PET Imaging of Receptor Tyrosine Kinases in Cancer Weijun Wei1,2,Dalongni2, Emily B

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Review Molecular Cancer Therapeutics PET Imaging of Receptor Tyrosine Kinases in Cancer Weijun Wei1,2,DalongNi2, Emily B. Ehlerding3, Quan-Yong Luo1,andWeiboCai2,3,4

Abstract

Overexpression and/or mutations of the receptor tyrosine and visualize RTKs with noninvasive diagnostic tools will kinase (RTK) subfamilies, such as epidermal growth factor greatly reﬁne clinical treatment strategies for cancer patients, receptors (EGFR) and vascular endothelial growth factor facilitate precise response prediction, and improve drug devel- receptors (VEGFR), are closely associated with tumor cell opment. Positron emission tomography (PET) agents using growth, differentiation, proliferation, apoptosis, and cellular targeted radioactively labeled antibodies have been developed invasiveness. Monoclonal antibodies (mAb) and tyrosine to visualize tumor RTKs and are changing clinical decisions for kinase inhibitors (TKI) speciﬁcally inhibiting these RTKs have certain cancer types. In the present review, we primarily focus shown remarkable success in improving patient survival in on PET imaging of RTKs using radiolabeled antibodies with an many cancer types. However, poor response and even drug emphasis on the clinical applications of these immunoPET resistance inevitably occur. In this setting, the ability to detect probes. Mol Cancer Ther; 17(8); 1625–36. 2018 AACR.

Introduction epidermal growth factor receptor (EGFR) belongs to another family of RTKs and includes three other members (erbB2/HER- Due to their complex heterogeneity and tendency to spread 2, erbB3/HER-3, and erbB4/HER-4). We previously showed throughout the body, treating cancers is very challenging. Over that HER-kinase–targeted imaging agents would enable maxi- the past quarter century, progress in cancer biology has led to mum benefit(i.e.,patientstratification, therapeutic response the discovery and identification of receptor tyrosine kinases monitoring, and new drug development) in cancer patient (RTK), which control many fundamental cell behaviors and management (4). c-Met, another RTK for hepatocyte growth drive tumor initiation, maintenance, and progression (1). factor (HGF), has been found overexpressed or aberrantly These discoveries have fueled the development of effective activated in a variety of cancers (5). targeted therapeutics such as monoclonal antibodies (mAb) Although mAbs targeting the above-mentioned RTKs have and tyrosine kinase inhibitors (TKI), shifting cancer patients' become a standard of care for patients with certain mutation- care from traditional empirical treatments to an era of person- positive tumors, the efficacy of the currently approved mAbs as alized treatment. single agents is very limited; therefore, synergistic regimens Human RTKs contain 20 subfamilies and only approximately containing conventional chemotherapeutic agents and the halfoftheRTKfamiliesarewellunderstood(2).RTKsare mAbs are applied (6). In addition, drug resistance almost anchored in the cytoplasmic membrane, and gain-of-function invariably occurs in cancer patients treated with these thera- mutations and/or overexpression of these RTKs are closely peutic antibodies. Traditionally, biopsy and immunohis- related to growth and proliferation in malignant tissues (2). tochemistry are performed to determine the RTK status of Of them, vascular endothelial growth factor A and VEGFRs have cancer tissues and to guide subsequent treatment. However, implicated roles in tumor angiogenesis, and visualization of spatial expression levels of RTKs can vary over time and among VEGFR expression in vivo with a radiopharmaceutical may be lesions (1), indicating the urgent need for novel noninvasive a viable clinical option for imaging angiogenesis (3). The approaches to visualize RTKs' plasticity throughout the whole body. Moreover, noninvasive methods that can select patients who will potentially benefit from combinational therapy and 1Department of Nuclear Medicine, Shanghai Jiao Tong University Affiliated Sixth predict treatment response will greatly optimize management People's Hospital, Shanghai, China. 2Department of Radiology, University of strategies for cancer patients. Wisconsin–Madison, Wisconsin. 3Department of Medical Physics, University of In recent years, molecular imaging has rapidly developed and 4 Wisconsin–Madison, Wisconsin. University of Wisconsin Carbone Cancer Cen- has been widely used both in clinical setting and in preclinical ter, Madison, Wisconsin. arenas (7–9). Single-photon emission computed tomography W. Wei and D. Ni contributed equally to this article. (SPECT) and positron emission tomography (PET) are radionu- Corresponding Authors: Weibo Cai, University of Wisconsin–Madison, Room 7137, clide molecular imaging techniques that enable noninvasive 1111 Highland Avenue, Madison, WI 53705. Phone: 608-262-1749; Fax: 608-265- evaluation of biochemical changes and expression of molecular 0614; E-mail: [email protected]; and Quan-Yong Luo, Department of Nuclear targets within living subjects. Although a SPECT probe, 111In- fi Medicine, Shanghai Jiao Tong University Af liated Sixth People's Hospital, 600# labeled trastuzumab, has been used in clinical trials to detect Yishan Road, Shanghai 200233, China. E-mail: [email protected] HER2-positive metastatic breast cancer (10), the sensitivity of doi: 10.1158/1535-7163.MCT-18-0087 SPECT is several orders of magnitude lower than that of PET (11). 2018 American Association for Cancer Research. In comparison, PET imaging is of high sensitivity and can be used

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to investigate physiological and molecular mechanisms of human ward strategy for generating RTK-speciﬁc PET radiotracers has diseases (11). PET imaging of RTKs with radiolabeled mAbs, mostly, but not entirely, involved the radiolabeling of therapeutic denoted as immunoPET, may provide a noninvasive method for mAbs. Antibody fragments, nanobodies, and smaller molecule assessing the dynamics of RTKs, selecting patients for personal- inhibitors have also been investigated for in vivo visualization of ized treatment, predicting response to RTK inhibition therapy, RTKs. With shorter biological half-lives, these probes are well and facilitating drug development (7). suited for fast imaging protocols when labeled with rapidly 68 18 Most PET imaging clinical trials have been focused on using decaying radioisotopes such as Ga (t1/2, 68 minutes) and F radiolabeled FDA-approved antibodies (12), such as trastuzumab (t1/2, 110 minutes). Our review focuses mainly on the current for breast cancer and esophagogastric adenocarcinoma (EGA; research in the ﬁeld of antibody-based immunoPET probes and refs. 13–17), cetuximab for colorectal and lung cancers (18– highlights potential clinical implications of immunoPET in visu- 20), and bevacizumab for several indications (21–26). PET alizing RTKs and in guiding clinical decisions (Fig. 1). 64 imaging using the positron emitters Cu (t1/2, 12.7 hours) and 89 Zr (t1/2, 78.4 hours) for antibody labeling has been extensively studied over the last decade (8, 27). 89Zr has a longer half-life, PET Imaging of the HGF/c-Met Pathway which generally matches the serum half-life of most mAbs and Dysregulation of HGF/c-Met signaling is implicated in a num- therefore is suitable for antibody imaging (28, 29). A straightfor- ber of malignancies (30), and synergistic effects of EGF and HGF

Figure 1. RTKs are deregulated in most human cancers and control many fundamental cell behaviors by regulating biochemical signals. Representative antibody-based PET probes targeting corresponding RTKs (or pathways) are shown. MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol-3-kinase; JAK, Janus kinase; PKC, protein kinase C; PLCg, phospholipase C-g; STAT, signal transducer and activator of transcription.

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were noted in non–small cell lung cancer (NSCLC; ref. 31). probe showed high affinity for human c-Met in 15 subjects with Moreover, c-Met amplification can drive HER3-dependent PI3K high risk of colorectal cancer (49). In addition to optical imaging signaling, thereby mediating NSCLC resistance to EGFR inhibitors in assessing c-Met (49, 50), 18F-AH113804 is a peptide-based (32, 33). Currently, c-Met targeted therapy involves small- c-Met–specific PET imaging probe that has been used to visualize molecule inhibitors (crizotinib, capmatinib, etc.; refs. 31, 34), locoregional recurrence of breast cancer in a clinical trial (51). and antibodies (emibetuzumab, ficlatuzumab, rilotuzumab, These results suggest that HGF/c-Met–specific PET imaging may onartuzumab, etc.; refs. 35, 36). Phase II trials in patients with correctly identify patients most likely to benefit from c-Met– advanced NSCLC demonstrated that the addition of onartuzu- targeted therapies or from EGFR inhibition therapy, as it has been mab to the EGFR inhibitor erlotinib resulted in an improvement reported that c-Met is implicated in acquired resistance to EGFR in progression-free and overall survival for c-Met–positive inhibitors in NSCLC (31, 33). patients (37); however, a phase III study failed to observe the additive therapeutic effect of onartuzumab to erlotinib (38). PET Imaging of the VEGF/VEGFR Pathway Despite these disappointing results, suppressing the HGF/c-Met pathway may still have an antitumor effect in other primary The VEGF/VEGFR signaling pathway plays an important role in tumors and/or overcome the resistance of molecularly targeted the regulation of angiogenesis. VEGFs bind to three associated therapies (30, 39). Noninvasive PET visualization of c-Met transmembrane RTKs known as VEGFR-1, VEGFR-2, and VEGFR- dynamics could therefore potentially facilitate patient stratifica- 3. Among them, VEGFR-2 is a key receptor involved in the devel- tion and guide c-Met–directed therapies. opment of blood vasculature and is an attractive target for anti- Initial attempts to develop nuclear medicine imaging probes angiogenic tumor therapy (52). Approved antiangiogenic drugs for detecting c-Met expression used murine mAbs (40, 41). Based such as bevacizumab, lenvatinib, sorafenib, sunitinib, and pazo- on these studies, taking the step to evaluate immunoPET probes panib target this pathway and are associated with modest survival using humanized or fully human anti-c-Met mAbs is relatively advantages in certain kinds of cancers (53). Bevacizumab and straightforward. Using the humanized therapeutic mAb onartu- ramucirumab are IgG1 antibodies directed against vascular endo- zumab, which inhibits HGF binding and therefore the HGF/MET thelial growth factor A (VEGF-A) and VEGFR2, respectively. To date, signaling pathway (35), Jagoda and colleagues initially synthe- clinical PET studies using 89Zr-Df-bevacizumab were performed in sized 89Zr-DFO-onartuzumab and 76Br-onartuzumab and found patients with breast cancer, neuroendocrine tumors, renal cell that the former probe exhibited higher uptake and tumor-to- carcinoma (RCC), NSCLC, and glioma (21, 22, 26, 54, 55). muscle ratios in MKN-45 gastric carcinoma models (42). Li and Based on fundamental preclinical studies (3, 9, 56, 57), in a colleagues developed c-Met–targeting bivalent cys-diabodies clinical feasibility study, Gaykema and colleagues demonstrated and radiolabeled one of the candidates with 89Zr. The authors that 89Zr-Df-bevacizumab could be used to detect primary breast found that uptake of 89Zr-labeled H2 cys-diabody was higher in cancer (22). Another pilot clinical study in patients with meta- the gefitinib-resistant NSCLC model than in the low c-MET– static RCC demonstrated that 89Zr-Df-bevacizumab PET visual- expressing NSCLC model, indicating that immunoPET could be ized renal tumor lesions with striking heterogeneity between used to assess c-MET expression levels for both therapeutic and lesions, therefore reflecting differences in vascular characteristics diagnostic purposes (43). Pool and colleagues then reported that (24). Everolimus, an inhibitor of mammalian target of rapamycin the readily translatable probe 89Zr-Df-onartuzumab could effec- (mTOR), also inhibits VEGF-A expression, but there are not tively discriminate erlotinib-induced c-Met upregulation in reliable biomarkers to predict efficacy of everolimus in patients NSCLC models (44). This study herein highlighted the potential with metastatic RCC at the moment. van Es and colleagues value of 89Zr-Df-onartuzumab PET in assessing c-Met upregula- investigated the value of 89Zr-Df-bevacizumab as a tool to identify tion-mediated erlotinib resistance in patients with NSCLC. More everolimus efficacy in 13 patients with metastatic RCC, and importantly, c-Met–directed treatment using NVP-AUY-922, a reported that everolimus decreased 89Zr-Df-bevacizumab tumor heat shock protein 90 (HSP90) inhibitor (45), could reduce uptake in 10 patients who received continuous everolimus treat- 89Zr-Df-onartuzumab uptake in HCC827 models (Fig. 2A). Rilo- ment and achieved stable disease at 3 months (Fig. 2D; ref. 26). tumumab (AMG102) is an HGF-binding antibody and can bind Jansen and colleagues reported that 89Zr-Df-bevacizumab had to and neutralize HGF, thus preventing its binding to c-Met. poor uptake in mice bearing diffuse intrinsic pontine glioma However, a phase III trial failed to observe the therapeutic effects (DIPG; ref. 23), implying that treatment with bevacizumab in of rilotumumab in patients with c-Met–positive gastric or gastro- DIPG patients is justified only after 89Zr-Df-bevacizumab immu- esophageal adenocarcinoma (46). Previously, there were no tools noPET demonstrates positive VEGF expression in tumor tissue. In to noninvasively determine the levels of HGF present in the local spite of these disappointing preclinical data, the same team tumor microenvironment. To this end, 89Zr-DFO-AMG102 was further reported that, in children with DIPG, five of seven primary developed by Price and colleagues, and this probe could selec- tumors showed focal 89Zr-Df-bevacizumab uptake while no sig- tively accumulate in tumors with high levels of HGF protein (Fig. nificant uptake was seen in the healthy brain (58). A further 2B and C; ref. 47). study by Veldhuijzen van Zanten and colleagues reported that Our team produced a recombinant human HGF and labeled 89Zr-Df-bevacizumab uptake was correlated with microvascular the agent with 64Cu. PET imaging revealed specific and prominent proliferation in a patient with DIPG and highlighted that 89Zr-Df- uptake of the tracer in c-Met–positive U87MG tumors but signif- bevacizumab PET could be used to delineate intralesional het- icantly lower uptake in c-Met–negative MDA-MB-231 tumors erogeneity (Fig. 2E; ref. 59). These results indicate that 89Zr-Df- (48). One concern for these tracers based on the HGF ligand is bevacizumab immunoPET is feasible in children with DIPG and their potential to stimulate tumor growth by activating c-Met may help select patients with the greatest chance of benefiting (48). Burggraaf and colleagues initially developed a fluorescently from bevacizumab treatment. Notably, 89Zr-Df-bevacizumab labeled peptide (GE-137) for optical imaging of c-Met, and this could also offer a tool to refine clinical management of patients

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Figure 2. Representative c-Met and VEGF pathway–specific PET probes. A,89Zr-Df-onartuzumab is a c-Met targeting PET probe. While in vehicle-treated HCC827 xenografts uptake of 89Zr-Df-onartuzumab did not differ before treatment and after treatment (left), the corresponding uptake of the radiotracer in the NVP-AUY- 922–treated mice decreased more than 30% after treatment (right), indicating that 89Zr-Df-onartuzumab PET may provide a powerful tool for visualizing c-Met dynamics. B, 89Zr-DFO-AMG102 is another HGF/c-Met signaling pathway–specific PET probe. PET maximum intensity projection (MIP) images in HGF and MET double–positive U87MG tumor models at 24 and 120 hours after injection of 89Zr-DFO-AMG102. C, Corresponding MIP images in MKN45 tumor models that had negative HGF and positive MET in the local tumor microenvironment. These results imply that 89Zr-DFO-AMG102 may act as a companion diagnostic tool for selection of patients more likely to respond to any HGF-targeted therapy. D, 89Zr-Df-bevacizumab PET MIP image before everolimus treatment showed multiple local and distant metastases from a left kidney tumor. 89Zr-Df-bevacizumab PET demonstrated that tumor uptake decreased significantly after 6 weeks of treatment using everolimus. E, In vivo performance of 89Zr-Df-bevacizumab PET in the diagnosis and localization of DIPG. Gadolinium-enhanced T1 MR images (top) showed the enhanced primary pontine glioma and metastatic gliomas (white arrows) in the right ventricular trigone and cervicomedullary junction, and all these glioma lesions were clearly visualized by 89Zr-Df-bevacizumab PET (bottom). Panels reproduced with permission from ref. 26, 47, 59, SNMMI and ref. 44, Springer.

with von Hippel–Lindau disease (25), and neuroendocrine Ranibizumab is a mAb Fab derivative of bevacizumab and has a tumors (55). From a clinical perspective, a patient's baseline higher affinity for all soluble and matrix-bound human VEGF-A plasma VEGF-A level is an independent prognostic factor for isoforms than bevacizumab (62). Nagengast and colleagues ini- certain cancer types (60), and resistance to antiangiogenic ther- tially created 89Zr-Df-ranibizumab and reported that VEGF PET apies ultimately leads to patients' relapse and poor survival (53, imaging using 89Zr-Df-ranibizumab allowed serial analysis of 61). Therefore, it would be significant to develop personalized angiogenic changes in ovarian tumor models following treatment therapeutic strategies through immunoPET imaging of angiogen- with the kinase inhibitor sunitinib (63). In addition, several esis biomarkers that might help select proper patients and reflect studies have been performed to image tumor vasculature by the response of tumor cells to antiangiogenic drugs. visualizing VEGFR-2. Meyer and colleagues engineered and

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radiolabeled single-chain VEGF and further validated the feasi- help select proper patients for subsequent therapy and elucidate bility of imaging VEGFR-1 and VEGFR-2 in an orthotopic murine the underlying resistance mechanisms (4, 67–69). tumor model (64). We synthesized and characterized a ramucir- Trastuzumab was the first clinically approved anti-HER2 anti- umab-based PET imaging agent, 64Cu-NOTA-RamAb, for map- body for breast cancer patients. In preclinical studies, trastuzu- ping VEGFR-2 expression in vivo (65), and found that this tech- mab has been used as a targeting agent to map HER2 expression nology could visualize VEGFR-2 in nude mice bearing HCC4006 and distribution either by PET imaging or by SPECT imaging and A549 NSCLC tumor models. Based on our results, a study (4, 13, 70, 71). In addition, 89Zr-Df-trastuzumab PET, rather from Eric and colleagues further suggested that a three-time- than 18F-FDG PET, successfully assessed the pharmacodynamic point method could be used to assess the in vivo performance of effects of afatinib (an EGFR-HER2 dual inhibitor) in HER2- 64Cu-NOTA-RamAb (66). positive gastric cancer models (Fig. 3A and B; ref. 72). In clinical practice, HER2 status has been determined by immunohis- tochemistry, and fluorescence in situ hybridization has been PET Imaging of HER2 validated to predict the efficacy of the HER2-targeting anti- The human epidermal growth factor receptor 2 (HER2) trans- body–drug conjugate trastuzumab emtansine. Because SPECT membrane oncoprotein is overexpressed in many human tumors, imaging using 111In-trastuzumab discovered new HER2-positive and several HER2-targeting agents have entered clinical practice lesions in 13 of 15 patients with breast cancer (10), Dijkers and (4). However, despite this success, responses to these antibodies colleagues developed 89Zr-Df-trastuzumab (13), and reported and small molecules have been hampered by resistance or poor that PET imaging using this probe in breast cancer patients was responses. In this setting, molecular PET imaging techniques can able to detect both previously known metastatic lesions and

Figure 3. Representative HER2-, EGFR-, and HER3-targeting PET probes and their potential clinical applications. A, 89Zr-Df-trastuzumab PET specifically imaged HER2-positive NCI-N87 tumors (red circle). On the contrary, 18F-FDG and 18F-FLT PET nonspecifically detected both the HER2-positive NCI-N87 tumor (red circle) and HER2-negative MKN-74 tumor (black circle). B, Afatinib therapy in HER2-positive xenograft models reduced uptake of 89Zr-Df-trastuzumab in a time- dependent manner, justifying the potential value of 89Zr-trastuzumab PET in measuring the pharmacodynamic effects of afatinib in the clinical setting. C, 64Cu-PCTA- cetuximab PET imaging clearly visualized EGFR-positive TE-8 xenograft models (white dotted circle). D, 177Lu-cetuximab radioimmunotherapy, rather than saline or cetuximab treatment, markedly reduced 18F-FDG uptake in TE-8 models. E, 89Zr-Df-lumretuzumab is a HER3-specific immunoPET probe. PET/CT scanning performed 4 days after injection of 89Zr-lumretuzumab detected one lung metastasis (white arrow) from ampullary cancer. Panels reproduced with permission from ref. 72, 99, SNMMI and ref. 115, AACR.

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lesions which had been previously undetected (73). Recently, 64Cu-NOTA-pertuzumab/89Zr-DFO-pertuzumab are effective 89Zr-Df-trastuzumab PET has been investigated as a useful tool tools for imaging HER2 expression. More recently, a first-in- to predict response in breast cancer patients treated with trastu- human study in patients with HER2-positive breast cancer zumab emtansine, and to improve the understanding of tumor showed that 89Zr-DFO-pertuzumab PET/CT was safe and dem- heterogeneity in breast cancers (14). Indeed, the heterogeneity onstrated optimal imaging 5 to 8 days after administration of within and across tumor lesions in a single patient or among the tracer. Additionally, 89Zr-DFO-pertuzumab PET/CT was different patients is increasingly being recognized, with signif- able to image multiple sites of HER2-positive malignancy icant therapeutic implications (74). In support of this concept, including HER2-positive brain metastases (86). Further clinical Ulaner and colleagues demonstrated that 89Zr-DFO-trastuzu- studies are still needed to validate the efficacies of these two mab PET/CT detected unsuspected HER2-positive metastases in probes, especially in improving patient stratification. patients with HER2-negative primary breast cancer (15, 75). It is Compared with intact antibodies, antibody fragments and quite difficult for conventional biopsy techniques, which gen- single-domain antibodies (sdAb) have superior imaging charac- erally obtain samples from a limited number of lesions, to teristics, such as rapid clearance, high target-to-background ratios, identify these unique cohorts. This exciting finding may facilitate reduced radiation dose, and engineered sites for site-specific better management of HER2-negative breast cancer patients, as conjugation (87, 88). However, compared with clinically avail- about 10% to 15% of patients with HER2-negative primary able antibodies, the immune responses, safety profiles, and effi- breast cancer may still benefit from HER2-targeted therapy cacy of these novel sdAbs in humans are largely unknown. (76). Furthermore, early changes in 89Zr-Df-trastuzumab uptake Positron emitter–labeled nanobodies as probes for evaluating in breast cancer metastases following treatment with heat HER2 status have been reported (89–92). One strength of such shock protein 90 inhibitor NVP-AUY922 correlated with probes, for example, [18F]RL-I-5F7 and [18F]RL-I-2Rs15d (89, 90), changes of lesion size measured on CT images (77). In addition is their ability to rapidly cross the intact blood–brain barrier and to 89Zr-labeled trastuzumab, 64Cu-DOTA-trastuzumab is anoth- detect brain metastases from breast cancers 1 hour after injection er alternative for optimizing trastuzumab treatment (16). of the tracers. In comparison, the best time points for detecting HER2 is overexpressed in EGA and trastuzumab has been brain metastases using either 89Zr-Df-trastuzumab or 89Zr-DFO- approved by the FDA to treat EGA patients with positive HER2 pertuzumab were 4 to 5 days for the former tracer and 5 to 8 days expression (78). Considering the fact that only a subset of patients for the latter following tracer administration (73, 86). Another with HER2-positive EGA respond to trastuzumab (79), O'Dono- strength is the higher tumor-to-blood and tumor-to-muscle ratios ghue and colleagues studied the value of 89Zr-DFO-trastuzumab which will result in higher contrast PET imaging (89, 93). A phase I in assessing HER2 status in primary and metastatic EGA (17), and study from Keyaerts and colleagues showed PET imaging using a reported that this imaging method detected local and metastatic 68Ga-HER2-nanobody is a safe procedure and tracer accumula- lesions in 80% of the imaged patients. This study indicated that tion in HER2-positive breast carcinoma metastases is high, which 89Zr-DFO-trastuzumab PET has superior advantages over single- warrants further assessment in a phase II trial (94). site biopsy, because this noninvasive imaging technology can assess variation in levels of HER2 and target engagement in both the primary and metastatic tumor lesions simultaneously. Nota- PET Imaging of EGFR (HER1) bly, bifunctional chelators have certain impact on the in vitro EGFR and its relatives (i.e., ErbB2/HER2, ErbB3/HER3, and stability and in vivo performance of 89Zr-labeled trastuzumab and ErbB4/HER4) are well-known oncogenic drivers in several types antibody–drug conjugates (80). of cancers such as lung cancer, breast cancer, and glioblastoma. Pertuzumab is another HER2-targeting mAb approved by EGFR was among the first RTKs for which the ligand binding the FDA after a survival benefit was achieved for patients with mechanism was studied (95). Inhibitors for this specific RTK, HER2-positive metastatic breast cancer. Importantly, pertuzumab including antibody therapeutics (e.g., cetuximab and panitumu- binds to a HER2 binding site distinct from that of trastuzumab, mab) and small-molecule TKIs (e.g., erlotinib, gefitinib, lapatinib, augmenting the binding and treatment efficacy of each other (81). and osimertinib), have been the most successful examples of Thus, it is of great importance to noninvasively detect the biodis- molecularly targeted therapies in cancers (95, 96). tribution of pertuzumab-specific binding sites when synergistic Cetuximab is a chimeric EGFR-specific IgG1 mAb and functions regimens containing trastuzumab and pertuzumab are given for by preventing ligand activation and receptor dimerization (97). breast cancer patients. In this setting, Marquez and colleagues It was shown in a phase I first-in-human study that 89Zr-Df- conducted an in vivo study in breast cancer models using 89Zr- cetuximab was safe and well tolerated (20). PET imaging using DFO-pertuzumab and observed significant tracer uptake in MDA- 89Zr-labeled cetuximab may also select NSCLC patients, head and MB-231 xenografts (82). More importantly, pretargeting strate- neck cancer patients, and colorectal cancer patients who will gies using unlabeled trastuzumab could further enhance 89Zr- potentially benefit from cetuximab treatment (18, 19, 98). In DFO-pertuzumab accumulation in tumors. Combination treat- recent years, theranostic approaches using mAbs have represented ments with trastuzumab and pertuzumab have also shown a rapidly expanding component of cancer treatments (12). Song enhanced antitumor effects in xenograft models of human and colleagues prepared 64Cu/177Lu-labeled versions of cetuxi- ovarian cancer (83), and the addition of pertuzumab to che- mab and assessed the theranostic efficacy in an esophageal squa- motherapy in patients with platinum-resistant ovarian carci- mous cell carcinoma model (99). The authors elaborately found noma demonstrated favorable trends in progression-free sur- that 64Cu-PCTA-cetuximab immunoPET evaluated EGFR expres- vival (84). Based these preclinical and clinical data, our group sion levels in tumors, and 177Lu-cetuximab radioimmunotherapy synthesized 64Cu-NOTA-pertuzumab and broadened the appli- effectively inhibited tumor growth much more thoroughly than cation of the probe to detect both subcutaneous and orthotopic that of cetuximab alone (Fig. 3C and D). Thus, the pair of ovarian cancer models (85). These solid data demonstrated that 64Cu/177Lu-cetuximab may provide a tailored treatment strategy

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for EGFR-positive patients by immunoPET imaging of EGFR administration of 64Cu-DOTA-patritumab is safe, the diagnostic expression and by selective therapeutic radiation delivery. efficacy was limited in solid tumors (116). Comparatively, 89Zr- Panitumumab is a fully humanized mAb that binds to EGFR Df-lumretuzumab PET could visualize intra- and inter patient and is FDA approved for use in receptor-expressing colorectal tumor heterogeneity and target accessibility, and the most opti- cancers without KRAS mutations (100). Wei and colleagues mal PET conditions were found to be 4 and 7 days after admin- synthesized 89Zr-DFO-panitumumab and this probe accumulat- istration of 89Zr-Df-lumretuzumab with 100 mg cold antibody ed in various subcutaneous athymic nude female xenograft mod- (Fig. 3E; ref. 115). In the absence of side effects of lumretuzumab els (101). Lindenberg and colleagues then calculated the maxi- in treating HER3-expressing solid tumors (122), 89Zr-Df-lumre- mum dosing for effective imaging with 89Zr-DFO-panitumumab tuzumab may have potential value in guiding lumretuzumab in 3 patients, and results from this study indicated that injection of treatment of patients with HER3-positive solid tumors. More approximately 1 mCi (37 MBq) of 89Zr-DFO-panitumumab recently, Warnders and colleagues constructed a HER3 signal- intravenously was safe and detected lung metastases from colo- ing–specific probe, 89Zr-MSB0010853, and reported that 89Zr- rectal cancer, but imaging of the included 3 patients failed to MSB0010853 PET could detect HER3-overexpressing H441 demonstrate significant radiotracer accumulation in tumors for NSCLC cancer models in vivo (123). Although development of all time points and failed to detect tumors in the liver due to therapeutic HER3 antibodies is still in an early phase (122, 124), physiologic hepatic excretion of the probe (102). Future studies these preliminary results shed light on the feasibility of nonin- may add unlabeled panitumumab to optimize the radiotracer's vasive HER3-specific PET imaging for mapping HER3 expression uptake in tumors (103). More recently, imgatuzumab (GA201), and distribution of HER3 antibodies. a novel humanized anti-EGFR IgG1 isotype mAb, has been foundtoefficiently inhibit EGFR pathway activation in head PET Imaging of Other RTKs and neck cancer patients and may serve as a potential probe for Apart from the above RTKs, there are 17 more RTK subfamilies visualizing EGFR-expressing tumors after radiolabeling with and nearly half of these RTK families are poorly understood (2). isotopes (104, 105). One example is the platelet-derived growth factor receptor-alpha Repebody, a novel nonantibody protein scaffold (106), has (PDGFR-a), which has been reported to be involved in tumor also been engineered to monitor EGFR expression (107, 108). angiogenesis and maintenance of several cancer types including One advantage of repebody-based PET imaging probes is their thyroid cancer (125–127). Importantly, PDGFR-a is associated small molecular weight (30 kDa), which may enable early with lymph node metastases in papillary thyroid carcinoma (PTC; visualization of various EGFR-expressing solid tumors as early ref. 127). Michael and colleagues described a PDGFRa-specific as 1 hour after injection of the probes. Furthermore, due to immunoPET probe—64Cu-NOTA-D13C6—for PTC and demon- superior circulation clearance and tissue penetration, those strated that PET imaging using this agent could delineate PDGFRa probes could be labeled with positron emitters with shorter expression in PTC models in vivo and could therefore potentially half-lives, such as 64Cu (t , 12.7 hours) and 18F(t ,110 1/2 1/2 serve as a novel and promising radiotracer for imaging PDGFRa- minutes; ref. 109). positive metastasis from PTC (128). Another example is the As we previously reviewed (110), PET imaging using bispecific insulin-like growth factor-1 receptor (IGF-1R), which has a peptide- and antibody-based heterodimers may demonstrate well-established role in malignant tumors and has become a higher targeting efficacy and specificity than their monospecific promising target for cancer therapy and imaging (129, 130). As peers. We generated a bispecific immunoconjugate (denoted as R1507 is a mAb directed against IGF-1R, Heskamp and colleagues Bs-F(ab) ) by linking a cetuximab Fab and a TRC105 Fab (target- 2 developed 111In-R1507 and 89Zr-Df-R1507 and reported that ing CD105), and confirmed that 64Cu-NOTA-Bs-F(ab) had a 2 both of the tracers could be applied to noninvasively determine synergistic improvement on both affinity and specificity in visu- IGF-1R expression in vivo in breast cancer xenografts (131). Our alizing small U87MG tumor nodules (<5 mm in diameter; group also developed an IGF-1R–specific PET probe (denoted as ref. 111). Similar results were reported by Kwon and colleagues 89Zr-DFO-1A2G11) and demonstrated highly specific uptake of who developed an immunoconjugate targeting HER2 and EGFR the tracer in IGF-1R–positive pancreatic tumor models (132), and verified its diagnostic efficacy in breast cancer models (112), implying the potential value of this probe in identifying patients and by Mahmood and colleagues who prepared PET probes that may benefit from anti–IGF-1R therapy. specific to EGFR and HER3 and monitored resistance to PI3K In addition, RTKs are important drug targets, and several and protein kinase B (PKB, also known as Akt) inhibitors using small-molecule inhibitors have been developed and approved breast cancer models as well (66). by the FDA in addition to mAbs. Actually, development of radiolabeled TKIs and their analogues is under preclinical and PET Imaging of HER3 clinical translational research (133), and various radiolabeled TKIs have been developed to image tumors in both preclinical Unlike other members of the family, HER3 has relatively weak and clinical studies for several RTKs, such as EGFR (134, 135), kinase activity but still can form highly activated heterodimers HER2 (136), VEGFR (137), tropomyosin receptor kinase (Trk; with EGFR and HER2. Activating mutations in HER3 have been ref. 138), stem cell growth factor receptor (SCFR; ref. 139), and identified in multiple cancer types and therefore HER3 is now IGF-1R (129, 140, 141). being examined as a direct therapeutic target (113). Preclinical and clinical PET imaging has also been developed using anti- HER3 antibodies, such as lumretuzumab (114, 115), patritumab Conclusion and Future Perspectives (116), and mAb3481 (117), or using peptides (118), and affi- Currently, the role of spatial deregulation of RTKs in tumori- bodies (119–121). In a pilot clinical trial that included 11 genesis may be vastly underappreciated, and the development of participants, Lockhart and colleagues reported that although high-resolution immunoPET techniques for detecting RTK

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activity in normal and tumor tissues will be essential for studying demonstrated the success of 68Ga-HER2-nanobody in a clinical the role of spatial RTK patterning in the heterogeneous tumor setting (94), and it is easier for immunoPET probes derived from environment. In this state-of-the-art review, we presented typical sdAbs to traverse the blood–brain barrier (90), future studies may examples of deregulated RTKs in cancers and summarized strat- further design noninvasive means to detect RTKs through the use egies to prepare noninvasive PET imaging probes specific for these of sdAbs (150). targets. The current review demonstrates that noninvasive immu- In the development of antibody-based diagnostic, therapeu- noPET could accurately delineate RTK status within a tumor or tic, or theranostic probes, we should pay attention to the fact across multiple tumors within a patient. Visualization of RTK that the immunodeficient strains of animals we use in preclin- phenotype using immunoPET could facilitate selection of ical studies may impact the in vivo fate and performance of the patients for targeted therapies and response assessment/predic- investigated immunoPET probes. For example, a recent study tion. In addition, a greater understanding of the spatial expression reported that immunoPET radiotracers had inefficient tumor of RTKs with the help of immunoPET could affect ongoing efforts targeting and high off-target binding to the spleen in highly to develop therapeutic drugs targeting RTKs. immunodeficient mouse models (151). This was consistent In 2017, the development of antibody therapeutics pro- with a previous study which reported that antibody–drug ceeded at a fast pace, and this is expected to continue in the conjugates had limited antitumor activity in NSG mice coming years. Several RTK-targeting therapeutic antibodies (152). In this setting, pretargeting strategies may also be har- [such as margetuximab, (vic-)trastuzumab duocarmazine, and nessed for optimizing both the imaging quality and therapy DS-8201 for HER2, and depatuxizumab mafodotin for EGFR] effects in the coming future (151, 153, 154). are undergoing evaluation in late-stage clinical studies of Besides assessing RTKs and selecting proper patients for patients with cancer (142). For mAb-based immunoPET subsequent molecularly targeted therapy using either small probes, 89Zr is utilized in clinical trials much more extensively molecules or antibodies, noninvasive imaging of RTKs could than any other radiometals. DFO and DFO-based bifunctional facilitate image-guided radionuclide therapy. Actually, therapy chelators are the most commonly used chelators for 89Zr- of B-cell lymphomas with radiolabeled mAbs has produced radiolabeling (143), and there are two simple protocols that impressive clinical results because two radiolabeled anti-CD20 canbefollowedtodo89Zr radiolabeling (144, 145). However, antibodies, 131I-tositumomab and 90Y-ibritumomab tiuxetan, DFO is not an optimal chelator for 89Zr coordination because were approved by FDA more than a decade ago (155, 156). of 89Zr transchelation, which will cause high bone uptake of RTKs are ideal candidates for investigating and performing 89Zr. This was exemplified by a recent study by Vugts and radioimmunotherapy (157, 158). Therefore, radiolabeled anti- colleagues, in which the authors reported that, when compared bodies or antibody fragments targeting markers included in the with DFO, the bifunctional isothiocyanate variant of desfer- current review and other potential neoantigens may provide rioxamine (denoted as octadentate DFO) was advantageous another therapeutic option for patients with cancer in the era of for 89Zr labeling because 89Zr-DFO-trastuzumab had molecularly targeted therapy and precision medicine. enhanced stability in in vitro studies and superior performance We firmly believe that immunoPET will allow for better cancer in in vivo studies over 89Zr-DFO-trastuzumab (80). Therefore, patient management in the clinical setting with the fast develop- there is still room for future studies to optimize radiolabeling ment of this field. Considering most data with immunoPET strategies when developing 89Zr-immunoconjugates (143). probes have been generated in preclinical stages or in small groups With the advent and maturation of click chemistry (146), future of patients, further studies are still needed to push clinical trans- studies may further harness this powerful method to synthesize lation of some of these promising probes and to confirm the value and assess antibody-based PET imaging probes with improved of clinically reported probes. in vivo performance. In addition to singly targeted imaging probes, antibody-based heterodimers or dual-targeting probes Disclosure of Potential Conflicts of Interest may have higher targeting efficacy and superior specificity than No potential conflicts of interest were disclosed. their corresponding monospecific peers (110, 147). Generally, adequate tissue penetration ability, which is inverse- Acknowledgments ly proportional to the size of an imaging probe, and high signal- This work was partially sponsored by the Ph.D. Innovation Fund of Shanghai to-noise ratio, which is closely related to the circulation of a probe, Jiao Tong University School of Medicine (No. BXJ201736) and the China are two important properties of an immunoPET probe. sdAbs, Scholarship Council (No. 201706230067) to W. Wei, the Shanghai Key Dis- also known as nanobodies or heavy chain–only antibodies bear- cipline of Medical Imaging (No. 2017ZZ02005) to Q.Y. Luo, the American Cancer Society (125246-RSG-13-099-01-CCE), and the NIH (P30CA014520) ing a variable region (VHH), hit the sweet spot and have emerged to W. Cai. E.B. Ehlerding was partially sponsored by the NIH (T32CA009206 as substitutes for their full-size counterparts in diagnostic or and T32GM008505). therapeutic applications (87). Enzymatic methods such as sortase have been used to append metal chelators to enable labeling Received February 5, 2018; revised April 19, 2018; accepted June 4, 2018; sdAbs with 64Cu or 89Zr (148, 149). Because a first-in-human trial published first August 1, 2018.

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1636 Mol Cancer Ther; 17(8) August 2018 Molecular Cancer Therapeutics

Weijun Wei, Dalong Ni, Emily B. Ehlerding, et al.

Mol Cancer Ther 2018;17:1625-1636.

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