantitative and comprehensive comparison studies are needed to evaluate the value of 89 Zr-based immuno-PET. Additionally, the radiation dose for patients undergoing a  This research was originally published in [103]. © by the Society of Nuclear Medicine and Molecular Imaging, Inc. radiation dose for patients will limit repeated application of 89 Zr-based immuno-PET [104]. However, introducing new PET/CT scanners to allow better-quality immuno-PET images to be obtained with a lower 89 Zr radioactivity (37 MBq) dose have reduced the radiation dose [102,103]. Furthermore, research is focusing on combining 89 Zr-based immuno-PET with other imaging modalities. For example, the use of 89 Zr-immuno-PET in combination with nearinfrared fluorescence (NIRF) imaging has been reported by several groups [106][107][108]. The ongoing development of new 89 Zr-labeled antibodies directed against novel tumor targets is believed to rapidly expand applications of 89 Zr-labeled immuno-PET to a valuable method in the medical imaging.