University of Groningen Antibody imaging as biomarker in ...anticancer therapeutics currently in...

University of Groningen

Antibody imaging as biomarker in early cancer drug development and treatmentLamberts, Laetitia Elisabeth

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Citation for published version (APA):Lamberts, L. E. (2016). Antibody imaging as biomarker in early cancer drug development and treatment.Rijksuniversiteit Groningen.

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Antibody Positron Emission Tomography Imaging inAnticancer Drug DevelopmentLaetitia E. Lamberts, Simon P. Williams, Anton G.T. Terwisscha van Scheltinga, Marjolijn N. Lub-de Hooge,Carolien P. Schröder, Jourik A. Gietema, Adrienne H. Brouwers, and Elisabeth G.E. de Vries

Laetitia E. Lamberts, Anton G.T.Terwisscha van Scheltinga, Marjolijn N.Lub-de Hooge, Carolien P. Schröder,Jourik A. Gietema, Adrienne H. Brouw-ers, and Elisabeth G.E. de Vries, Univer-sity of Groningen, University MedicalCenter Groningen, Groningen, the Neth-erlands; and Simon P. Williams, Genen-tech, South San Francisco, CA.

Published online ahead of print atwww.jco.org on March 16, 2015.

Terms in blue are defined in the glos-sary, found at the end of this articleand online at www.jco.org.

Authors’ disclosures of potentialconflicts of interest are found in thearticle online at www.jco.org. Authorcontributions are found at the end ofthis article.

Corresponding author: Elisabeth G.E. deVries, MD, PhD, Department of MedicalOncology, University of Groningen,University Medical Center Groningen,P.O. Box 30.001, 9700 RB Groningen,the Netherlands; e-mail: [email protected].

© 2015 by American Society of ClinicalOncology

0732-183X/15/3313w-1491w/$20.00

DOI: 10.1200/JCO.2014.57.8278

A B S T R A C T

More than 50 monoclonal antibodies (mAbs), including several antibody–drug conjugates, are inadvanced clinical development, forming an important part of the many molecularly targetedanticancer therapeutics currently in development. Drug development is a relatively slow andexpensive process, limiting the number of drugs that can be brought into late-stage trials.Development decisions could benefit from quantitative biomarkers, enabling visualization of thetissue distribution of (potentially modified) therapeutic mAbs to confirm effective whole-bodytarget expression, engagement, and modulation and to evaluate heterogeneity across lesions andpatients. Such biomarkers may be realized with positron emission tomography imaging ofradioactively labeled antibodies, a process called immunoPET. This approach could potentiallyincrease the power and value of early trials by improving patient selection, optimizing dose andschedule, and rationalizing observed drug responses. In this review, we summarize the availableliterature and the status of clinical trials regarding the potential of immunoPET during earlyanticancer drug development.

J Clin Oncol 33:1491-1504. © 2015 by American Society of Clinical Oncology

INTRODUCTION

Cancer remains a major cause of death worldwide,with annual mortality predicted to reach 11.5 mil-lion by 2030.1 The mainstay of systemic treatmenthas long been DNA-damaging chemotherapy, butmolecular insights have facilitated the developmentof more selective drugs, which are increasingly be-coming part of standard care.2 Many of these arebased on monoclonal antibodies (mAbs), becausethey can combine exquisite target specificity withdesirable safety profiles.3 Even inert mAbs can bemade potent as delivery vehicles with cytotoxins inantibody–drug conjugates (ADCs) or radionuclidesin radioimmunotherapy (RIT).4

More than 200 anticancer mAbs have been inclinical trials, resulting in 13 mAbs, three ADCs, andtwo RIT conjugates being granted marketing ap-proval by the US Food and Drug Administrationand European Medicines Agency. In 2013, morethan 300 mAbs were in clinical development, in-cluding 50 anticancer mAbs, 10 of which are inphase II/III trials.5

Clinical drug development requires enormousresources, but it often disappoints; only 8% of first-in-human anticancer drugs obtained regulatory ap-proval between 1990 and 2006, while clinicaldevelopment time averaged 9 years.6 Knowing

which therapeutic antibody candidates to advanceinto clinical development depends on understand-ing both target and antibody biology in patients. Akey aspect of this biology—target-dependent anti-body tumor uptake—can be quantified throughmolecular imaging in patients.7 The combination ofantibody-based imaging reagents and positronemission tomography (PET), known as immuno-PET, greatly improves on antibody imaging withsingle-photon emitters. ImmunoPET offers supe-rior image quality, exquisite sensitivity, and accuratequantification.7-9 The technology has great potentialto inform decisions in early clinical development byaddressing fundamental questions of drug delivery,dose finding, and target modulation, thereby prior-itizing the right agents.10 In this review, we summa-rize the available literature and the status of clinicaltrials regarding the potential of immunoPET duringearly anticancer drug development.

MABS AND THEIR EFFECTS ON TUMORS

mAbs are multimeric binding proteins (approxi-mately 150 kDa) that are highly specific ligands fortheir cognate antigens (Fig 1A). They can exert ther-apeutic effects in three ways: directly through ago-nistic or antagonistic effects on receptor signalingand turnover, indirectly through effects on tumor

JOURNAL OF CLINICAL ONCOLOGY B I O L O G Y O F N E O P L A S I A

VOLUME 33 � NUMBER 13 � MAY 1 2015

© 2015 by American Society of Clinical Oncology 1491

129.125.175.213Information downloaded from jco.ascopubs.org and provided by at Bibliotheek der Rijksuniversiteit on June 1, 2016 from

Copyright © 2015 American Society of Clinical Oncology. All rights reserved.

http://www.jco.org

http://www.jco.org

mailto:[email protected]

mailto:[email protected]

http://dx.doi.org/10.1200/JCO.2014.57.8278

vasculature or stroma (Fig 1B), or through recruitment of the immunesystem, with subsequent complement-dependent cytotoxicity orantibody-dependent cellular cytotoxicity (Fig 1C).11,12

More powerful approaches to activate the immune system—immunotherapies—rely on antibodies manipulating specific receptor–ligandinteractionsintheimmunosuppressivetumormicroenvironment.Ipilimumab, for example, blocks cytotoxic T-cell lymphocyte–associatedantigen 4 (CTLA-4) to overcome T-cell anergy. After being registered foruse in patients with melanoma, it resulted in impressive durable re-sponses13 and sparked interest in antibodies against targets such as pro-grammed death receptor 1 (PD-1) and its ligand PD-L1, which have alsoshown durable responses in phase I studies.14-16

Many antibody variants and conjugates, all amenable to study byimmunoPET, have been recognized or engineered, which may have asignificant impact in oncology.17,18 Through a linker moiety, antibodyconjugates combine selective antigen binding with a potent payload:cytotoxic drugs for ADCs or radionuclides for RIT (Figs 1D and 1E).4

The target antigen helps concentrate the potent payload to elicit directcell destruction. Currently, three ADCs are registered: gemtuzumabozogamicin and brentuximab vedotin for treatment of various hema-tologic malignancies and adotrastuzumab emtansine (T-DM1)for human epidermal growth factor 2 (HER2) –positive breast

cancer.19-21 Several RIT agents have been registered, namely iodine-131 (131I) –labeled tositumomab and yttrium-90 (90Y) –ibritumomabtiuxetan in the treatment of non-Hodgkin lymphoma.22 Table 1 listsall currently approved mAbs and conjugates.

The effect of antibodies on tumors typically depends on the tissueconcentration, but this is often unknown. Moreover, proper doseselection can be challenging in situations where blood pharmacoki-netics are a poor predictor of tissue kinetics because of tumor burden,immune status, and other factors.11 ImmunoPET can help directlydetermine antibody tissue kinetics in patient tumor lesions to refineour understanding of dose-response relationships during preclinicaland early clinical trials.

MONITORING TARGET EXPRESSION

Patients likely to benefit from a targeted therapy should exhibittumor-selective expression of the molecular target. Developing andvalidating diagnostic strategies for expression analysis has thereforebecome an important part of drug development. Typically, this is donethrough immunohistochemistry (IHC) or quantitative polymerasechain reaction on tumor biopsies. However, this may be unreliable,

Fc fragment

Fab fragment

Light chain

Heavychain

Variable Hypervariable Constant Regions:

Antibody

Cancercell

Cancercell

Antibody

Cellgrowth

Cellsurvival

Proliferation

NK cell

Attack cancer cell

Granzyme and perforin release

ADC binds to receptor

ADC in plasma

ADC-receptor complexis internalized

Cytotoxic agentis released

Apoptosis

177Lu

177Lu

177Lu

RITRITADC

Linker DM1

LinkerDM1 Linker DM1

A

E

DCB

Fig 1. Design and mode of action of monoclonal antibodies (mAbs). (A) Immunoglobulin G antibodies consist of four polypeptide chains: two identical heavy and twoidentical light chains. Each chain contains variable, antigen-binding regions (Fab), which bind to antigen (drug target), and a constant region (Fc) involved in immunesystem activation. (B) Binding of antibody to antigen (target), causing modulation of downstream signaling by agonistic receptor engagement or antagonistic blocking,eventually leading to inhibition of cell growth, proliferation, and survival. (C) Immune-related cell destruction by antibody-dependent cellular cytotoxicity. Fc part of mAbbinds to Fc receptor on effector cells of immune system (natural killer [NK] cells/lymphocytes), causing immune activation and thereby apoptosis of tumor cell. (D)Structure of antibody–radionuclide (for radioimmunotherapy [RIT]) and antibody–drug conjugates (ADCs). (E) Mode of action of ADC, binding to receptor and causinginternalization of ADC–receptor complex. In lysosomes, ADC and receptor are then degraded, releasing cytotoxic agent, which induces apoptosis of target cell.

Lamberts et al

1492 © 2015 by American Society of Clinical Oncology JOURNAL OF CLINICAL ONCOLOGY



Tabl

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Antibody PET Imaging in Anticancer Drug Development

www.jco.org © 2015 by American Society of Clinical Oncology 1493



Tabl

e1.

Mon

oclo

nalA

ntib

odie

s,A

ntib

ody–

Dru

gC

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gate

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tion

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g/kg

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Lamberts et al




because previously sampled and stored tissue may not representthe current tumor status, and heterogeneity exists between andwithin lesions.24-26

With immunoPET, the presence of an accessible target can bedemonstrated noninvasively in whole-body imaging early in clinicaldevelopment. This permits exploration of the relationships betweenorgan- and lesion-level antibody uptake, tumor heterogeneity, drugeffect, and more conventional biomarkers, which may be taken intolater-stage trials.

Monitoring target expression across all lesions may be partic-ularly pertinent for ADC/RIT agents. This is because their deliveryto the target is based especially on tissue expression patterns, whichmay not represent the oncogenic behavior of the tumor. Conse-quently, lesions without a sufficient target may show poor uptakewhile remaining aggressive and contribute to poor clinical out-comes even when the ADC/RIT agent is locally efficacious in le-sions with sufficient uptake. Comprehensive lesion assessment istherefore required, but this approach (especially serial assessment)is impractical with invasive techniques. As a noninvasive proce-dure, immunoPET avoids this problem.

DEVELOPMENT OF ANTIBODY IMAGING: LABELSAND DETECTION

Early nuclear imaging relied on two-dimensional scintigraphy, whichwas difficult to quantify. In the early 1980s, three-dimensional single-photon emission computed tomography (SPECT) offered significantimprovements.27 SPECT still has a large installed equipment base andis widely used in the clinic for diagnostics in several disciplines inaddition to oncology.28-32 Temporal resolution and quantification ofSPECT was recently improved by the combination with computedtomography (CT). The adoption of PET cameras driven by the utilityof fluorine-18 (18F) fluorodeoxyglucose (18F-FDG) PET in oncologyhas resulted in another leap in spatial and temporal resolution basedon the physics of detecting coincident pairs rather than single gammarays.33-35 Most PET cameras routinely used with 18F-FDG are easilyadapted to work with other PET isotopes. Nuclear imaging of antibod-ies requires matching the radioactive half-life of the isotope to thetimescale of tumor antibody uptake (ie, using isotopes with half-lifeon order of days).36,37

Single-photon gamma-emitting radiometals including technetium-99m (99mTc), lutetium-177 (177Lu), yttrium-88 (88Y), and especiallyindium-111 (111In) have all been used to label mAbs for imaging,along with iodine isotopes (123I, 125I, and 131I). PET imaging of anti-bodies has been reported with isotopes including iodine-124 (124I),bromine-76 (76Br), yttrium-86 (86Y), and copper-64 (64Cu), but nonehave been as widely used as the radiometal zirconium-89 (89Zr).10,38

Our group at the University Medical Center Groningen has de-veloped to date five 89Zr-labeled antibodies for human use.39-43 Theprocedure has been robust, delivering more than 260 patient doses ondemand, with few labeling or logistic failures.

Depending on the radioisotope, different labeling methods areused. Iodines are often labeled directly to the antibody through simpleand widely available procedures, whereas radiometal ions are intro-duced indirectly by first conjugating a suitable chelator moiety to theantibody (typically using lysine groups) and then noncovalentlychelating the metal ion. Once antibodies have been internalized into

the tumor cells, they are subject to catabolism through lysosomaldegradation. The catabolites of radiometal ion chelates remaintrapped (residualized) inside the cells, leading to an accumulation ofradiometal (and PET signal) in the target tumor tissue over time. Incontrast, most iodine-containing catabolites are nonpolar moleculesthat are rapidly lost from the tumor cells, resulting in reduced tumorimage contrast at later time points.7 Iodine labels may thus be bestsuited for imaging noninternalizing targets.44,45 Characteristics ofSPECT and PET radionuclides regarding half-life, radiation exposure,and residualization properties are summarized in Table 2.

FROM SPECT AND PRECLINICAL PET TOCLINICAL IMMUNOPET

SPECT showed early success in assessing target presence and identify-ing tumor lesions both preclinically and clinically. Trastuzumab, bev-acizumab, the DR4 mAb mapatumumab, capromab penditide, andcG250 have all been labeled and imaged successfully with 111In inhumans (Table 2).

Many preclinical small animal studies have been performed with89Zr-labeled mAbs to determine their tumor targeting characteristics.Appendix Table A1 (online only) provides an overview of the 89Zr-labeled mAbs for preclinical research including scanning days andtumor types assessed.

Using knowledge obtained in these studies, translation toclinical tracers has been possible. Interestingly, with epidermalgrowth factor receptor (EGFR) – directed mAbs, there has been aninteresting disparity between efficacy, EGFR expression deter-mined by IHC, and tumor uptake as determined by the EGFR-targeting radiotracers.67-71

These studies point to the potential utility of complementingorgan-level PET imaging with microscopic-level fluorescence imagingto help explain these disparities. The first clinical imaging trial using89Zr-cetuximab PET imaging showed differential uptake among pa-tients. Larger studies are needed to determine the relation betweencetuximab dosing, efficacy, and tumor uptake.72

The clinical feasibility of using radiolabeled mAbs for tumorlocalization with PET was first demonstrated in nine patients withbreast cancer receiving 124I-labeled HMFGI, a murine mAb against amucin target on breast cancer cells. Scans at 1 to 4 days after injectionrevealed a higher uptake in the tumor compared with normal breasttissue.73 Subsequently, many mAbs have been labeled with PET iso-topes for clinical imaging. For example, 124I-labeled cG250 was ad-ministered to identify aggressive clear-cell renal cell carcinoma (RCC)before surgery in a phase I trial of 26 patients with a renal masssuspected to be malignant. In 15 of 16 patients with proven clear-cellcarbonic anhydrase IX (CAIX) –positive RCC histology, immunoPETidentified the tumor.61 A large number of patients (n � 195) withrenal masses underwent 124I-cG250 PET to identify the clear-cell RCCphenotype before surgery. Both sensitivity and specificity improvedcompared with conventional CT: 86% and 86% for immunoPETcompared with 75% and 47% for CT, respectively.62

On the basis of early successes, 89Zr has now been coupledwith 14 different mAbs for clinical use. For all mAbs, signal- andtumor-to-background ratios seem to be optimal 3 to 7 days aftertracer injection. Table 3 provides an overview of these mAbs andtheir clinical trials.





Tabl

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An interesting early example was a tumor visualization study in14 patients with HER2-positive metastatic breast cancer. Tracer dosesof 89Zr-trastuzumab were administered with 10 mg of unlabeled (ie,cold) trastuzumab and appeared in the gut of trastuzumab-naivepatients but yielded excellent tumor images in patients already receiv-ing much higher cold trastuzumab doses for treatment. Increasing the

cold dose to 50 mg was necessary to acquire good images intrastuzumab-naive patients. Excellent 89Zr-trastuzumab tumor up-take revealed known HER2-positive tumor lesions in liver, lung, andbone. Moreover, unknown brain metastases were detected, demonstrat-ing delivery of antibody to brain metastases across a locally compro-mised blood–brain barrier.39 Trastuzumab clearance is dose

Table 3. 89Zr-Labeled Antibodies in Finalized or Ongoing Clinical Studies

Antibody Site Status ClinicalTrials.gov Identifier

BevacizumabPrimary BC UMCG Finished75 NCT00991978Metastatic BC UMCG Finished79 NCT01081613Inflammatory BC Dana-Farber Cancer Institute, Brigham and Women’s Hospital Boston Ongoing NCT01894451Metastatic RCC UMCG Finished40 NCT00831857

UMCG Ongoing NCT01028638Neuroendocrine tumors UMCG Finished85 NCT01338090VHL UMCG Finished NCT00970970MM UMCG Ongoing NCT01859234

TrastuzumabMetastatic BC UMCG Finished39 —

UMCG, Royal Marsden Hospital Finished79 NCT1081600Jules Bordet Institute, UZA, UMCG, VUmc, UMCN Ongoing89 NCT01565200Jules Bordet Institute Ongoing NCT01420146UMCG Ongoing NCT01832051UMCG, VUmc, UMCN Ongoing NCT01957332

BC Washington University School of Medicine Ongoing NCT02065609Esophagastric cancer MSKCC Ongoing NCT02023996

FresolimumabGlioblastoma UMCG Finished41 NCT01472731

U36HNSCC VUmc Finished91

CetuximabStage IV cancer Maastricht Radiation Oncology, VUmc Ongoing NCT00691548Metastatic colorectal cancer VUmc Finished72 NCT01691391

VUmc, UMCG, UMCN Ongoing NCT02117466HNSCC NKI-AVL, Maastricht University Medical Center, Karolinska Institutet,

Institut Catala de la Salut, Gustave Roussy, UMC UtrechtOngoing NCT01504815

RituximabB-cell lymphoma VUmc Ongoing NTR3392

OfatumumabB-cell lymphoma VUmc Ongoing NTR3392

HuJ591Metastatic prostate cancer MSKCC Finished73a NCT01543659

IAB2MMetastatic prostate cancer MSKCC Ongoing NCT01923727

MSTP2109A (STEAP1)Metastatic prostate cancer MSKCC Ongoing73b NCT01774071

RO5479599HER3-positive solid tumors UMCG Ongoing42 NCT01482377

RO5429083CD44-positive metastatic solid tumors VUmc, UMCN Finished NCT01358903

MMOT0530AOvarian or pancreatic cancer UMCG, VUmc Finished43 NCT01832116

Ibritumomab tiuxetanB-cell lymphoma VUmc Finished66 —

PanitumumabRefractory GI and urothelial

carcinomas, NSCLC, sarcomasNCI Ongoing NCT02192541

Abbreviations: BC, breast cancer; HER3, human epidermal growth factor receptor 3; HNSCC, head and neck squamous cell carcinoma; MM, multiple myeloma;MSKCC, Memorial Sloan-Kettering Cancer Center; NCI, National Cancer Institute; NSCLC, non–small-cell lung cancer; NKI-AVL, Nederlands Kanker Instituut Antonivan Leeuwenhoek; NTR, Nederlands Trial Register; RCC, renal cell cancer; STEAP1, six transmembrane epithelial antigen of prostate 1; UMCG, University MedicalCenter Groningen; UMCN, Radboud University Medical Center Nijmegen; UZA, Universitair Ziekenhuis Antwerpen; VHL, von Hippel–Lindau disease; VUmc, VrijeUniversity Medical Center; 89Zr, zirconium-89.

Lamberts et al




http://ClinicalTrials.gov

dependent. In this imaging study, we noticed that an additionalamount of predosed cold (ie, unlabeled) antibody was needed foradequate tumor visualization with immunoPET.39 89Zr-trastuzumabimaging has been used to resolve a clinical dilemma, in which HER2status was important, but a biopsy was impractical.74 A clinical trial isunder way to demonstrate utility in a larger patient population. Figure2 shows a representative 89Zr-trastuzumab PET scan. In another trial,patients receiving trastuzumab therapy were imaged with 64Cu-trastuzumab PET, showing primary breast, lymph node, andmetastatic lung lesions.65 Additional trials are ongoing with 64Cu-trastuzumab to determine the optimal dose and to assess the co-rrelation of tumor tracer uptake with IHC HER2 expression (ClinicalTrials.gov identifier NCT01093612 and NCT00605397). No head-to-head comparisons are yet available between 64Cu-trastuzumaband 89Zr-trastuzumab PET in patients, but the principal rationalefor using 64Cu rather than 89Zr would be that its shorter half-life(12.7 v 78.4 hours) could lower the patient radiation burden. Thismay be attainable where the optimal imaging time point is within 2days after injection.

Other whole-body patient data support the target-specificnature of the immunoPET signal; in histologically proven clear-cellRCC, all 124I-cG250 PET–positive lesions were CAIX positive.61 Inpatients with primary breast cancer, 89Zr-bevacizumab PET uptakecorrelated with vascular endothelial growth factor A (VEGF-A)expression determined by enzyme-linked immunosorbent assay(ELISA).75

TARGET MODULATION ASSESSED BY ANTIBODY IMAGING

Development of potential therapeutic agents can benefit greatly frombiomarkers of target modulation. This typically requires assessmentsbefore and after treatment. Given the timescales of radioactive decay,antibody pharmacokinetics, biodistribution, and tumor uptake, using111In-SPECT or 89Zr-immunoPET as a pharmacodynamic biomarker

allows a second imaging mAb injection approximately 14 days afterthe first.40,66 In 14 patients with RCC, 111In-bevacizumab SPECTvisualized tumors 7 days after tracer injection; reduced 111In-bevacizumab uptake was noted in all nine patients after 4 weeks ofneoadjuvant sorafenib treatment.48 111In-bevacizumab SPECT de-tected all known lymph node lesions in nine patients with stage III toIV melanoma at baseline. After one therapeutic dose of bevacizumab,a 21% reduction in tumor tracer uptake was observed. The tumoruptake in the second scan series correlated with VEGF-A levels mea-sured by IHC in the tumor tissue.49

Several imaging studies have examined the effects of modulatingheat shock protein 90 (HSP90) on its client proteins. Changes in HER2induced by HSP90 inhibitors in xenografts have been imaged withseveral reagents: F(ab=)2 trastuzumab fragments, full-length trastu-zumab, and a fast-clearing Z-domain PET tracer.75,77

Another effect of HSP90 inhibition—reduced VEGF secretion—was monitored by 89Zr-bevacizumab PET in VEGF-expressing xeno-grafts. Treatment resulted in a 44% signal decrease 6 days after tracerinjection. This reduction correlated with lowered tumor levels ofVEGF-A as measured by ELISA.78 Similar studies of HSP90 inhibitorshave been taken into the clinic, where patients with HER2-positivemetastatic breast cancer received 89Zr-trastuzumab scans before andafter three once-per-week doses of NVP-AUY922. Heterogeneoustumor uptake was observed at baseline, and a variety of effects wereseen in the post-treatment 89Zr- trastuzumab scans.79

MODULATION OF ANTIBODY DELIVERY TO TUMOR

Treatment with the VEGF mAb bevacizumab resulted in reducedVEGF activity and consequent reductions in vessel density and mac-romolecular permeability in preclinical models.80 Consistent withthese processes, 89Zr-PET in mouse xenograft models showed thatbevacizumab treatment reduced acute tumor uptake of 89Zr-labeledtrastuzumab, bevacizumab, and nonspecific immunoglobulin G.81

Fig 2. Zirconium-89–trastuzumab positronemission tomography/computed tomographyimages in patient with human epidermalgrowth factor receptor 2 –positive breast can-cer with multiple bone metastases in verte-brae and a large liver metastasis (transverse,sagittal, and coronal views).







Similar reductions in trastuzumab tumor uptake were reported inHER2-expressing xenografts in mice administered the anti-VEGFmAb B20-4.1, which cross reacts with both human and murineVEGF.82 Acute vascular effects of bevacizumab were also apparent inclinical studies. PET scans with 15O-H2O in patients with non–small-cell lung cancer showed rapid reduction in tumor perfusion after atherapeutic dose of bevacizumab, as well as a 34% lower 11C-docetaxeltumor uptake.83

In serial 89Zr-bevacizumab PET scans, patients with metastaticRCC receiving bevacizumab/interferon showed 47% less tumor up-take after 2 weeks of treatment, whereas a modest variable decreasewith sunitinib suggested different modes of action in these antiangio-genic therapies.40 A third antiangiogenic drug, the mammaliantarget of rapamycin inhibitor everolimus, also caused lower 89Zr-bevacizumab tracer uptake in mice bearing human ovarian cancerxenografts.84 In patients with neuroendocrine tumors, everolimustreatment also showed a decline in 89Zr-bevacizumab uptake by35%.85 These studies together suggest that 89Zr-bevacizumab PETcould be used as a biomarker of combined vascular permeability andVEGF levels after antiangiogenic therapy to determine tumor accessi-bility for subsequent treatment.

ANTIBODY IMAGING TO SUPPORT ANTIBODY DOSE FINDING

Pharmacokinetic studies of trastuzumab have shown that the clear-ance from blood is dose dependent and elevated in patients with hightumor burden.86 ImmunoPET with 89Zr-trastuzumab in patients hasadded to knowledge about tumor tissue kinetics and shown thatcurrent clinical practice may be underdosing trastuzumab. A patientwith metastatic breast cancer with extensive tumor load underwent89Zr-trastuzumab immunoPET before and during trastuzumab ther-apy. Initial scans showed high tracer uptake in liver metastases butinadequate visualization of known bone lesions and fully depletedblood levels. Imaging during trastuzumab therapy showed bothliver and bone metastases and persistence in the blood pool. Thisillustrates that a higher dose of mAb may be needed to optimizetumor delivery in patients with a high burden of antibody-internalizing tumor.87

Scouting experiments determine antibody distribution with animaging isotope to enable patient-specific radiation dosimetry calcu-lations before therapeutic RIT. 90Y is a high-energy beta-emittingisotope for RIT applications, whereas 86Y may be the ideal surrogateisotope for scouting because it is chemically identical to 90Y. In prac-tice, 89Zr-immunoPET may suffice; it effectively predicted the biodis-tribution of 90Y- and 177Lu-labeled cetuximab in a study of xenograftsexpressing EGFR.88 Clinical data indicating the utility of 89Zr in scout-ing come from immunoPET studies of 89Zr–ibritumomab tiuxetanbefore RIT with 90Y–ibritumomab tiuxetan. Seven relapsed pa-tients with CD20-positive B-cell non-Hodgkin lymphoma re-ceived 68 MBq 89Zr-ibritumomab tiuxetan and showed specifictumor uptake after 3 to 6 days. This was followed after 14 days by atherapeutic dose of 15 or 30 MBq/kg of 90Y–ibritumomab tiuxetanwith a coinjection of 89Zr–ibritumomab tiuxetan. The highest ab-sorbed 90Y dose, calculated from the 89Zr biodistribution, wasfound in the liver (3.2 � standard deviation of 1.8 mGy/MBq), andbiodistribution of 89Zr was not influenced by the simultaneous90Y–ibritumomab tiuxetan treatment.66

The utility of immunoPET for scouting RIT doses shouldextend to ADC therapies. Molecular imaging of unconjugatedmAbs could quantify tumor uptake and confirm adequate dosing.The processes driving tumor signal for imaging are the same asthose for drug delivery by an ADC—a combination of tissue expo-sure, tissue penetration, expression of target receptor, and anti-body internalization. Understanding these factors in patients willlikely encourage further use of whole-body 89Zr-immunoPET inthe development of ADC therapeutics. Recently, interim patient-based analysis during an ongoing trial in patients with HER2-positive metastatic breast cancer showed a negative predictivevalue of 89Zr-trastuzumab imaging for RECIST response to theHER2 ADC adotrastuzumab emtansine of 88%. When combinedwith 18F-FDG PET after one cycle of adotrastuzumab emtansine,the negative predictive value was 100%.89 A phase I study of anADC comprising a mesothelin mAb armed with the antimitoticdrug MMAE is being accompanied by a parallel trial using baseline89Zr scans with the naked antimesothelin antibody to quantifyantibody uptake in tumor lesions and ultimately to relate this toantitumor effects.43

COSTS AND CONSEQUENCES OF IMMUNOPET IMAGING

ImmunoPET can be costly, and its routine use requires appropriatejustification. Antibody labeling and a series of PET scans can costseveral thousand US dollars (highly dependent on country and insti-tution, with United States being more expensive than Europeansites).90 However, immunoPET is valuable for making good decisionson drug development investments and is valuable for providingwhole-body all-lesion information in patients. This informationwould be difficult to obtain even from multiple biopsies, which entailtheir own risks and costs.

ImmunoPET also seems to be safe. A dosimetry study ofionizing radiation, the principal hazard to which immunoPETpatients are exposed, was performed in 20 patients with head andneck squamous cell carcinoma who received 74.9 MBq (� stan-dard deviation of 0.6 MBq) of 89Zr-labeled U36 mAb without anyadverse events. The mean radiation whole-body dose in patientswas 40 mSv, which is comparable to two abdominal CTs.91 How-ever, new PET/CT scanners are more sensitive, so most clinicalstudies now use only 37 MBq 89Zr injected activity, with scansperformed 2 to 8 days later. Concerning tolerability, patient scan-ning times with 89Zr-labeled mAbs typically increase from 45 min-utes soon after injection to 90 minutes 1 week later.

FUTURE IMAGING APPLICATIONS IN DRUG DEVELOPMENT

Nuclear imaging will always have limitations imposed by the inev-itable radiation burden to patients, clinical staff, and caregivers andthe relatively high cost of dealing with nuclear reagents. Although itis not an alternative for whole-body nuclear imaging, optical im-aging with fluorescent dyes may acquire an important role in thenear future.

The utility of molecular-guided optical surgery was assessed in afeasibility study of patients with ovarian cancer who underwent deb-ulking surgery after injection of fluorescein isothiocyanate (FITC)

Lamberts et al




–labeled folate to target the folate receptor alpha (FR-�). Intraopera-tive imaging at 520 nm showed uptake in all FR-�–expressing tumorlesions (even tumor deposits � 1 mm).92 To reduce autofluorescencenoise, near-infrared fluorophores (NIRFs), which emit light at wave-lengths between 700 and 900 nm, are preferred.93

IRDye800CW is an NIRF suitable for producing conjugates un-der good manufacturing practices for patient injection. With bevaci-zumab and trastuzumab, it has yielded highly specific images ofxenograft tumors in mice using a real-time intraoperative clinicalprototype camera system. Results were compared with those obtainedwith 89Zr-immunoPET.94

The use of an IRDye800CW conjugate as the imaging agentopens up possibilities for multimodality imaging— both to imagein vivo and to look directly at microscopic mAb distribution inrelation to blood vessels, tumor cells, and its antigen targetdistribution. An ongoing trial in patients with primary breastcancer is evaluating safety, uptake, quantification, and localiza-tion of bevacizumab-IRDye800CW in tumor and normal tissues(ClinicalTrials.gov identifier NCT01508572). Dual-labeled anti-bodies (detectable by SPECT, PET, or NIRF) have been developedand used to facilitate the development and validation of multimo-dality imaging.95,96

Understanding cancer immunotherapies such as ipili-mumab and anti-PD1/PD-L1 mAbs may require dual-labeledreagents in patients, because target expression at the organ level(imaged by immunoPET) and distribution at the microscopiclevel (imaged by NIRF) are both fundamentally important tounderstanding the effects of these drugs.97 This will allow effi-cient use and better interpretation of valuable— but inherentlyvariable—patient tissue samples by enabling both overall tumoruptake measurement and histopathologic follow-up in thesame lesions.

Furthermore, small antibody fragments, such as nanobodies,have become interesting for in vivo imaging of cell activity, andthey have been proposed as theranostic tools for both diagnosticand targeted radionuclide therapy in oncology.98 Pretargeting isanother interesting technique in preclinical development, in whichantibody and radionuclide delivery are separated to reducesystemic radioactivity exposure. A bispecific antibody is firstadministered to target the tumor cell with one epitope, followedby administration of a radioisotope-bound peptide that bindsto the second isotope.99,100

DISCUSSION

This review demonstrates that immunoPET, especially 89Zr-immunoPET, is showing real promise as a biomarker in early clinicalmAb, ADC, and RIT trials. Complementing traditional histologicassessments of static target expression, immunoPET may reveal targetpresence, engagement, and internalization and directly demon-strate antibody uptake in tumor tissue in patients at the time oftheir treatment. This has the potential to provide prognostic andpredictive information, helping in the decision between antibody-based drug candidates and dosing regimens and in the selection ofappropriate patients for enrollment onto early clinical trials. Thewhole-body, all-lesion nature of immunoPET lends itself to ad-

dressing the increasingly recognized complexities of between- andwithin-lesion tumor heterogeneity.

Furthermore, pharmacologic proof of principle has been shownin some cases by measuring the mAb tumor uptake before and duringtreatment using immunoPET as a biomarker of response. Dose-finding studies could therefore be based on quantification of antibodyuptake as a function of antibody dose. For ADC candidates, immuno-PET studies in early development may inform the choice of target andantibody by revealing unexpected pharmacokinetic characteristics.For example, such studies could identify an organ that functions as asink (ie, collects large amount of tracer) and make predictions aboutorgan toxicity, because the premise of molecular targeting is to obtainstrongly selective tumor uptake. In addition, the biology of targetexpression and antibody uptake might be studied in the context ofacquired resistance and combination treatment strategies.

As with the adoption of other clinical technologies, the standard-ization of protocols for producing the reagents and acquiring, process-ing, and analyzing the 89Zr-immunoPET images will need robustmulticenter trials. Appropriate interpretation will depend on thought-ful comparisons of imaging results against traditional gold-standardmetrics of target expression, such as IHC obtained subsequent tobiopsies of imaged lesions.

Some potential confounding factors exist, particularly in tumorswith low target expression levels, in which vasculature and nonspecificuptake may contribute to the observed low-level signals. This places alower limit on the amount of antibody uptake that can be meaning-fully studied. Unraveling these contributions may require dynamicstudies in which the temporal patterns of uptake can be monitored;the internalization-driven signal is likely to increase progressively overtime, while static components such as blood volume remain relativelyconstant, and noninternalizing binding will plateau relatively early.Several recent studies have emphasized the collection of tumor biop-sies for measurement of protein expression of the target to correlatewith the PET signal, a step that should be encouraged to develop ourunderstanding of the imaging results.61,62,75

The half-life and stability of 89Zr mAb– based radiopharma-ceuticals lend themselves to centralized production and distribu-tion, facilitating collaborative multicenter studies. ImmunoPETtracers are powerful and practical tools for gaining more insightinto the mechanism of action, response, and treatment outcome ofantibody-based therapeutics.

AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTSOF INTEREST

Disclosures provided by the authors are available with this article atwww.jco.org.

AUTHOR CONTRIBUTIONS

Conception and design: Laetitia E. Lamberts, Simon P. Williams,Elisabeth G.E. de VriesCollection and assembly of data: Laetitia E. Lamberts, Simon P.Williams, Elisabeth G.E. de VriesData analysis and interpretation: All authorsManuscript writing: All authorsFinal approval of manuscript: All authors






http://www.jco.org

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■ ■ ■





GLOSSARY TERMS

antibody-drug conjugate: an antibody chemically linkedto a therapeutic cytotoxic agent providing targeted delivery of thecytotoxic agent preferentially to cancer cells expressing the anti-gen recognized by the antibody.

antigen: a substance that promotes, or is the target of, an im-mune response.

immunohistochemistry: the application of antigen-antibody interactions to histochemical techniques. Typically, atissue section is mounted on a slide and incubated with antibod-ies (polyclonal or monoclonal) specific to the antigen (primaryreaction). The antigen-antibody signal is then amplified using asecond antibody conjugated to a complex of peroxidase-antiperoxidase, avidin-biotin-peroxidase, or avidin-biotinalkaline phosphatase. In the presence of substrate and chro-mogen, the enzyme forms a colored deposit at the sites ofantibody-antigen binding. Immunofluorescence is an alternateapproach to visualize antigens. In this technique, the primaryantigen-antibody signal is amplified using a second antibodyconjugated to a fluorochrome. On ultraviolet light absorption,the fluorochrome emits its own light at a longer wavelength(fluorescence), thus allowing localization of antibody-antigencomplexes.

monoclonal antibody: an antibody that is secreted from a singleclone of an antibody-forming cell. Large quantities of monoclonal anti-bodies are produced from hybridomas, which are produced by fusingsingle antibody-forming cells to tumor cells. The process is initiatedwith initial immunization against a particular antigen, stimulating theproduction of antibodies targeted to different epitopes of the antigen.Antibody-forming cells are subsequently isolated from the spleen. Byfusing each antibody-forming cell to tumor cells, hybridomas can eachbe generated with a different specificity and targeted against a differentepitope of the antigen.

pharmacodynamics: the study of the biochemical and physiologiceffects of a drug on the body.

pharmacokinetics: a branch of pharmacology that studies the rela-tionship between drug exposure level, time course of exposure, and theoverall response of an organism. Although pharmacokinetics is largelyapplied to drugs, it is also applicable to other compounds such as nutri-ents, toxins, hormones, etc. Pharmacokinetics is subdivided into ab-sorption and disposition (distribution, metabolism, and excretion) andis generally referred to as ADME (absorption, distribution, metabolism,excretion). With respect to drugs administered, all processes occur intandem once a drug dose is administered. In clinical trials, phase I stud-ies will typically study pharmacokinetics and safety of the drug.

Lamberts et al




AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

Antibody Positron Emission Tomography Imaging in Anticancer Drug Development

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships areself-held unless noted. I � Immediate Family Member, Inst � My Institution. Relationships may not relate to the subject matter of this manuscript. For moreinformation about ASCO’s conflict of interest policy, please refer to www.asco.org/rwc or jco.ascopubs.org/site/ifc.

Laetitia E. LambertsNo relationship to disclose

Simon P. WilliamsEmployment: GenentechStock or Other Ownership: RochePatents, Royalties, Other Intellectual Property: Genentech holds IP onsite-specific antibody labeling technologies (Inst)

Anton G.T. Terwisscha van ScheltingaNo relationship to disclose

Marjolijn N. Lub-de HoogeNo relationship to disclose

Carolien P. SchröderNo relationship to disclose

Jourik A. GietemaResearch Funding: Roche (Inst), Abbvie (Inst), Siemens (Inst)

Adrienne H. BrouwersNo relationship to disclose

Elisabeth G.E. de VriesResearch Funding: Roche (Inst), Genentech (Inst), Novartis (Inst),Amgen (Inst)


www.jco.org © 2015 by American Society of Clinical Oncology



http://www.asco.org/rwc

http://jco.ascopubs.org/site/ifc

Appendix

Table A1. Preclinical Studies Evaluating Specific-Tumor Accumulation and Biodistribution of Different 89Zr-Labeled mAbs in Human Tumor–Bearing Mice or Rats

mAb Target

Model

Tumor Assessment

Scanning MomentsTumor Target–Specific

UptakeAnimalModel Tumor Location Type Location

Bevacizumab (Nagengast WB,et al: J Nucl Med 48:1313-1319, 2007)

VEGF Mice Subcutaneous SKOV3 human ovariancancer cell line

VEGF positive Days 1 to 7 (optimaldays 3 and 7)

Present; 89Zr-IgG controlshows lower uptake

Trastuzumab (Dijkers EC, et al:J Nucl Med 50:974-981,2009)

HER2 Mice Subcutaneous SKOV3 human ovariancancer cell line

HER2 positive Days 1 to 6 Present; HER2-negativecell line GLC4 showslower uptake

Cetuximab67 EGFR Mice Subcutaneous Different EGFR-expressing humancell lines

1 to 120 hours (optimalat 72 hours)

Higher uptake inintermediate-expressing cell linescompared with high�

Panitumumab (Nayak TK, et al:J Nucl Med 53:113-120,2012)

EGFR Mice Subcutaneouspulmonary andintraperitoneal

EGFR-expressing humancolorectal cancer cellline LS-174T

Days 1 to 6 Present; higher uptakein LS-174T than innegative cell lineA375 (humanmalignant melanoma)

R1507 (Heskamp S, et al: JNucl Med 51:1565-1572,2010)

IGF-1R Mice Subcutaneous SUM1249 human triple-negative breastcancer cell line

Days 1 to 7 No comparison with IgGor IGF-1R–negativecell line

Capromab pendetide (7E11;Ruggiero A, et al: J NuclMed 52:1608-1615, 2011)

PSMA Mice Subcutaneous PSMA-positive humancancer cell lines

1 to 120 hours (optimalat 72 hours)

No comparison with IgGor PSMA-negativecell line

DN30 (Perk LR, et al: Eur JNucl Med Mol Imaging35:1857-1867, 2008)

c-MET Mice Subcutaneous GLT-16 human gastriccarcinoma cell line;FaDu mouse-cellcarcinoma

c-MET positive andnegative

Days 1 to 4 Present; higher uptakein GLT-16 with 89Zr-DN30 than with 124I-DN30 and comparedwith c-Met–negativecell line

Fresolimumab (Oude MunninkTH, et al: J Nucl Med 52:2001-2008, 2011)

TGF-� Mice Subcutaneous andmetastaticmodel

CHO clones with TGF-�MDA-MB-231

Days 1 to 7 No difference in 89Zr-fresolimumabcompared with 111In-IgG uptake becauseof presence of onlylatent TGF-� insteadof active

RO5323441 (Oude MunninkTH, et al: J Nucl Med 54:929-935, 2013)

PlGF Mice Huh-7; ACHN PlGF positive andnegative

Days 1 to 7 Present; compared with111In-IgG andcompared with PlGF-negative cell line

AMA (Ter Weele EJ, et al:104th Annual Meeting ofthe American Associationfor Cancer Research,Washington, DC, April 6-10, 2013 [abstr 2659])

MSLN Mice Subcutaneous HPAC and CAPAN-2 MSLN positive Days 1 to 6 (optimalday 6)

Present; higher uptakeof 89Zr-AMA than111In-IgG

RG711676 HER3 Mice Subcutaneous Four different HER3-expressing cell lines

Days 1 to 6 (optimalday 6)

Present; higher uptakein higher HER3-expressing tumors

cG250 (girentuximab; BrouwersA, et al: Cancer BiotherRadiopharm 19:155-163,2004)

CAIX Nude rats Subcutaneous RCC cell line SK-RC-52 5 minutes and 1, 2,and 3 days

Increasing tumor uptakeover time; nocomparison with IgGor CAIX-negativetumor

Abbreviations: AMA, antimesothelin antibody; CAIX, carbonic anhydrase IX; c-MET, anti-mesenchymal epithelial transition factor; 64Cu, copper-64; EGFR, epidermalgrowth factor receptor; HER2, human epidermal growth factor receptor 2; HER3, human epidermal growth factor receptor 3; 124I, iodine-124; IGF-1R, insulin-likegrowth factor 1 receptor; IgG, immunoglobulin G; 111In, indium-111; mAb, monoclonal antibody; MSLN, mesothelin; PlGF, placental growth factor; PSMA,prostate-specific membrane antigen; RCC, renal cell cancer; TGF-�, transforming growth factor beta; VEGF, vascular endothelial growth factor; 86Y, yttrium-86; 89Zr,zirconium-89.

�Disparity between efficacy, EGFR expression determined by immunohistochemistry, and tumor uptake is known as determined by EGFR-targeting radiotracers89Zr-cetuximab, 64Cu-cetuximab, 64Cu-panitumumab, 86Y-panitumumab, and 111In-panitumumab (Dijkers EC, et al: J Nucl Med 50:974-981, 2009; Nayak TK, et al:J Nucl Med 53:113-120, 2012; Heskamp S, et al: J Nucl Med 51:1565-1572, 2010).58,67

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