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Review Theranostic applications of antibodies in oncology

Emmy D.G. Fleurena,*, Yvonne M.H. Versleijen-Jonkersa, Sandra Heskampb, Carla M.L. van Herpena, Wim J.G. Oyenb, Winette T.A. van der Graafa, Otto C. Boermanb aDepartment of Medical Oncology, Radboud University Medical Centre, Nijmegen, The Netherlands bDepartment of Nuclear Medicine, Radboud University Medical Centre, Nijmegen, The Netherlands

ARTICLE INFO ABSTRACT

Article history: Targeted therapies, including antibodies, are becoming increasingly important in cancer Received 14 January 2014 therapy. Important limitations, however, are that not every patient benefits from a specific Accepted 10 March 2014 antibody therapy and that responses could be short-lived due to acquired resistance. In Available online 21 March 2014 addition, targeted therapies are quite expensive and are not completely devoid of side- effects. This urges the need for accurate patient selection and response monitoring. Keywords: An important step towards personalizing antibody treatment could be the implementation Theranostics of theranostics. Antibody theranostics combine the diagnostic and therapeutic potential of Antibody an antibody, thereby selecting those patients who are most likely to benefit from antibody Personalized medicine treatment. This review focuses on the clinical application of theranostic antibodies in Biomarker oncology. It provides detailed information concerning the suitability of antibodies for Molecular imaging theranostics, the different types of theranostic tests available and summarizes the efficacy of theranostic antibodies used in current clinical practice. Advanced theranostic applications, including radiolabeled antibodies for non-invasive functional imagining, are also addressed. Finally, we discuss the importance of theranostics in the emerging field of personalized medicine and critically evaluate recent data to determine the best way to apply antibody theranostics in the future. ª 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction speciﬁcally interfere with aberrant targets or pathways of cancer cells, which is in contrast to the generalized cytotoxic ef- Targeted therapies are becoming increasingly important for fects of standard chemotherapy (Teng et al., 2013). The two the treatment of cancer. These therapies are designed to main types of targeted therapy are the monoclonal antibodies

* Corresponding author. Department of Medical Oncology (internal postal code: 452), Radboud University Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: þ31 (0)24 3618897; fax: þ31 (0)24 3540788. E-mail address: [email protected] (E.D.G. Fleuren). http://dx.doi.org/10.1016/j.molonc.2014.03.010 1574-7891/$ e see front matter ª 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. 800

Table 1 e Registered unconjugated therapeutic antibodies for cancer treatment.

Antibody Antibody type Target antigen Antigen category FDA-approved indicationsa Theranostic antibody test

Trastuzumab (Herceptin) Humanized HER2 Growth factor Breast cancer (HER2-positive); as single agent or HER2 IHCc (e.g. HercepTest) combined with chemotherapy for adjuvant or palliative treatment Gastric or gastro-oesophageal junction carcinoma (HER2-positive); as first-line treatment in combination with cisplatin and capecitabine or 5-fluorouracil OEUA NOOY8(2014) 799 8 ONCOLOGY MOLECULAR Cetuximabb (Erbitux) Chimeric EGFR Growth factor Head and neck cancer; combined with radiation Colorectal cancer: EGFR IHCc therapy for initial treatment of locally or regionally (e.g. EGFR pharmDx kit) advanced disease or as single agent for patients who failed prior platinum-based therapy Colorectal cancer (EGFR-positive); palliative treatment of pretreated metastatic disease Panitumumabb (Vectibix) Human EGFR Growth factor Colorectal cancer (EGFR-positive); as single EGFR IHCc (e.g. EGFR agent for pretreated metastatic disease pharmDx kit) Bevacizumab (Avastin) Humanized VEGF Vascular target Colorectal cancer; for first-line and second-line No (under development) treatment of metastatic disease, in conjunction with 5-fluorouracil-based chemotherapy Non-small cell lung cancer (NSCLC); first-line treatment of advanced disease, in combination with carboplatin and paclitaxel, in patients who have not yet received chemotherapy Glioblastoma; as single agent in adult patients for second line treatment Renal cell carcinoma (RCC); in conjunction e

with IFNa to treat metastatic disease 812 Rituximab (Rituxan) Chimeric CD20 Hematopoetic Non-Hodgkin’s lymphoma (NHL) (CD20-positive); NHL: CD20 ELISA/ differentiation for B cell NHL and maintenance therapy for ﬂow cytometry antigen untreated follicular NHL Chronic lymphocytic leukemia

EGFR: Epidermal Growth Factor Receptor; VEGF: Vascular Endothelial Growth Factor, IHC: immuno-histochemistry, ELISA: enzyme-linked immunosorbent assay. a Based on cancer.gov (January 2014) and (Scott et al., 2012). b Not recommended for colorectal cancer patients whose tumors express mutated KRAS. c In addition to IHC, the predictive value of HER2 and EGFR mRNA expression or gene amplification as measured by polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH) has also been studied in the clinic. Because this review focuses specifically on the theranostic applications of antibodies in oncology, we did not further consider these applications. Table 2 e Conjugated therapeutic antibodies for cancer treatment.

Antibody Target antigen Antibody type Conjugate type Emittera Indicationsb,c Theranostic antibody test

90 Y-Ibritumomab CD20 Murine Radionuclide b FDA: non-Hodgkin’s lymphoma (NHL); Safety: biodistribution and dosimetry 111 tiuxetan (Zevalin) (yttrium-90) for relapsed or refractory, low-grade or prior to therapy ( In- Ibritumomab tiuxetan) (2014) 799 8 ONCOLOGY MOLECULAR follicular B cell NHL or previously untreated follicular NHL in patients achieving a partial or complete response to first-line chemotherapy 131 I-Tositumomab CD20 Murine Radionuclide b and g FDA: NHL (CD20-positive); for relapsed CD20 ELISA/flow cytometry (Bexxar) (iodine-131) or refractory, low-grade, follicular or Safety: biodistribution and dosimetry transformed NHL prior to therapy (low-dose Bexxar) 177 Lu/90 Y -J591 PSMA Humanized Radionuclide b and g/b Research: prostate cancer Research: 111 In-J591 for (lutetium-177/yttrium-90) response prediction 177 Lu eGirentuximab CAIX Chimeric Radionuclide b and g Research: renal cell carcinoma (RCC) Research: 111 In-Girentuximab (cG250) (lutetium-177) for response prediction Gemtuzumab CD33 Humanized Toxin (calicheamicin) n.a. FDA withdrawn: acute myeloid leukemia CD33 ELISA/flow cytometry ozogamicin (Mylotarg) (AML) (CD33-positive); for relapsed patients >60 years not suited for other chemotherapy Trastuzumab emtansine HER2 Humanized Toxin (emtansine) n.a. FDA: breast cancer (HER2-positive); HER2 IHC (e.g. HercepTest) (T-DM1, Kadcyla) metastatic Research: 89 Zr-trastuzumab

to exclude non-responders e 812 a b-emission is solely used for radioimmunotherapy, while g-emission can be used for imaging. b FDA-approved indications are speciﬁcally pointed out (FDA), others are under clinical investigation (research). c Based on cancer.gov (January 2014) and (Scott et al., 2012). 801 802 MOLECULAR ONCOLOGY 8 (2014) 799e812

Table 3 e Characteristics of theranostic antibody tests.

Theranostic antibody assay Standard use Advantages Disadvantages

Immunohistochemistry Solid tumors Simple to perform Requires invasive biopsies Low costs No whole body target expression Routinely available No information about target accessibility Variability (tissue handling/fixation) Semi-quantitative Subjective interpretation Membranous expression often hard to define Enzyme-linked Hematological Minimally invasive No whole body immunosorbent tumors (blood sampling) target expression assay (ELISA) Simple to perform No information about Low costs target accessibility Routinely available Subjective interpretation Can define (arbitrary thresholds) membranous expression Quantitative Flow cytometry Hematological Minimally invasive No whole body tumors (blood sampling) target expression Relatively simple to perform No information about Relatively low costs target accessibility Available in most laboratories Subjective interpretation Can define (arbitrary thresholds) membranous expression Very accurate Quantitative Functional imaging Solid and Minimally invasive Specific equipment required (immuno-PET/ hematological (IV injection) Specific expertise required immuno-SPECT) tumors Can define High costs membranous expression Tracers must be prepared Very accurate shortly before injection Quantitative Whole body target expression Target accessibility taken into account Can be performed repetitively

PET: positron emission tomography, SPECT: single photon emission computed tomography, IV: intravenous.

(mAbs) and tyrosine kinase inhibitors (TKIs). The major ad- This review focuses on the clinical application of theranos- vantages of these targeted molecules are their tumor speci- tic antibodies in oncology. It provides detailed information ficity and low toxicity profile. Important limitations, concerning antibody suitability for theranostics, the different however, are that (i) not every patient benefits from a specific types of theranostic tests available and summarizes the effi- targeted treatment and (ii) responses could be short-lived due cacy of theranostic antibodies currently used in clinical prac- to acquired resistance (Gerber, 2008). This urges the need for tice. In addition, more advanced theranostic applications, accurate patient selection and response monitoring. including the use of radiolabeled antibodies are addressed. A promising strategy towards improving personalized Finally, we discuss the importance of theranostics in the medicine is the implementation of theranostics. Theranostics emerging field of personalized medicine and critically eval- combine the therapeutic (“thera”) and diagnostic (“nostic”) po- uate recent data to determine the best way to apply antibody tentials of a certain compound. Prior to therapy, the com- theranostics in the future. pound is used in a diagnostic test to determine whether the drug will (potentially) exert a therapeutic effect, making it a powerful tool for personalizing cancer treatment. Because it 2. Antibodies used for theranostic applications operates on the molecular level, it could be more accurate for response prediction and/or monitoring than more general To date, several mAbs have been approved by the Food and tumor characteristics such as tumor morphology (e.g. CT and Drug Administration (FDA) for the treatment of cancer. Anti- MRI) or metabolism (e.g. 18F-FDG-PET). bodies specifically target antigens expressed on the cell MOLECULAR ONCOLOGY 8 (2014) 799e812 803

tumor progression. But even when this is the case, tumors only rarely depend on a single regulatory pathway for growth and survival because of their molecular complexity and heterogeneity. Parallel activation of other receptors or mutations in downstream pathways are not rare events. This could at least in part explain the lack of response in tumors with apparent oncogenic target expression (De Palma and Hanahan, 2012). Because physiological factors (e.g. vasculari- zation, hypoxia, intratumoral pressure) can impede adequate antibody targeting to the tumor, these should also be taken into account when predicting response (Heldin et al., 2004; Jain, 1999). In addition, the antibody Fc-portion can evoke an additional anti-tumor response by triggering antibody- dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) processes. This underscores the complexity of finding a suitable predictive biomarker and indicate that theranostic applications should be interpreted with caution. Not every overexpressed membranous tumor protein is a Figure 1 e Whole body biodistribution of a radiolabeled antibody. potential candidate for treatment with unconjugated anti- Whole-body coronal PET/CT images of 124I-girentuximab, acquired bodies as functional involvement is also required. Neverthe- 5 days after antibody infusion. The specific uptake of the antibody can less, high target expression on tumor cells may be sufficient be seen in the left renal tumor (arrow), which is expressing the CAIX for predicting response for conjugated antibodies. However, antigen. The concentration of the radiolabeled antibody in a tumor also with conjugated antibodies, target accessibility should can be determined quantitatively. Left: CT, middle: PET, right: be taken into account and for these constructs it is of utmost fused PET/CT. This figure was originally published in Nat Rev importance to also determine target expression levels on non- Cancer; reprinted with permission (Scott et al., 2012) (adapted). tumor tissue to avoid side-effects. membrane or ligands (e.g. bevacizumab, Section 4.3) and can 3. Types of theranostic antibody tests either be administered unconjugated (Table 1) or conjugated to cytotoxic moieties such as radionuclides or toxins to in- Because target expression is usually a prerequisite for crease efficacy (Table 2)(Gerber, 2008). Because target expres- response, it seems a logical first step to base patient selection sion is usually a prerequisite for antibody response, one could on target expression. Although several comments can be base patient selection on the expression of the target antigen made on this design (e.g. involvement of other pathways), (Fleuren et al., 2011; Heskamp et al., 2013b). However, for detection of the target protein is expected to at least exclude many therapeutic antibodies mere target expression appeared primarily resistant patients. At present, several different diag- not sufficient to predict response in all patients. For unconju- nostic methods exist to screen for target expression, which all gated or naked antibodies it is essential that, in addition to its have their specific advantages and limitations as discussed presence on the tumor, the target antigen is also involved in below and summarized in Table 3.

Figure 2 e Heterogeneous distribution of 124I-girentuximab in RCC lesions. (A and B) Axial, sagittal, and coronal CT (top), PET (middle), and fused PET/CT (bottom) images of a patient with clear cell renal cancer with relatively homogeneous intratumoral distribution of the radiolabeled antibody (arrows) (A) and a patient with large, centrally necrotic clear cell renal cancer with marked heterogeneity (arrows) (B). (C) Axial CT (top), PET (middle), and fused PET/CT (bottom) images of a patient with advanced clear cell renal cancer. Antibody distribution within the primary tumor is heterogeneous (arrows), whereas distribution within metastatic nodes is relatively homogeneous (arrowheads). This ﬁgure was originally published in JNM; reprinted with permission (Pryma et al., 2011) (adapted). 804 MOLECULAR ONCOLOGY 8 (2014) 799e812

Figure 3 e 111In-bevacizumab imaging in a RCC patient before and after anti-angiogenic treatment. Anterior and posterior 111In-bevacizumab scans at baseline (A) and after 4 weeks of treatment with the TKI sorafenib (angiogenesis inhibitor; 400 mg twice daily) (B). A marked decrease of 111In-bevacizumab uptake is observed (arrows). This ﬁgure was originally published in JNM; reprinted with permission (Desar et al., 2010) (adapted).

3.1. Immunohistochemistry antigen expression in single cells from a suspension. For biomarker detection with flow cytometry, antibodies can be Immunohistochemistry (IHC) is currently the most commonly used to determine the expression of a particular antigen on used procedure to perform antibody theranostics for solid tu- blood cell subtypes (Brown and Wittwer, 2000). A well- mors. The antibody can be used to detect target expression on known example of a hematological theranostic assay is tumor tissue by IHC. When the section is scored positive for CD20 testing by ELISA or flow cytometry prior to rituximab target expression, the same antibody can subsequently be therapy (Section 4.4). ELISAs are in general quick and easy to used for therapy. A well-known example of this approach is perform, readily available, can define membranous expres- the HercepTest (Section 4.1). IHC has the advantages of being sion levels and can be analyzed quantitatively. Flow cytome- simple to perform, cheap, and readily available in most pa- try measures single cells, is quantitative, very accurate and thology laboratories. There are however numerous variables, can measure specific membranous target expression. Disad- such as tissue handling and fixation and antibody sensitivity vantages of both techniques include the subjective interpreta- and specificity. Because slides are not scored by automated tion of results due to arbitrary threshold settings, so there is computer systems, inter-observer interpretation and vari- no straightforward difference between positive and negative ability can also affect the accuracy of test results (Cuadros samples. Also with these techniques, only a fraction of tumor and Villegas, 2009). Because antibodies can only target cells is measured thus no whole body tumor expression extracellularly-expressed antigens, it would be appropriate pattern is determined. to specifically screen for membranous expression. This is however difficult to determine by conventional IHC, often 3.3. Immuno-PET/immuno-SPECT due to high cytoplasmic receptor expression. Other limitations include the lack of information obtained about in vivo For both solid and hematological tumors, target expression target accessibility and the need for invasive biopsies. More- levels can be determined in vivo using molecular imaging over, receptor expression is determined on a limited number methods, such as immuno-positron emission tomography of sections of a single biopsy, often formalin-fixed paraffin- (PET) and immuno-single photon emission computed tomog- embedded (FFPE) archival tissue. Because tumors are usually raphy (SPECT). For both techniques, an antibody is labeled very heterogeneous, patients often present with multiple le- with a radionuclide (positron or g-emitter) and injected intra- sions and target expression levels can change over time (for venously. The 3D distribution of the radiolabeled antibody is instance during therapy), multiple biopsies would be required subsequently visualized with a SPECT or PET scanner. With for adequate antigen testing, which in most cases is clinically these molecular imaging techniques, several limitations of unfeasible. conventional IHC and serum assays can be circumvented. Ad- vantages include non-invasive screening and visualization of 3.2. Hematological analysis specific membranous target expression. Moreover, these whole body scans enable target visualization in the whole tu- For hematological cancers, immuno-assays are commonly mor, in multiple lesions simultaneously and take target acces- used for theranostic approaches, such as enzyme-linked sibility into account (Figures 1 and 2). These scans can be immunosorbent assay (ELISA) or flow cytometry. An ELISA performed repetitively to asses target modulation over time, uses antibodies to determine the amount of antigen in serum, for instance during (targeted) treatment. Because theranostic plasma or other body fluids (Kenny and Dunsmoor, 1983). molecular imaging approaches are relatively new, they have Flow cytometry is a laser-based technology measuring not yet been implemented as standard diagnostic tests. MOLECULAR ONCOLOGY 8 (2014) 799e812 805

Results from clinical trials are however encouraging, as for instance shown with indium-111 (111In)-labeled-girentuximab imaging prior to lutetium-177 (177Lu)-girentuximab radioimmunotherapy (RIT) (Section 5.3). Disadvantages include the use of speciﬁc equipment and expertise, tracers should be prepared shortly before injection and costs are higher than with conventional methods (Heskamp et al., 2013b).

4. Theranostic antibodies in current clinical practice

4.1. Trastuzumab (Herceptin)

One of the most widely used theranostic approaches in clinical practice is the HercepTest to determine HER2 expression levels in breast cancer (BC) prior to trastuzumab therapy. HER2, also known as Neu or ErbB2, is a member of the epidermal growth factor receptor (EGFR) family and plays an important role in BC pathogenesis. HER2 is overexpressed in 20e30% of all BCs and trastuzumab showed activity only against HER2-overexpressing tumors. An increasing number of studies suggest that HER2 status is more important than Figure 4 e 111In-R1507 immuno-SPECT and immunohistochemical the histologic BC type (Cuadros and Villegas, 2009; Heskamp IGF-1R expression in mice with subcutaneous bone sarcoma et al., 2013b). Notably, trastuzumab would not have been xenografts. 111In-R1507 immuno-SPECT/CT scans (A) and IGF-1R approved for BC treatment if studies were performed upon a expression levels of corresponding tumors (B) in mice with bone general population of BC patients without prior identification sarcoma xenografts. Xenografts with high (OS-1), intermediate (EW- of HER2 expression. HER2 is also overexpressed in subsets of 5), or no (EW-8 and OS-33) response to treatment with the IGF-1R ovarian, lung, gastric and oral cancers, and all newly diag- antibody R1507 were selected. Images were acquired 3 days post nosed gastric cancers are also tested for HER2 by IHC to select injection of 111In-R1507. This figure was originally published in Clin patients eligible for trastuzumab therapy (de Mello et al., 2013; Cancer Res; reprinted with permission (Fleuren et al., 2011) (adapted). Meric-Bernstam and Hung, 2006). Unfortunately, however, only 30% of HER2-positive BCs actually respond to trastuzumab therapy. This may be explained by either inaccurate HER2 assessments or that these tumors not strictly depend for EGFR a diagnostic IHC test (EGFR pharmDX Kit) was devel- on HER2-mediated growth signaling, for instance due to muta- oped and approved to aid in identifying colorectal cancer tions in downstream pathways (e.g. PI3K). Furthermore, HER2 (CRC) patients eligible for cetuximab or panitumumab treat- expression can differ between primary tumors and metasta- ment (Ensinger and Sterlacci, 2008). However, although EGFR ses, and this technique does not take target accessibility into is broadly expressed in metastatic CRC (mCRC), only 10% of account (De Palma and Hanahan, 2012; Heskamp et al., 2013b). patients respond to cetuximab. In those tumors with apparent Consequently, several approaches have been developed to EGFR expression but no response to cetuximab or panitumu- determine HER2 expression levels in vivo, with promising re- mab, additional signal transduction pathways can be active. sults so far. Several clinical studies indicated that 111In-trastu- Clinical studies indicated that tumors harboring mutations zumab SPECT and copper-64 (64Cu)-labeled or zirconium-89 in KRAS, a downstream effector protein in the EGFR- (89Zr)-labeled trastuzumab PET are feasible to identify HER2- signaling cascade, show negligible responses to cetuximab positive lesions in patients with primary and metastatic BC despite elevated EGFR levels. Consequently, current clinical (mBC) (Dijkers et al., 2010; Perik et al., 2006; Tamura et al., practice in CRC is to screen for KRAS mutations, and only 2013). Although the exact predictive value of these imaging KRAS wildtype tumors are considered for EGFR-targeted ther- modalities for trastuzumab response remains to be investi- apies (De Roock et al., 2010). Other studies further demon- gated, they have great potential. In mice, 111In-pertuzumab strated that the effect of cetuximab is independent of the (a HER2 dimerization inhibitor) SPECT sensitively imaged degree of EGFR expression in the tumor. Remarkably, cetuxi- HER2 downregulation during trastuzumab treatment in an mab even showed activity in EGFR-negative CRC patients in vivo human BC model, illustrating the feasibility of moni- (Chung et al., 2005; Han, 2011). Thus, EGFR overexpression is toring HER2 expression levels during HER2-mediated treat- not a reliable predictor of EGFR-antibody efficacy. One poten- ment (McLarty et al., 2009). tial explanation for this discrepancy could simply be inconsistent methodology and interpretation of EGFR IHC expression 4.2. Cetuximab (Erbitux) and panitumumab (Vectibix) (section 3.1.). Several studies reported variability in EGFR staining depending on the tissue fixation technique used, Cetuximab and panitumumab both target EGFR, also known possibly leading to false-negative samples (Liu and as HER1. EGFR plays an important role in proliferation, migra- Carpenter, 1993; Ong et al., 1990; Stein and Staros, 1996). tion and survival of tumor cells. Similar to HER2-testing, also Also, most EGFR analyses have been performed on archival 806 MOLECULAR ONCOLOGY 8 (2014) 799e812

primary tumor specimens that may have been kept for survival (PFS) and/or overall survival (OS) upon bevacizumab months to years before assessing EGFR levels. A dramatic treatment (Lambrechts et al., 2013). Also in advanced gastric decline in EGFR staining intensity has been reported with cancer, patients with high baseline plasma VEGF-A levels increased storage time of tissue samples, resulting in many exhibited improved OS compared to patients with low VEGF- more EGFR-negative scores in older specimens (Atkins et al., A levels (Van Cutsem et al., 2012). In randomized CRC, NSCLC, 2004). An additional concern is that EGFR expression in meta- and RCC studies, no such correlation was observed, possibly static tumor specimens can differ significantly from EGFR due to differences in sampling techniques (Lambrechts et al., expression in the corresponding primary tumor (Johnson 2013). These results indicate that circulating VEGF levels et al., 1997). Because of tissue availability, the primary tumor may be of importance towards predicting bevacizumab is frequently used to establish the patient’s EGFR status, but response, but stress the importance of applying the appro- it is the metastases that are being treated with cetuximab priate methodology to assess target expression levels. (Chung et al., 2005). Several studies investigated the feasibility to visualize tu- Also for EGFR, several attempts were made to visualize this mor VEGF expression in patients using molecular imaging receptor in vivo with molecular imaging techniques. Promising techniques, and some even tried to link this to bevacizumab results were obtained with 111In-cetuximab SPECT and 64Cu- (or other angiogenesis inhibitors) response (Figure 3). 111In- cetuximab and 89Zr-cetuximab PET in in vivo cancer models, bevacizumab SPECT visualized VEGF expression in various although liver and skin uptake was also observed (Aerts malignancies, including CRC, RCC and melanoma and 89Zr- et al., 2009; Corcoran and Hanson, 2013; Hoeben et al., 2011). bevacizumab PET visualized primary BC (Desar et al., 2010; Moreover, skin toxicity appeared a surrogate marker for Gaykema et al., 2013; Nagengast et al., 2011; Scheer et al., cetuximab activity, implying an anti-tumor effect when skin 2008). There was however no straightforward correlation be- EGFR saturation occurs (Burtness et al., 2005). Accordingly, tween radiolabeled bevacizumab uptake and tumor VEGF-A with 111In-cetuximab SPECT in lung carcinoma patients, tu- expression as determined by conventional methods (in situ mors were only imaged when higher doses of cetuximab hybridization/ELISA), suggesting that other factors play a were administered (Divgi et al., 1991). A phase I trial investi- role in bevacizumab targeting, such as vascular volume, gating the predictive value of 89Zr-cetuximab PET to cetuxi- vascular permeability and/or interstitial fluid pressure. In mab therapy in stage IV cancer patients was initiated melanoma patients, bevacizumab treatment resulted in a recently, including a loading dose of 400 mg/m2 cetuximab decrease in 111In-bevacizumab tumor targeting, which could (NCT00691548). Another recent study investigates the predic- be due to both therapeutic VEGF-blocking and bevacizumab- tive value of 89Zr-cetuximab PET prior to chemoradiation induced vascular changes, since bevacizumab-induced with cetuximab or cisplatin in head and neck cancer patients vascular changes can hamper tumor uptake of antibodies (Heukelom et al., 2013)(NCT01504815). (Arjaans et al., 2013; Heskamp et al., 2013a; Nagengast et al., 2011). Although these results indicate that bevacizumab imag- 4.3. Bevacizumab (Avastin) ing might aid in optimizing bevacizumab therapies, to date no studies have been performed to determine whether bevacizu- Bevacizumab is an antibody directed against Vascular Endo- mab imaging is predictive to bevacizumab therapy. thelial Growth Factor-A (VEGF-A), which plays a central role in inducing the formation of new blood vessels (angiogenesis). 4.4. Rituximab (Rituxan) VEGF is a ligand that specifically binds to the VEGF receptor (VEGFR). Bevacizumab is currently approved (alone or com- Rituximab was the first therapeutic mAb approved by the FDA bined with other drugs) for the treatment of mCRC, non-small for the treatment of cancer. Rituximab is directed against the cell lung cancer (NSCLC), renal cell carcinoma (RCC) and recur- cell surface marker CD20, which is expressed on B-lympho- rent glioblastoma. Response to bevacizumab is also observed cytes and approved for the treatment of non-Hodgkin’s lym- in only a subset of patients and the overall clinical benefit is phoma (NHL). Beneficial effects have also been reported in limited. Consequently, extensive clinical biomarker studies chronic lymphocytic leukemia (CLL) in combination with have been performed (Lambrechts et al., 2013). Many studies chemotherapy (Okroj et al., 2013). Also for rituximab, patients addressed the possible predictive role of circulating VEGF-A are selected based on CD20 expression levels, which can be levels by conventional methods, but inconsistent results measured by ELISA or flow cytometry. Again, its predictive were found. In NSCLC, breast, ovary, and endometrium cancer value is not straightforward (Smith, 2003; Tran et al., 2010). studies response rates in patients with high VEGF-A levels While rituximab is effective in more than 50% of CD20- were significantly higher in the bevacizumab arm than in positive follicular NHL patients, it is less effective in other the placebo arm. Many other studies however failed to CD20-positive lymphoma subtypes, with activity in only observe such an effect (Lambrechts et al., 2013). A recent 10e15% of CD20-expressing small lymphocytic lymphomas extensive meta-analysis evaluating 1816 patients from phase (McLaughlin et al., 1998). This may partially be explained by III CRC, NSCLC, and RCC trials confirmed that pretreatment interpretation of diagnostic results. For example, a positive VEGF-A levels are a prognostic rather than a predictive marker flow cytometric sample is typically determined as having (Hegde et al., 2013). However, a novel ELISA with a preference >30% of cells above the cut-off. Thus even in a CD20- to detect short VEGF-A isoforms could be superior for positive lymphoma, many cells may have low or no expres- response prediction. mBC and pancreatic cancer patients sion. No or loss of CD20 expression is associated with rituxi- with high baseline VEGF-A expression levels, as measured mab resistance (Sugimoto et al., 2009). CLL and small with this novel ELISA, exhibited improved progression-free lymphocytic lymphoma have faint CD20 staining, MOLECULAR ONCOLOGY 8 (2014) 799e812 807

corresponding to a lower rituximab response than follicular xenografts in mice, showing the feasibility of MET-imaging lymphoma. Mantle cell and diffuse-large-cell lymphoma, and encouraging further clinical (biomarker) studies (Hay however, have similar or even higher CD20 expression levels et al., 2003; Perk et al., 2008). than follicular lymphoma, but lower response rates, thus staining intensity alone does not predict response (Smith, 2003). Therefore, other mechanisms must be involved affecting rituximab response, although the exact underlying 5. Conjugated theranostic antibodies in current resistance mechanisms are yet unknown. clinical practice Rituximab has been labeled with various radioactive com- pounds for either imaging or therapeutic RIT (Section 5.1), 5.1. 90Y-Ibritumomab tiuxetan (Zevalin) and 131I- depending on the radioisotope used. Iodine-131 (131I)-labeled Tositumomab (Bexxar) rituximab SPECT visualized CD20-positive cells in NHL patients, although these studies primarily focused on hemato- Zevalin and Bexxar are, like rituximab, antibodies directed logic toxicity due to subsequent rituximab-RIT (Section 5.1) against the CD20 antigen (Section 4.4) and approved for NHL (Boucek and Turner, 2005; Leahy et al., 2006). In four patients treatment. Unlike rituximab, these are murine antibodies with highly CD20-positive primary central nervous system and conjugated to therapeutic b-emitting radionuclides lymphomas (PCNSL), iodine-123 (123I)-labeled rituximab (yttrium-90 (90Y) and 131I, respectively) for RIT. Although there SPECT revealed very low tumor accumulation up to 48 h are differences between Zevalin and Bexxar, such as different post-injection, most likely because rituximab cannot pass murine mAbs, radioisotopes and in vivo behavior, they share the blood brain barrier (BBB), thus these tumors are unlikely similar toxicity and efficacy profiles (Nowakowski and to respond to rituximab treatment despite apparent CD20 Witzig, 2006). Zevalin or Bexxar RIT is combined with unla- expression (Dietlein et al., 2005). This exemplifies the impor- beled rituximab treatment to prevent excessive uptake of tance of target accessibility instead of solely investigating the radiolabeled antibodies in the spleen and bone marrow. target expression levels with conventional methods. Although One advantage of RIT over regular immunotherapy is its ca- there is at present no clinical study specifically linking pre- pacity to actually kill tumor cells instead of solely inhibiting treatment radiolabeled-rituximab tumor uptake levels to rit- proliferation. Secondly, the radioisotope delivers radiation uximab therapy response, clinical imaging results so far not only to tumor cells binding the antibody, but also to neigh- indicate that molecular imaging approaches could be more boring cells inaccessible to the antibody or with insufficient predictive of response than conventional methods. antigen expression (cross-fire effect). The additive efficacy of Zevalin and Bexxar RIT over unconjugated rituximab has 4.5. Antibodies against novel targets been demonstrated in various studies. In a randomized con- trol trial in patients with recurrent NHL, Zevalin RIT was Two novel promising targets for various cancers are the well-tolerated and had a higher rate of overall and complete Insulin-like Growth Factor-1 Receptor (IGF-1R) and MET responses compared to rituximab alone (Witzig et al., 2002b). (Fleuren et al., 2013; Pollak, 2012). Numerous clinical trials Zevalin RIT was also effective in rituximab-refractory NHL pa- have been performed with various IGF-1R antibodies in carci- tients (Witzig et al., 2002a). Similar to rituximab, patients nomas and sarcomas. Unfortunately, despite some spectac- eligible for Zevalin or Bexxar treatment are primarily selected ular responses, the majority of patients are resistant to IGF- based on CD20 expression levels assessed by conventional 1R-targeted antibody therapy (Basu et al., 2011). However, methods. However, particularly for RIT, molecular imaging these studies were performed upon a heterogeneous group seems more appropriate, not only for assessing lymphoma of patients without prior identification of a predictive targeting but also for evaluating the distribution of CD20- response biomarker. Nevertheless, IGF-1R expression as positive cells in non-tumor tissue (e.g. bone marrow) to esti- determined by IHC cannot be an accurate predictor of mate toxicity levels. Because 90Y is a pure therapeutic b- response, since in Ewing sarcomas IGF-1R is expressed in emitter, it requires a surrogate radioisotope (g-emitter), such virtually all tumors, but the majority of patients are IGF-1R- as 111In, for imaging. Thus for Zevalin imaging, ibritumomab therapy resistant (Scotlandi et al., 2011; van de Luijtgaarden tiuxetan can be labeled with 111In, while for Bexxar the 131I-la- et al., 2013). Molecular IGF-1R imaging might be more bel can be used for both imaging and therapy since it emits adequate as exemplified in a preclinical bone sarcoma study. both b and g radiation. However, also for RIT, the predictive Here, 111In-R1507 SPECT was able to differentiate the degree value of CD20 imaging is not straightforward. In eight NHL pa- of response to the IGF-1R antibody R1507, while IGF-1R IHC tients with biopsy-proven CD20-positivity, 111In-ibritumomab could not, highlighting the importance of selecting the appro- tiuxetan scans revealed that patients with the highest quanti- priate predictive test and warranting further clinical studies tative concentration of lymph node radioactivity had a clinical (Figure 4)(Fleuren et al., 2011). and FDG-PET/CT response upon Zevalin treatment (Jacobs Although the majority of MET-targeting drugs are TKIs, the et al., 2009). However, another study in 19 NHL patients MET antibody ornatuzumab (MetMab) showed promising (pre) showed no correlation between 111In-ibritumomab tiuxetan clinical results (Catenacci et al., 2011). High immunohisto- uptake and Zevalin response (Gokhale et al., 2005). In 4 out chemical MET expression was an indicator for ornatuzumab of 6 patients with PCNSL, tumor 111In-ibritumomab tiuxetan efficacy in combined trials (Sharma and Adjei, 2011). At pre- accumulation was observed up to 5 days post-injection, in sent, two MET-antibodies have been radiolabeled (89Zr-DN30 contrast to another study using 123I-rituximab (Dietlein and 125I-met3) and clearly visualized MET-expressing et al., 2005; Maza et al., 2009) (Section 4.4). The discrepancy 808 MOLECULAR ONCOLOGY 8 (2014) 799e812 was ascribed to delayed antibody accumulation in some 5.3. 177Lu-girentuximab PCNSL patients due to some remaining BBB function, which could only be visualized by 111In-ibritumomab tiuxetan due Girentuximab binds to carbonic anhydrase IX (CAIX), a heat- to a longer physical half-life, pointing out the importance of sensitive transmembranous glycoprotein, and is developed selecting the appropriate radionuclide. The study showed for the treatment of RCC. Previous studies reported almost however no correlation between 111In-ibritumomab tiuxetan ubiquitous expression (>90%) of CAIX in clear cell RCC tumor uptake and Zevalin response. Similar discordances (ccRCC). Girentuximab has been successfully labeled to 111In, have been reported for Bexxar scans (Sgouros et al., 2003). 131I and 124I, though intra-patient comparisons revealed Because Zevalin and Bexxar are murine antibodies that might higher uptake of residualizing 111In-girentuximab prepara- induce human anti-mouse antibodies (HAMA), in general tions (Brouwers et al., 2005; Pryma et al., 2011). Thus for RIT, these agents are not administrated repeatedly. To obviate higher radiation doses can be guided to tumor lesions with HAMA induction and allow repeated administration, several residualizing radionuclides, such as 177Lu and 90Y. A phase I studies radiolabeled the chimeric antibody rituximab (131I-rit- study in metastatic ccRCC patients reported that 177Lu-giren- uximab and 177Lu-rituximab) and investigated its RIT efficacy. tuximab RIT is well-tolerated, can be infused multiple times In a phase I/II 177Lu-rituximab trial of 29 lymphoma patients, and may stabilize previously progressive metastatic ccRCC activity (overall response and stable disease (SD)) was re- (Stillebroer et al., 2013). 111In-girentuximab was used for diag- ported in 79% of patients (Forrer et al., 2013). In relapsed or re- nostic imaging, and only patients with visible CAIX-targeting fractory NHL studies, 131I-rituximab RIT achieved high overall received high-dose 177Lu-girentuximab RIT (n ¼ 23). Within and complete response rates with minimal toxicity and this group, 78% of patients responded (1 partial response repeated 131I-rituximab RIT increased the response rate and and 17 SD), which is superior over another study adminis- duration compared to a single treatment (Kang et al., 2013; tering two consecutive 131I-girentuximab treatments and indi- Leahy et al., 2006; Turner et al., 2003). Prior to 131I-rituximab cates the predictive value of 111In-girentuximab imaging RIT, imaging of a tracer activity of 131I-rituximab was per- (Brouwers et al., 2005). formed for individualized dosimetry to monitor 131I-rituximab distribution in these studies. Although this was primarily per- 5.4. Gemtuzumab ozogamicin (Mylotarg) formed to assess expected toxicity, these scans often visualized target areas very clearly. Mylotarg is a CD33-mAb (gemtuzumab) linked to a toxin belonging to the class of calicheamicins. CD33 is expressed on the majority of myeloid leukemia cells, making it an attrac- 5.2. 177Lu-J591 and 90Y-J591 tive target for acute myeloid leukemia (AML). Mylotarg was developed because lintizumab, an unconjugated CD33 anti- The J591 antibody is directed against the prostate-specific body, showed only modest clinical activity with or without membrane antigen (PSMA). PSMA expression is highly con- chemotherapy. Mylotarg improved survival in a subset of stricted to prostate epithelium and is upregulated in prostate AML patients when combined with standard chemotherapy, cancer. Virtually all prostate cancers express PSMA and it is but the FDA requested withdrawal from the market due to considered a prostate-cancer restricted target. Although the results of a phase III trial in AML (Jurcic, 2012). In that J591 can be administered unlabeled for regular immuno- study, the addition of mylotarg to standard therapies resulted therapy, best responses are seen at relatively high J591 doses in no survival benefits and more toxicity (Petersdorf et al., (100 mg) and the current opinion is that conjugation of J591 2013). Because CD33-targeting might still be of interest in with toxins or radionuclides is a more promising approach certain AML subsets, the introduction of predictive response (Morris et al., 2005). For RIT purposes, J591 has been labeled biomarkers could aid in selecting those patients that could with 90Yor177Lu in phase I studies in metastatic benefit from this treatment (Jurcic, 2012). castraction-resistant prostate cancer (CRPC) patients with promising results (Tagawa et al., 2013; Vallabhajosula et al., 5.5. Trastuzumab emtansine (T-DM1, Kadcyla) 2005b). 177Lu-J591 can be used for both imaging and therapy T-DM1 consists of the HER2-antibody trastuzumab (Section because it emits both b and g radiation, while for 90Y-J591 im- 4.1) linked to the potent toxin mertansine (DM1). In phase II aging a surrogate radionuclide is required. A phase II study of studies, T-DM1 was active in HER2-positive mBC patients re- 177Lu-J591 in CRPC patients demonstrated accurate tumor tar- fractory to trastuzumab and lapatinib and led to improved geting and prostate specific antigen (PSA) responses. Impor- PFS compared to the combination of first-line trastuzumab tantly, non-invasive assessment of PSMA expression by and docetaxel (Hurvitz et al., 2013; Krop and Winer, 2014). In imaging proved to be a predictive biomarker, since patients a recent phase III trial in HER2-positive mBC patients who pre- with poor PSMA-imaging were less likely to respond to viously received trastuzumab and a taxane, T-DM1 improved 177Lu-J591 therapy (Tagawa et al., 2013). For 177Lu-J591 and the time to symptom worsening compared to capecitabine 90Y-J591 RIT patient selection, 111In-J591 imaging can be per- and lapatinib (7.1 vs. 4.6 months) (Verma et al., 2012). Based formed since distribution patterns are generally comparable on the latter study, T-DM1 was recently FDA-approved for and in this way no patient is exposed to a b-emitter in the HER2-positive mBC patients previously treated with trastuzu- screening process (Kawano et al., 2010; Vallabhajosula et al., mab. Similar to trastuzumab monotherapy, patients were 2005b). Multiple administrations of 177Lu-J591 or 90Y-J591 selected based on HER2-expression by conventional methods, were tolerated (Vallabhajosula et al., 2005a). although again HER2-positivity is at best a prerequisite. MOLECULAR ONCOLOGY 8 (2014) 799e812 809

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