receptor occupancy imaging studies in oncology drug ... · including imaging studies in early drug...

Review ArticleTheme: Advances and Applications of In Vivo Medical Imaging in Drug Development and RegulationGuest Editors: Peng Zou, Doanh Tran, and Edward Bashaw

Receptor Occupancy Imaging Studies in Oncology Drug Development

Ingrid J. G. Burvenich,1,2 Sagun Parakh,1,2,3 Adam C. Parslow,1,2 Sze Ting Lee,2,4

Hui K. Gan,1,2,3 and Andrew M. Scott1,2,3,4,5,6,7

Received 25 October 2017; accepted 12 February 2018; published online 8 March, 2018

Abstract. The selection of therapeutic dose for the most effective treatment of tumours isan intricate interplay of factors. Molecular imaging with positron emission tomography(PET) or single–photon emission computed tomography (SPECT) can address questionscentral to this selection: Does the drug reach its target? Does the drug engage with the target ofinterest? Is the drug dose sufficient to elicit the desired pharmacological effect? Does the dosesaturate available target sites? Combining functional PET and SPECT imaging withanatomical imaging technologies such as magnetic resonance imaging (MRI) or computedtomography (CT) allows drug occupancy at the target to be related directly to anatomical orphysiological changes in a tissue resulting from therapy. In vivo competition studies, using atracer amount of radioligand that binds to the tumour receptor with high specificity, enabledirect assessment of the relationship between drug plasma concentration and targetoccupancy. Including imaging studies in early drug development can aid with dose selectionand suggest improvements for patient stratification to obtain higher effective utility from adrug after approval. In this review, the potential value of including translational receptoroccupancy studies and molecular imaging strategies early on in drug development isaddressed.

KEY WORDS: drug development; positron emission tomography (PET); receptor imaging; receptoroccupancy; single–photon emission tomography (SPECT).

INTRODUCTION

In drug development, pharmacokinetic (the time courseof drug concentrations in plasma resulting from a particulardrug dose) and pharmacodynamic (the relationship betweendrug concentrations in the plasma and a pharmacologicaleffect) parameters need to be established. Typically, dosing

schedule selection in drug development is based on preclin-ical studies as well as results of phase I and II studies, butthese studies may not show the relationship between theamount of drug administered and the occupancy of targetreceptors in the tumour. However, when targeting receptorswith a drug, a correlation of receptor occupancy withtherapeutic effect is usually required to achieve maximumpharmacological effect. Therefore, quantifying receptor occu-pancy early in drug development (preclinical and phase Istudies) can aid in choosing the correct dose for the phase IIand phase III studies (Fig. 1) (1).

Molecular imaging is a technique that can be used duringdrug development in oncology to assess receptor occupancyof drug non-invasively, and in all target lesions of a patient.Receptor occupancy studies analyse whether the drug hasreached the target, but more importantly provide a directquantitative measurement of how much of a given dose of thedrug is engaged with its target. Linking the receptoroccupancy curve to drug efficacy (e.g. tumour growthinhibition, reduction of blood biomarker levels) can providecrucial insights during drug development in the relationshipbetween dose levels given and drug efficacy. Based onreceptor occupancy analysis, a decision can then be made asto whether the dose level used has achieved a certain receptor

Guest Editors: Peng Zou, Doanh Tran, and Edward Bashaw

Ingrid J. G. Burvenich and Sagun Parakh have equal contribution.1 Tumour Targeting Laboratory, Olivia Newton-John Cancer Re-search Institute, Melbourne, Australia.

2 School of Cancer Medicine, La Trobe University, Melbourne,Australia.

3 Department of Medical Oncology, Austin Health, Melbourne,Australia.

4 Department of Molecular Imaging and Therapy, Austin Health,Melbourne, Australia.

5 Department of Medicine, University of Melbourne, Melbourne,Australia.

6 Tumour Targeting Laboratory, Olivia Newton-John Cancer Re-search Institute, 145 Studley Road, Heidelberg, Victoria 3084,Australia.

7 To whom correspondence should be addressed. (e–mail:[email protected])

The AAPS Journal (2018) 20: 43DOI: 10.1208/s12248-018-0203-z

1550-7416/18/0200-0001/0 # 2018 American Association of Pharmaceutical Scientists

http://crossmark.crossref.org/dialog/?doi=10.1208/s12248-018-0203-z&domain=pdf

occupancy level necessary to elicit drug benefit, and whetherbinding the target is effective to treat the tumour.

Molecular imaging techniques include nuclear medicineimaging modalities (positron emission tomography (PET) andsingle-photon emission computed tomography (SPECT) aswell as optical imaging. Quantification of tumour cell-surfacereceptor via optical imaging using fluorescently labelledprobes can be very challenging because of the poor tissuepenetration and heterogeneous optical properties of tissuesleading to reduced detection of signal dependent on the depthof the tissue. Preclinical studies have shown the potential ofoptical imaging to quantify receptor occupancy for differenttargets such as alphavbeta3 integrin (αvβ3) (2), humanepidermal growth factor receptor 2 (HER2) (3) and epider-mal growth factor receptor (EGFR) (4). However, becausenuclear medicine approaches are readily translatable intoclinical practice, this review will focus on principles of PET orSPECT imaging to quantify receptor expression and occu-pancy in drug development, including preclinical and clinicalstudies.

KEY PROCESSES OF RECEPTOR IMAGING STUDIES

There are several key steps involved in the developmentof a radiolabelled imaging probe before receptor studies areperformed in preclinical and clinical phase I molecularimaging studies. The initial phase involves understanding ofthe disease and the receptor characteristics used to target thedisease. The following questions need to be addressedthrough biochemical, pathological, and in vitro assays: whatis the expression level of the target in the tumour versusnormal tissue? Does the receptor internalise? Does thereceptor level change because of drug engagement? As anexample, only breast cancer patients expressing estrogenreceptor (ER) in the tumour will benefit from anti-estrogenhormone therapy. As a result of treatment, the tumour mightbecome resistant and lose overexpression of ER through thecourse of treatment. Although primary tumour screening viaimmunohistochemistry techniques can assess ER expressionin primary tumour and lymph node lesions, this cannot beapplied when the disease has spread through the body unlessmultiple biopsies are performed. Therefore, developing animaging agent that could detect and quantify the ER receptorstatus through the course of drug therapy is valuable (5).

Imaging modalities such as PET and SPECT can both beused. PET uses compounds labelled with positron (β+)emitting radioisotopes as molecular probes to image and

quantify targets in vivo. Positrons emitted from the nucleus ofthe radioisotope are anti-electrons that travel a short distanceand combine with an electron, a process called annihilation.When annihilation occurs, their masses convert into theirenergy equivalent through emission of two 511-keV photonsthat travel in opposite direction, 180° apart. The two 511-keVphotons are electronically detected as a coincidence eventwhen they strike opposing detectors simultaneously (6).SPECT uses compounds labelled with gamma (γ) emittingradioisotopes as molecular probes to image targets in vivo.Because the gamma rays travel in all directions, a collimatoris placed in front of the detectors to detect gamma rays fromone direction only. This allows accurate identification of thesource of emission (7). Although imaging probes are avail-able for both imaging modalities, PET is more commonlyused for quantitative analysis of receptor concentrations dueto its inherent advantages for quantitation and resolution. Inaddition, multimodality imaging combines two or moreimaging modalities to combine the strengths of individualimaging modalities. Anatomical imaging technologies that areused in combination with PET and SPECT include computedtomography (CT) and magnetic resonance imaging (MRI).CT measures differences in X-ray attenuation by tissues togenerate images that reflect the anatomy of the body (8).MRI uses a magnet field to measure the different magneticdipoles of nuclei with unequal number of neutrons andprotons (e.g. hydrogen (1H)) in different tissues (9). Thestate of hydrogen in tumour tissue may differ from healthytissue of the same type, making MRI suitable for identifyinganatomical changes that represent tumour tissue. Usingmultimodality imaging in receptor occupancy studies providesan immediate link between amount of drug reaching thereceptor imaged via PET and SPECT and drug responsemeasured by tumour size changes through anatomical imag-ing via MRI and CT.

Table I shows examples of various types of radioligandsthat are currently being used in clinical studies to imagereceptor expression. These radioligands include small mole-cule ligands (e.g. 18F–FDHT, 18F–FES), peptides (e.g.somatostatin analogues, arginine-glycine-aspartic acid(RGD) analogues, cholecystokinin (CCK)/gastrin derivatives,bombesin (BBN)), antibody fragments and antibodies. Thesynthesis of imaging probes needs to be time and cost-effective. Imaging probes must be pure, stable and havesuitably high specific activity (amount of radioactivity to non-radioactive molecules). In preclinical in vitro and in vivo tests,the target specificity of the imaging probe, strength of target

Fig. 1. Receptor occupancy studies enhance the drug development pipeline. The inclusion of receptor imagingexperiments in the clinical development pipeline can assist in the selection of appropriate dosing prior to thedevelopment of phase II and phase III protocols

43 of 16 The AAPS Journal (2018) 20: 43

TableI.

Current

Clin

ical

Recep

torIm

agingStud

iesin

Oncolog

y

Targe

tAge

ntTyp

eof

agen

tTum

ortype

Pha

seClin

icalTrials.go

vIden

tifier

Ref

gpA33

124 I–h

uA33

Antibod

yColon

cancer

INCT00199862

(10,11)

AR

18F–F

DHT

Smallmolecule

Breastcancer

IINCT02697032

(12,13)

αvβ

368Ga-NOTA-B

BN-R

GD

Pep

tide

Prostatecancer

Breastcancer

I INCT02747290

NCT02749019

(14–16)

68Ga-NOTA-3PTATE-R

GD

Pep

tide

NSC

LC

INCT02817945

68Ga-NODAGA-R

GD

Pep

tide

Glio

ma,

melan

oma,

cancer

ofup

perrespiratorytract,breast

cancer,

bone

metastasis,

ovariancancer,lung

cancer,no

n-Hod

gkin

malig-

nant

lymph

oma,

NET,pa

ncreatic

canc

er,oe

soph

agus

canc

er,

stom

achcancer

INCT02666547

(17)

18F-R

GD-K

5Pep

tide

Lym

phom

a,lung

,he

adan

dne

ck,no

n-seminom

atou

sge

rmcell

tumou

rs,m

etastasis

II II II

NCT02490891

NCT02325349

NCT02317393

(18,19)

68Ga-BNOTA-PRGD2

Pep

tide

Glio

ma

Lun

gcancer

I INCT01801371

NCT01527058

(20,21)

18F-A

l-NOTA-PRGD2

Pep

tide

Tum

ours,bron

chog

enic

carcinom

a,breast

carcinom

a,he

adan

dne

ckcancer,lym

phom

a,softtissue

neop

lasm

sI

NCT02441972

(22–24)

18F-FPPRGD2

Pep

tide

Breastcancer,lung

cancer,glioblastomamultiform

e(G

BM)an

dothe

rcancersrequ

iringan

ti-an

giog

enesistreatm

ent

I/II

NCT01806675

(25)

CA6

64Cu-DOTA-B

-Fab

Antibod

yfragmen

tOvarian

cancer,b

reastcancer

INCT02708511

(26)

CA9

111 D

OTA-giren

tuximab

-IRDye800C

WAntibod

yRCC

IINCT02497599

(27,28)

124 I-G

iren

tuximab

Antibod

yRCC

III

NCT01762592

*(29)

CCKBR

177 L

u-PP-F11N

Pep

tide

Med

ullary

thyroidcarcinom

a0/I

NCT02088645

(30)

111 In-PP-F11

(CP04)

Pep

tide

Med

ullary

thyroidcarcinom

aI

NCT03246659

(30)

CEA

68Ga-IM

P-288

/TF2

Pep

tide

/antibod

yfragmen

tBreastcancer

I/II

NCT01730612

(31,32)

111 In-IM

P-288

/TF2

90Y-IMP-288

/TF2

Pep

tide

/antibod

yfragmen

tMetastaticcolorectal

cancer

I/II

NCT02300922

(33,34)

EGFR

89Zr-ABT806

Antibod

yGlio

ma

INCT03058198

(35,36)

18F-O

DS2

004436

Smallmolecule

NSC

LC

0/I

NCT02847377

(37)

18F-M

PG

Smallmolecule

NSC

LC

INCT02717221

(38)

18F-IRS

Smallmolecule

NSC

LC

INCT03031522

(39,40)

89Zr-Pan

itum

umab

Antibod

yGastrointestina

lcarcinom

as,NSC

LC,urothe

lialcarcinom

as,an

dsarcom

asI

NCT02192541

(41,42)

89Zr-Cetux

imab

Antibod

ycolon

I/II

NCT02117466

(43)

68Ga-NODAGA-A

c-Cys-Z

EGFR:1907

Affibo

dyNSC

LC

INCT02916329

(44)

ER

18F-FES

Smallmolecule

Desmoidtumou

rsBreastcancer

0 I I/II

II

NCT02398773

NCT02374931

NCT01957332

NCT00816582

(45)

(46)

(5,47)

(48)

Eph

A2

89Zr-DS-8895a

Antibod

yEph

A2po

sitive

cancers

INCT02252211

(49)

GRPR

68Ga-NOTA-B

BN-R

GD

Pep

tide

Breastcancer

Prostatecancer

0 INCT02747290

NCT02749019

(14)

68Ga-RM2

Pep

tide

Prostatecancer

II IINCT02559115

NCT03

1136

17(50,51)

The AAPS Journal (2018) 20: 43 of 16 43

http://clinicaltrials.gov

TableI.

(con

tinu

ed)

Targe

tAge

ntTyp

eof

agen

tTum

ortype

Pha

seClin

icalTrials.go

vIden

tifier

Ref

II/III

NCT02624518

68Ga-NOTA-A

ca-B

BN

Pep

tide

Glio

ma

INCT02520882

(52)

HER2

89Zr-Trastuzum

abAntibod

yEsoph

ago-ga

striccancer

Breastcancer

0/I

I I/II

II

NCT02023996

NCT02065609

NCT01957332

NCT01565200

(53–

55)

89Zr-Trastuzum

ab89Zr-Pertuzumab

Antibod

yAntibod

yBreastcancer

INCT02286843

(56,57)

64Cu-DOTA-trastuzum

abAntibod

yBreastcancer

0NCT02827877

(58,59)

131 I–S

GMIB

Anti-H

ER2VHH1

Nan

obod

yBreastcancer

INCT02683083

(60)

89Zr-DFO-pertuzumab

Antibod

yHER2-po

sitive

cancer

INCT03109977

(61)

IGF-1R

68Ga-NODAGA-Z

IGF-1R:4:40

Affibo

dyIG

F-1R

overex

pression

tumors(suchas

coloncancer,N

SCLC

andgliomas)

0NCT02916394

PD-1

89Zr-pe

mbrolizum

abAntibod

yMelan

oma

NSC

LC

I INCT02760225

NCT03065764

(62)

89Zr-nivo

lumab

Antibod

yNSC

LC

IEud

raCT00476011*

(63)

PD-L1

18F–B

MS-98

6192

Antibod

yNSC

LC

IEud

raCT00

4760

11*

(63)

PSM

A68Ga-HBED-C

CPSM

ASm

allmolecule

Prostatecancer

0 I I/II

I/II

II II II II III

NCT02940262

NCT03223064

NCT02611882

NCT02488070

NCT02796807

NCT03062254

NCT03204123

NCT02673151

NCT03001869

(64–

70)

18F-D

CFPyl

Smallmolecule

Prostatecancer

RCC

0 0 0 0

NCT02856100

NCT02523924

NCT02793284

NCT02420977

(71–

75)

0 0 I I I I I II II II II/III

NCT03149861

NCT02687139

NCT02825875

NCT02691169

NCT02151760

NCT03232164

NCT03253744

NCT03173924

NCT03001895

NCT03181867

NCT02981368

(71–

75)

18F-D

CFBC

Smallmolecule

I I/II

NCT02190279

NCT01815515

(76–

78)



TableI.

(con

tinu

ed)

Targe

tAge

ntTyp

eof

agen

tTum

ortype

Pha

seClin

icalTrials.go

vIden

tifier

Ref

89Zr-J591

Antibod

yGlio

blastomacancer

Prostatecancer

0 INCT02

4105

77NCT02

6938

60(79,80)

SSTR

68Ga-DOTATOC

Pep

tide

Carcino

idtumou

rsDLBCL,

GEP,

neu

roblastoma,

MCL,

med

ulloblastoma,

men

ingiom

a,NET,p

aragan

glioma,

pituitarytumou

rsSm

allbo

wel

carcinoidtumou

rRecurrent

disease

0 0 I I II II II II II III

III

NCT01

6198

65NCT03

0013

49NCT03

0575

09NCT03

1970

12NCT03

2737

12NCT02

1777

73NCT02

4410

62NCT02

4885

12NCT02

4410

88NCT02

4196

64NCT01

8421

65

(81)

68Ga-DOTATATE

Pep

tide

Apu

doma,

carcinoidtumou

r,isletcelltumou

rM

ese

nch

ymal

tumour,

NET,

onco

genic

osteomalacia,

paragang

liomas,p

heochrom

ocytom

as

0 0 0 0

NCT02

1746

79NCT02

1866

78NCT00

0048

47NCT02

7437

41

(82)

I I/II

I/II

I/II

II II II III

NCT01

5240

16NCT02

0387

38NCT01

8732

48NCT03

1458

57NCT01

9675

37NCT02

8106

00NCT03

2060

60NCT02

8401

49

(82)

177 L

u-DOTATATE

Pep

tide

NET

II II II II

NCT02

2369

10NCT01

8767

71NCT02

0679

88NCT02

7542

9768Ga-DOTANOC

Pep

tide

GEP,

NET,n

onfunction

alpa

ncreatic

neuroe

ndocrine

tumou

rs0 II/III

IV

NCT03

2885

97NCT02

6082

03NCT02

6215

41

(83–85)

68Ga-NOTA-3PTATE-R

GD

Pep

tide

NSC

LC

INCT02

8179

45

AR

androg

enreceptor,C

A6cancer

antig

en6,

CA9carbon

ican

hydraseIX

,CCKBR

cholecystokinin-2receptor,D

FO

desferriox

amine,

DLBCL

diffuselargeB

celllymph

oma,

ECD

extracellular

domain,

EGFR

epidermal

grow

thfactor

receptor,Eph

A2

Eph

rin

type

-Areceptor

2precursor,

ER

estrog

enreceptor,FES

16alph

a-fluo

roestrad

iol,

FDHT

16-beta-fluo

ro-5-alpha

-dihy

drotestosteron

e,GEPga

stroen

teropa

ncreatic,G

RPR

gastrin-releasingpe

ptidereceptor,IG

F-R

1Insulin

-likegrow

thfactor,MBC

metastaticbrea

stcancer,MCL

man

tlecelllymph

oma,

NET

neuroe

ndocrine

tumou

rs,N

SCLC

non-sm

all-celllung

carcinom

a,PETpo

sitron

emission

tomog

raph

y,RCC

rena

lcellcancer,S

STR

somatostatinreceptors

*Studies

areavailablefrom

https://w

ww.clin

icaltrialsregister.eu/



https://www.clinicaltrialsregister.eu

binding (affinity, Ka), in vivo biodistribution and pharmaco-kinetic properties, in vivo stability, metabolites and toxicity,and in vivo imaging studies can be assessed and includestudies in animal models via in vivo competition assays.Imaging studies involve injection of the imaging probe in ananimal model, image acquisition at different time points, dataprocessing and computer modelling. Before embarking onclinical studies, approval from regulatory entities will need tobe sought.

RECEPTOR IMAGING IN ONCOLOGY

A variety of molecular targets are exclusively expressedor overexpressed on cancer cells and often implicated intumour growth and progression. Developing biological agentsagainst these molecular targets has proven to be a successfultherapeutic strategy. These cellular targets include growthfactor receptors (e.g. epidermal growth factor receptor(EGFR), human epidermal growth factor receptor 2(HER2), human epidermal growth factor receptor 3(HER3), ephrin receptor A2 (EphA2)), members of thetumour necrosis factor receptor (TNFR) superfamily (e.g.death receptor 5 (DR5, TRAILR2)), immune-checkpointregulators (e.g. PD-1 and CTLA-4), steroid hormone recep-tors (estrogen receptor (ER), androgen receptor (AR)),proteins involved in angiogenesis (e.g. alphavbeta3 integrin(αvβ3)), growth factor receptors (GFRs, e.g. insulin-likegrowth factor 1 receptor (IGF-1R)) and G-protein coupledreceptors (GPCRs, e.g. gastrin releasing peptide receptor(GRPR), cholecystokinin receptors (CCKR) and somato-statin receptors (SSTR)). Targets also include glycoproteins(glycoprotein A33 (gpA33), mucin 1–sialoglycotope (CA6),carcinoembryonic antigen (CEA)) and enzymes (e.g. car-bonic anhydrase IX (CA9), prostate-specific membraneantigen (PSMA)). Imaging these receptors via molecularimaging can contribute in several ways to drug development(Table I).

One promising area of development has been the use ofreceptor imaging to guide therapy selection. Identifyingreceptor expression (individual and multiple lesions), or thelack of expression can be used to predict response inindividual lesions. As an example, imaging probes targetingHER2 have the potential to diagnose HER2-positive breastcancer, including distant metastases via one single, non-invasive procedure (86). Molecular imaging with 111In-labelled trastuzumab (anti-HER2 therapeutic antibody) hasshown that 11 out of 20 patients showing high 111In-trastuzumab uptake before therapy had objective responsesto trastuzumab (87). Of the nine women without 111In-trastuzumab tumour uptake, only one objective responsewas observed (87). Similarly, 89Zr-trastuzumab has shownantibody uptake in the majority of HER2-positive tumours,but some lesions only show low uptake of 89Zr-trastuzumab(88). Data has shown that more than 10% of patients withHER2-negative primary breast cancer may still benefit fromHER2-targeted treatment, indicating that HER2-negativepatients may have distant metastases that are HER2-positive (89). Therefore, 89Zr-trastuzumab molecularimaging could potentially be used to demonstrateheterogeneity of HER2 expression in different lesions, and

address issues of heterogeneity and discordance betweenprimary and metastatic disease in breast cancer.

Receptor imaging has also been explored to predicttherapeutic efficacy. This can be done directly by quantifica-t ion of markers of apoptos i s (e .g . imaging ofphosphatidylserine by annexin V-based probes (90–93)) orindirectly by quantification of cell-surface receptors that areknown to be altered in response to therapy. In mice bearing aHER2 amplified breast cancer cell line, mice showedsignificantly reduced tumour uptake of 125I-labelled anti-HER2 C6.5 diabody after 6 days of trastuzumab treatment(94). Upregulation of PSMA has been demonstrated afterandrogen-deprivation therapy (95). Evans et al. showed thatincreased PSMA expression in response to treatment with theanti-androgen drug MDV3100 can be quantitatively mea-sured in vivo in human prostate cancer xenograft modelsthrough PET imaging with 64Cu-J591, a fully humanisedradiolabelled antibody to PSMA (96). Similarly, Larson et al.assessed the uptake of 16β-18F-fluoro-5α-dihydrotestosterone(18F-FDHT) in patients with metastatic prostate cancer toassess AR expression. Treatment with testosterone resultedin diminished 18F-FDHT uptake at the tumour site, indicatingthat the uptake of 18F-FDHT in lesions can be measured toreflect post-treatment changes in the AR levels (12). Figure 2shows an example of 18F-FDHT imaging in a 68-year-old manwith metastatic prostate carcinoma. As another example,treatment of HER2-driven breast cancer with tyrosine kinaseinhibitor lapatinib can induce a compensatory HER3 in-crease, which may attenuate anti-tumour efficacy. In a recentstudy, Pool et al. evaluated the HER3 status in response toHER2 therapy with lapatinib using 89Zr-labelled anti-HER3antibody mAb3481 (97). Although in vitro lapatinib treat-ment increased HER3 expression in BT474, SKBR3 and N87cells, in vivo HER3 expression remained unchanged. 89Zr-mAb3481 PET imaging accurately reflected HER3 tumourstatus and might sensitively assess HER3 tumourheterogeneity and treatment response in patients (97).

With regard to the clinical development of antibodytherapeutics, molecular imaging has been proven effective indetermining the toxicity and therapeutic efficacy of theantibody either alone or in payload strategies (as a deliverysystem for radioisotopes or other toxic agents). The in vivospecificity is assessed by determining the biodistribution of anantibody (often radiolabelled) in patients to assess the ratioof antibody uptake in the tumour versus normal tissues (98–100). Quantitating normal tissue distribution allows therelationship of the loading dose to tumour concentration tobe accurately assessed, rather than relying on plasmaconcentration and clearance rates to establish an optimaldose. As an example, PET imaging using 124I-huA33 showedthat the uptake of radiolabelled huA33 in A33-expressingnormal and tumour tissues is driven primarily by the antigenconcentration, and such uptake was saturable (10,11,101). Asa second example, PET imaging with 124I-labelled anti-CA9chimeric cG250 has shown utility in identifying malignantclear cell renal carcinomas. The cG250 antibody targets amarker for hypoxia, carbonic anhydrase IX (CA9), expressedin 94% of clear cell carcinoma (102). A phase I clinical trial of25 patients identified 15 out of 16 patients with clear cellcarcinoma, and no uptake in non-clear cell carcinomas (29).The REDECT trial (phase III) identified 124 out of 143 clear


cell renal carcinoma patients resulting in a sensitivity andspecificity of 86% (103).

Quantification of receptor modulation can be achievedthrough a theranostic approach, where radiolabelled probesare used to determine a treatment strategy by combiningtherapeutics and diagnostics in the same agent. Although notlimited to antibodies, antibody-related therapeutics are par-ticularly suitable for this approach because they are designedagainst specific targets often on the cell-surface and arerelatively easy to radiolabel. As an example, radiolabellednaked trastuzumab (89Zr-trastuzumab) has been used toimage HER2-positive breast cancer patients that are likelyor unlikely to benefit from the antibody drug conjugatetrastuzumab emtansine (trastuzumab-DM1) (53). Othertheranostic applications include personalized radiotherapy,where a patient receives a diagnostically radiolabelled probefor dosimetry analysis prior to receiving the therapeuticallyradiolabelled probe, such as the FDA-approved 90Y-ibritumomab tiuxetan for the treatment of B cell non-Hodgkin lymphoma (104), radiolabelled IMP-288 peptidesfor pretargeted radioimmunotherapy using anti -carcinoembryonic antigen (CEA) × anti-histamine-succinyl-glycine (HSG)-humanised trivalent bispecific antibody (TF2)(NCT02300922, National Clinical Trial (NCT)) (33) anddosimetry-guided peptide receptor radiotherapy (PRRT)using 68Ga-DOTATATE (NETTER-1 trial, NCT01578239)(105).

RECEPTOR OCCUPANCY STUDIES

Receptor occupancy studies can provide vital informa-tion on dosing, receptor modulation and heterogeneity as wellas identifying and monitoring patients treated with targetedtherapies. Approaches to accurately quantify receptor ex-pression include kinetic-modelling techniques, paired-agentmethods and multiple-imaging-agent imaging technologies(106). Most receptor occupancy imaging studies involve

measuring the reduction of specific uptake of an imagingprobe after a given dose of drug under investigation isadministered. Two scans need to be performed per dose levelof the drug of interest: a baseline scan (before the drug ofinterest is administered) and a second post-drug administra-tion scan. Each pair of baseline and post-drug scans is used tocalculate receptor occupancy parameters (107). Althoughreceptor occupancy studies can be performed with all theprobes shown in Table I, smaller radiolabelled moleculesmight be more suitable for accurate evaluation of receptoroccupancy compared to intact radiolabelled antibodies, dueto more uniform penetration of tissue by smaller molecules(108). The section below provides examples of clinicalreceptor occupancy studies using molecular imaging.

FDHT and Androgen Receptor

Steroid hormone receptors are upregulated in a varietyof tumour types such as breast, gynecological and prostatecancers, providing vital information as predictive markers ofresponse to endocrine therapy and as prognostic biomarkers(109–112). 16β-[18F]fluoro-5α-dihydrotestosterone (FDHT),an analogue of endogenous 5α-dihydrotestosterone, hasbeen evaluated to determine androgen receptor (AR)expression and receptor occupancy in prostate cancer (113–116). AR binding selectivity of FDHT was shown in patientswith one or more foci of abnormally increased FDHTaccumulation by analyzing FDHT uptake both before andafter administration of an AR antagonist, flutamide. A dropin tumour FDHT uptake was seen in all lesions afterflutamide treatment, confirming findings seen in preclinicalstudies showing in vivo competition of FDHT by testosterone(116). Early phase studies evaluating enzalutamide, anapproved AR antagonist, used FDHT PET scans as anexploratory endpoint to measure the change in FDHT uptakebefore and after starting treatment (113). Enzalutamide wasshown to displace FDHT binding at all dosages evaluated.

Fig. 2. Sixty-eight-year-old man with metastatic prostate carcinoma. a Maximum Intensity Projection (MIP) 18F-FDHT images with androgenreceptor-positive lesions in the pelvis and sacrum (arrowheads), and physiologic activity in the biliary system and gallbladder (arrows). Similarrepresentation in the axial projection on CT (b), PET (c), and fused PET/CT (d) images


The degree and proportion of patients showing PSA declineswere dose-dependent from 30 to 150 mg/day, but reached aplateau between 150 and 240 mg/day, above which noadditional anti-tumour effects were seen. Interestingly, FDHTPET scans revealed that enzalutamide substantially displacedFDHT binding at all dosages evaluated, with an apparentmaximal effect seen at 150 mg. In another study treatmentwith apalutamide (ARN-509), another AR antagonist showeddecline in averaged maximum standardized uptake values(SUVmax-avg) in a dose-dependent fashion, indicating an on-target effect of AR inhibition (115). While a dose level of ≥120 mg resulted in 100% occupancy of AR binding sites viaFDHT PET uptake, a higher dose of 240 mg has been takenforward to phase III trials. The choice of a higher dose wasbased on the mean plasma trough levels in humans (2.5 μg/mL) associated with 120 mg dose level, which were at thelower end of the range that produced maximum tumourregression in the LNCaP/AR murine model of castration-resistant prostate cancer (3 to 6 μg/mL). Apalutamide iscurrently being evaluated in two phase III trials (ATLAS(NCT02531516) and SPARTAN (NCT01946204)) trials.

In all studies, reductions in FDHT uptake indicatedeffective targeting of the AR and support the use of 18F-FDHT to guide dose selection for anti-androgens in patients.FDHT response however did not predict for 18F-FDGresponse (113), and discordance in uptake of the two PETtracers may be due to intra and inter-tumoural receptorheterogeneity and altered metabolism of some tumoursindependent of AR expression. With an increasing role ofhormonal therapy in prostate cancer, the status of ARexpression can be used as a pharmacodynamic biomarkerand potentially predict for response/outcome to anti-androgen therapy.

FES and Estrogen Receptor

In breast cancer, estrogen receptor (ER)-positive tu-mours have a more favorable prognosis than ER-negativetumours. In addition, ER status determines the likelihood ofresponse to hormonal therapies (i.e. aromatase inhibitors(e.g. anastrazole), selective estrogen receptor modulators(e.g. tamoxifen), or estrogen receptor downregulators (e.g.fulvestrant)), with the response rate being roughly propor-tional to the concentration of ER in the tumour (117).16α–[18F]fluoro-17β-estradiol (FES) has been successfullyused for ER imaging and has been validated as a measureof ER expression (118–121). FES has a relative bindingaffinity of about 80% for the ER and 10% for the SHBGcompared to estradiol (122,123). In breast cancer, 18F-FESuptake has been shown to correlate with ER expression inbiopsy material assayed by in vitro radioligand binding and byimmunohistochemistry (119,120,124,125). FES uptake hasshown to be reduced in breast cancer metastases aftertreatment with anti-estrogen treatment (126); however, justless than half of ERs were unoccupied following fulvestranttherapy, suggesting the current dose of fulvestrant therapywas inadequate for complete block of ER in many of thesepatients (127). In a phase I study, FES PET/CT was used toevaluate ER occupancy and select a recommended phase IIdose for GDC-0810, a potent ER antagonist in patients withER-positive metastatic breast cancer (45). A median SUV

reduction of 98.5% was seen following treatment with GDC-0810 administered once or twice per day, with doses > 400 mgall demonstrating > 90% FES SUV suppression. A dose of600 mg was determined as the dose to take forward forfurther evaluation. While reduction was seen in all lesionswith FES uptake, there was heterogeneity in FES avidity inthe pre-treatment FES PET/CT scans as previously reported(45,109,128). These studies suggest a role for FES PET as apharmacodynamic biomarker for breast cancer, which canhelp determine the dosage of ER-targeted therapies neededfor maximal ER occupancy and/or downregulation.

Somatostatin Analogues and Therapeutic Peptides

Somatostatin analogues are approved cancer therapeuticpeptides for the treatment of neuroendocrine tumours(NET), which typically overexpress somatostatin receptors.Therapeutic approaches for metastatic NETs include phar-macological treatment with somatostatin analogues as well aspeptide receptor radionuclide therapy (PRRT) (105). Molec-ular imaging of somatostatin receptors has improved diagno-sis of NETs and has been investigated to establish dosimetryin PRRT. Although originally the use of 123I- and 111In-labelled octreotide (OctreoScan®) was successfullyestablished for imaging of somatostatin receptors, morerecently new chelators and PET isotopes (18F, 68Ga and64Cu) have further increased the sensitivity of somatostatinreceptor imaging. Two 68Ga-labelled peptides have beenapproved for clinical use in Europe (68Ga-DOTATOC,SomaKit®) and the US (68Ga-DOTATATE, NETSPOT®).The theranostic pairing of these peptides for 177Lu-DOTATATE (Lutathera®) PRRT patient selection hasrecently been approved in Europe and is currently underreview with the US Food and Drug Administration. Figure 3shows an example of somatostatin receptor imaging with68Ga-DOTATATE in a pat ient wi th metas ta t i cneuroendocrine tumours. Currently, multiple clinical trialsare evaluating 68Ga-DOTA-Tyr3-octreotide (DOTATOC)(81), 68Ga-DOTA-Tyr3-octreotate (DOTATATE) (82) and68Ga-DOTA-l-Nal3-octreotide (DOTANOC) (83) (Table I).A number of novel therapeutic peptides are being evaluatedin preclinical studies targeting a variety of key signalingpathways across many tumour types (129).

Preclinical models have shown that increased somato-statin receptor occupancy assessed quantitatively with 68Ga-DOTATOC PET results in decreased tumour proliferation(130). Quantitative somatostatin receptor imaging duringoctreotide therapy has therefore the potential to determinethe fractional receptor occupancy in NETs, allowingoctreotide dosing to be optimized in individual patients(130). Kratochwil et al. used 68Ga-DOTATOC to quantifythe expression of the somatostatin receptors (SSTR2) usingthe maximum standardized uptake value (SUVmax) in livermetastases of patients with NETs prior to PRRT and showedthat SUV analysis can be used to predict response probabilityof PRRT in NET (131).

HER2

HER2 over-expression and amplification have beenshown to be associated with treatment resistance and poorer


overall survival (132) and serves as a predictive biomarker foranti-HER2 treatment in a variety of tumour types (133).Given the predictive and prognostic importance of HER2,accurate determination of HER2 status to identify patientsthat will benefit from anti-HER2 therapy is vital. Currenttesting methods available can affect accurate interpretation ofreceptor status: trastuzumab has shown response in IHC3+/FISH-negative HER2 tumours (134) and in patients tradi-tionally classified as having HER2-negative disease (89).Developments in HER2 imaging have enabled non-invasivequantitative assessment of intra- and inter-tumour HER2expression, with potential to detect treatment resistance early(61,135).

In a study by Dijkers et al., 89Zr-labelled trastuzumabwas administered to trastuzumab-naive patients (10 or 50 mg)and to patients on trastuzumab treatment (10 mg). 89Zr-trastuzumab uptake was best seen 4 to 5 days after injectionand was demonstrated in known metastatic sites as well as inb ra in me ta s t a s e s p rev iou s l y no t ob se rved byfluorodeoxyglucose F18 (18F-FDG) (88). In a small pilotstudy by Ulaner et al., nine patients with metastatic breastcancer with confirmed HER2-negative primary diseaseunderwent 89Zr-trastuzumab PET/CT imaging (56). Uptakeof 89Zr-trastuzumab was seen in five patients and two of thesepatients had biopsy-proven HER2-positive metastases andwent on to benefit from HER2-targeted therapy (64). In theongoing phase II ZEPHIR trial (NCT01565200), 89Zr-trastuzumab is being evaluated as a predictive marker totrastuzumab-DM1 response. Additionally, the study aims toassess HER2 heterogeneity in patients with HER2-positivemetastatic breast cancer. Initial results found 29% of patientshad no tracer uptake and substantial receptor heterogeneitywas seen in 46% of patients (53). The IMPACT trial (Imaging

Patients for Cancer drug selection, (NCT01957332)) isevaluating the role of 18F-fluoroestradiol (FES)- and 89Zr-trastuzumab-PET/CT in determining HER2 receptorheterogeneity over time as well as the role of PET inpredicting early response in patients with newly diagnosedmetastatic breast cancer.

Trastuzumab labelled with the short-lived copper-64(64Cu)-radioisotope has minimal uptake in normal tissueand was able to detect HER2-positive lesions as well brainmetastases in patients with metastatic breast cancer (136,137).A clinical trial is currently underway evaluating 64Cu-DOTA-trastuzumab in predicting treatment response withtrastuzumab and pertuzumab before surgery in HER2-positive breast cancer (NCT02827877).

The novel anti-HER2 nanobody, VVH1, labelled withiodine-131 (131I) and the radio-iodinating reagent N-succinimidyl 4-guanidinomethyl 3-iodobenzoate (SGMIB) isbeing investigated as an imaging agent in an early phase Itrial (NCT02683083) (60).

CS-1008 and Death Receptor 5

CS-1008 is a humanised monoclonal antibody designedto activate apoptosis by targeting the cell surface receptordeath receptor 5 (DR5; also known as tumour necrosis factor(TNF)-related apoptosis-inducing ligand receptor 2(TRAIL-R2)), a member of the TNF receptor superfamily,capable of activating the extrinsic apoptotic pathway onligand stimulation (138,139). In a preclinical study, variousdose levels of 111In-labelled CS-1008 (111In-CS-1008) intumours with high and low levels of DR5 expression wereused to compare receptor occupancy rates with tumourgrowth inhibition (TGI). In both high and low expressing

Fig. 3. Fifty-five-year-old female with metastatic neuroendocrine tumour. 68Ga-DOTATATE PET scan demonstrates widespread somatostatinreceptor-positive bone metastases involving the imaged skeleton. Axial CT (a); PET (b) and fusion PET/CT (c) images, with correspondingcoronal CT (d) and PET (e); sagittal CT (f) and PET (g); and MIP (h) images. The arrowheads indicate representative lytic and sclerotic bonemetastasis in the sacrum


tumours, DR5 saturation occurred at dose levels of between1 and 3 mg/kg with this receptor saturation level alsopredictive of maximal therapeutic response. These studiesshowed a direct correlation between receptor occupancy andtherapeutic response as well as receptor saturation and doserequired for maximal response (140). Subsequent clinicalevaluation was performed in patients with metastatic colo-rectal cancer patients, where 111In-CS-1008 uptake in tumourwas found to be highly predictive of clinical benefit andsuperior to other biomarkers analysed (141). Figure 4 showsan example of 111In-CS-1008 uptake in a metastatic lunglesion of a metastatic colorectal patient that resulted insubstantial shrinkage after CS-1008 treatment as shown with18F-FDG PET/CT. 111In-CS-1008 detected tumour uptake inonly 63% of the patients, despite DR5 expressiondemonstrated by immunohistochemistry in all archivaltumour tissues, and was able to reveal inter- and intrapatientheterogeneity of uptake between DR5-expressing tumourlesions (141).

ABT-806 and Epidermal Growth Factor Receptor

ABT-806 is a humanised antibody that selectively targetsa conformationally exposed epitope on EGFR (142). The 806epitope is normally masked and only exposed in tumours withwild-type EGFR amplification, in tumours that express theEGFR mutant, EGFRvIII, or in tumours that have mutationsof the disulphide bond flanking the epitope (143,144).Preclinical studies using 111In-labelled ABT-806 showed adirect correlation of dose, receptor occupancy and responsein tumours that express wild-type EGFR and EGFRvIII. Inthe EGFRvIII-expressing xenograft model, a lower dose ofABT-806 (10 mg/kg) than cetuximab (28 mg/kg) achieved50% receptor occupancy (35). In contrast, ABT-806 requireda dose approximately twice that of cetuximab to achievesimilar receptor occupancy in the A431 wild-type EGFR-expressing tumour model. Targeting of the 806-epitope hasbeen extended to human trials with an 111In-labelled chimeric806 (111In-ch806) antibody (36) as well as 111In-labelled

Fig. 4. Phase I imaging of CS-1008 in patients with metastatic colorectal cancer. a Whole-body biodistribution of indium-111 labelled CS-1008(111In-CS-1008) in patient 014, showing gradual blood-pool clearance and no specific normal tissue uptake. b 111In-CS-1008 single-photonemission computed tomography and computed tomography (SPECT/CT) in patient 014 (left, SPECT; middle, CT; right, merged SPECT/CT),showing excellent uptake of 111In-CS-1008 in tumour (arrow) in right lung by day 7. c Axial images of maximum-intensity projection CT and18F-FDG PET images are displayed. Metastatic lesion in the right lung of patient 014 shows substantial shrinkage after treatment (reduction inmaximum standardized update value, 43%), with the shrinkage identified as early as 2 weeks after commencement of treatment with CS-1008.Originally published by the American Society of Clinical Oncology (Ciprotti M, et al. J Clin Oncol. 2015;33(24):2609–16) (141)


humanised ABT-806 (ABT-806i) (145). Figure 5 shows anexample of 111In-ch806 imaging in a glioma patient. In anearly phase study, patients with advanced tumours likely toexpress EGFR received 111In-labelled ABT-806 (ABT-806i).ABT-806i showed high, specific tumour uptake includingpatients with an intracranial tumour demonstrating thatABT-806i can cross the blood brain barrier (145). The lackof normal tissue uptake of ABT-806i confirmed the tumour-specific epitope of EGFR recognized by ABT-806i, anddemonstrated the feasibility of this approach in EGFRexpressing tumours.

Immune Checkpoint Inhibitors

Despite the therapeutic success of immune checkpointinhibitors (146), predictive biomarkers to these therapiesare severely lacking. Programmed death-ligand 1 (PD-L1)expression has been used as a biomarker in severalclinical trials (147); however, its use has been challengingdue to issues including differing IHC cutoffs, tissuepreparation, processing variability and intra- and inter-tumoural heterogeneity of PD-L1 expression (148). Deter-mining the appropriate dose for immune checkpointinhibitors in the clinic has been primarily based ontoxicity profiles and pharmacokinetic studies. Typically,the degree of receptor occupancy correlates with atherapeutic effect; however, this has not been shown withimmune checkpoint inhibitors where therapeutic benefitwas achieved at less than 100% receptor occupancy.Pharmacodynamic data for the immune checkpoint inhib-itors have been reported in a limited number of trials.Nivolumab, a human anti-PD-1 antibody, has shown tohave median programmed death-1 (PD-1) receptor occu-pancy of 64–70% for doses ranging from 0.1 up to 10 mg/kg (149,150). The fully human anti-PD-L1 antibody BMS-

936559 has a similar median receptor occupancy rate ofabout 65% depending on dose levels (151). In a phase Istudy of avelumab (MSB0010718C), an anti-PD-L1 humanmonoclonal antibody, receptor occupancy data showed thehigher dose of 10 mg/kg dose of avelumab achieves > 95%occupancy. Avelumab is being evaluated in a number ofphase II trials in solid and hematological malignancies(152).

The correlation between PD-1 receptor occupancyand response is not fully understood. Confounding this isthe unclear relationship of peripheral and intra-tumouralPD-1 receptor occupancy and the immune-modulatingactivity in the tumor microenvironment (153). As seenwith anti-PD1 antibodies, PD-L1 receptor occupancyappears to be dose-independent and does not correlatewith response; reasons for this poor correlation are beinginvestigated (151). Through the use of molecular imaging,real-time immune checkpoint receptor expression andoccupancy may be determined to identify patients thatwould likely benefit from these treatments as well asmonitor for potential toxicities. Supported by preclinicalstudies which evaluated various radiotracers for the non-invasive detection of PD-L1 expression (154,155), and PD-1 expression (62,156,157), radiotracers imaging PD-L1 orPD-1 expression are now being evaluated in early phaseclinical trials (NCT02453984; NCT02478099; EudraCTNumber: 2015-004760-11) (63). Preliminary data accumu-lated from the ongoing 2015-004760-11 clinical trial studyassessing whole body PD-1 and PD-L1 PET in patientswith NSCLC demonstrated substantial heterogeneityamong patients and among tumors within the samepatients (63). Interestingly, higher 18F-PD-L1 tumoruptake was seen in patients with ≥ 50% tumor PD-L1IHC and the highest 18F-PD-L1 SUV was measured in theresponding patient. Once this dataset is complete, this

Fig. 5. ch806 targeting of glioma. a–c Planar images of the head and neck of patient 8 obtained on day 0 (a),day 3 (b) and day 7 (c) after infusion of 111In-ch806. Initial blood pool activity is seen on day 0, and uptakeof 111In-ch806 in an anaplastic astrocytoma in the right frontal lobe is evident by day 3 (arrow) andincreases by day 7. d–f Specific uptake of 111In-ch806 is confirmed in SPECT image of the brain (d) (arrow),at the site of tumor (arrow) evident in 18F-FDG (FDG, fluorodeoxyglucose) PET (e), and MRI (f).Copyright (2007) National Academy of Sciences (Scott AM, et al. Proc Natl Acad Sci U S A.2007;104(10):4071–6) (36)


study may provide valuable insight into PD-1/PD-L1receptor occupancy and tumor response.

CONCLUSION

For any new drug, confirmation of drug uptake in thetarget tissue is essential for understanding whether bioavail-ability is sufficient for pharmacologic effect. In this context,direct measurement of drug concentration in target tissue viamolecular imaging aids in establishing the likelihood ofefficacy compared to preclinical studies, and is more accuratethan modelling uptake based on pharmacokinetic parameters.Multiple clinical studies are currently investigatingradiolabelled probes to determine drug target expression,measurement of drug concentration in target tissue andnormal tissues, tumour saturation and target heterogeneity.Molecular imaging probes can also be utilised to predict offtarget toxicity. This applies to antibodies, proteins, peptidesand other small molecules/drugs that have specific targets andpossible normal tissue target expression. Most progress todate has been achieved in imaging peptide receptors andsteroid receptors. It is possible that imaging studies maybecome more predictive of individual response and will play akey role in cancer management as currently highlighted byHER2 imaging as a predictive marker for anti-HER2therapies and the success of 68Ga-DOTATATE for 177Lu-DOTATATE peptide receptor radionuclide therapy.

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receptor occupancy imaging studies in oncology drug ... · including imaging studies in early drug...

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