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TECHNOLOGIES

DRUG DISCOVERY

TODAY

Drug Discovery Today: Technologies Vol. 8, No. 2–4 2011

Editors-in-Chief

Kelvin Lam – Blue Sky Biotech Inc., USA

Henk Timmerman – Vrije Universiteit, The Netherlands

Imaging techniques

89Zr-labeled compounds for PETimaging guided personalized therapyDanielle J. Vugts1,2,*, Guus A.M.S. van Dongen1,2

1Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, Amsterdam, The Netherlands2Department of Nuclear Medicine and PET Research, VU University Medical Center, Amsterdam, The Netherlands

89Zr-immuno-PET is an attractive option for the in vivo

evaluation of monoclonal antibodies (mAbs). For the

coupling of 89Zr to monoclonal antibodies several con-

jugation strategies are available all using desferrioxa-

mine as chelate. Here we discuss the production of89Zr, the available methods for coupling of 89Zr via

desferrioxamine to mAbs, and the evaluation of89Zr–mAb conjugates in preclinical and clinical studies.

*Corresponding author.: D.J. Vugts ([email protected])

1740-6749/$ � 2012 Elsevier Ltd. All rights reserved. DOI: 10.1016/j.ddtec.2011.12.004

Guest Editors:Nicolau Beckmann – Novartis Institutes for BioMedicalResearch, Basel, Switzerland.Bert Windhorst – VU Medical Center, Amsterdam,The Netherlands.

stages of mAb development and application for example, for

Introduction

The discovery of disease-specific tissue markers has boosted

the development of molecular therapeutics like monoclonal

antibodies (mAbs). To date 30 mAbs, mostly intact immu-

noglobulins, have been approved by the U.S. Food and Drug

Administration (FDA), of which 11 are for the systemic

treatment of cancer. These approved therapeutic antibodies,

including unmodified, biologically active chimeric and/or

human antibodies, as well as radio- and drug-immunocon-

jugates represent a multibillion-dollar market. Furthermore,

hundreds of new mAbs are in early clinical development and

in preclinical evaluation and next to intact mAbs also non-

traditional scaffolds like mAb-fragments, nanobodies, affibo-

dies and minibodies are being developed. Despite the com-

mercial successes of approved mAbs, the effectiveness is

limited and only a small number of patients is benefitting

from these expensive medicines. Additionally the approval of

new mAbs for novel targets is staggering [1,2].

Immuno-positron emission tomography (immuno-PET),

the tracking and quantification of mAbs with PET at high

resolution and sensitivity can be of great value in several

(i) understanding the mechanism of action of mAbs in vivo,

(ii) anticipating on non-target toxicity, (iii) optimization of

the mAb dose and administration scheduling, (iv) facilitating

patient selection and (v) speeding up drug development by

understanding the added value and distinctive features of the

mAb during early stage clinical trials. Ultimately, this will

result in personalized therapy in which the right drug is given

at the right time to the right patient. To enable visualization

of a mAb with a PET camera, a positron emitter (b+) has to be

stably and inertly coupled. PET isotopes that would be appro-

priate for labeling mAbs and mAb-fragments are 68Ga (t1/

2 = 68 min), 18F (t1/2 = 110 min), 64Cu (t1/2 = 12.7 hours), 90Nb

(t1/2 = 14.6 hours), 86Y (t1/2 = 14.7 hours), 89Zr (t1/2 = 78.4

hours) and 124I (t1/2 = 100.2 hours). Recent reviews describe

their physical characteristics, coordination chemistry and

labeling opportunities [3–6].

In this review we will focus on the use of immuno-PET with

zirconium-89 (89Zr) in mAb development and applications.89Zr is an ideal isotope for labeling intact mAbs because its

physical half-life of 78.4 hours matches with the biological

half-life of the mAbs and the time needed to reach optimal

e53

http://dx.doi.org/10.1016/j.ddtec.2011.12.004


mailto:[email protected]


Drug Discovery Today: Technologies | Imaging techniques Vol. 8, No. 2–4 2011

Physical Characteristics Production method

Purification

Zr for radiolabeling mAbs

Affinity chromatography using a

89Zr

89Y

IT

hydroxamate functionalized column

>99.99% radionuclidic and chemical

purity

Carrier free 89Zr in 1M oxalic acid with

nuclear reaction on Yttrium 78,4 h

15.7 s

EC, β+

IT= isomeric transition

Transition

EC

0.897

0.909 99.0

77.0

22.6β+

γ

Energy (MeV) Abundance (%)

EC= Electron Capture

γ

a) 89Y (p, n) 89Zr

b) 89Y (d, 2n) 89Zr

89

Drug Discovery Today: Technologies

Figure 1. Summary of the physical characteristics and production method of 89Zr.

target-to-non target ratios. 89Zr is a residualizing isotope and

is trapped inside the cell after internalization of the mAb,

while for example, 124I, another long-lived candidate radio-

isotope for immuno-PET with intact antibodies is released

from the tumor after internalization. Residualization also

occurs to some extent in organs of mAb catabolism like liver,

kidney and spleen. 89Zr emits a positron (b+ particle) with

23% efficiency and a concomitant gamma ray emission of

909 keV which decays 15.7 s later. Because PET is based on the

coincidence detection of two gamma photons of 511 keV,

which arise after combination of the b+ particle with an

electron, the delayed gamma photon of 909 keV does not

interfere with the overall image quality and accurate quanti-

fication of the PET images (Fig. 1).

General considerations regarding 89Zr-immuno-PET89Zr production89Zr can be produced through the irradiation of inexpensive

natural yttrium via the 89Y(p,n)89Zr reaction or the89Y(d,2n)89Zr reaction. The first method is mostly applied,

because a medium-to-small cyclotron can be used for this

production method. The average yield via this method is 38–

42 MBq/mAh resulting in batches of 6 GBq of 89Zr within four

to six hours, which is large taking into account that for

clinical immuno-PET studies patients receive 37 MBq. Pur-

ification of 89Zr from 89Y can be achieved via affinity chro-

matography using a hydroxamate functionalized column [7].89Zr can be eluted with 1 M oxalic acid, resulting in >99.99%

radionuclidic purity and>99.9% chemical purity. In the past,

the oxalic acid was removed by sublimation and radiolabel-

ing was performed in acetate buffer. Furthermore, H2O2 was

added to be absolutely sure that 89Zr was solely obtained in

the Zr(IV) oxidation state. Both steps, however, can be

omitted because it has been proven that without sublimation

e54 www.drugdiscoverytoday.com

of oxalic acid and the addition of H2O2 transchelation is still

perfect, while potentially toxic oxalic acid can be totally

removed during subsequent purification steps [8,9]. The

method of Verel et al. [8] at the VU University Medical Center

has been further improved and 89Zr in 1 M oxalic acid is

commercially produced by BV Cyclotron VU, (http://

www.cyclotron.nl), Amsterdam, The Netherlands, and dis-

tributed by IBA Molecular Benelux (http://www.iba-molecu-

lar.com), Louvain-la-Neuve, Belgium.

89Zr chelation and labeling

Zirconium is a hard Lewis acid and thus prefers hard Lewis

bases as donor groups. Furthermore, zirconium prefers 8-

coordination. Baroncelli and Grossi and Fouche et al. showed

that zirconium(IV) can form stable complexes with hydro-

xamates [10,11]. In the past attempts have been made to

complex zirconium with diethylenetriamine pentaacetic acid

(DTPA) and porphyrins, however, the only chelate identified

up to now that forms stable complexes with zirconium is

desferrioxamine (DFO). Because DFO is clinically used for the

neutralization of iron and aluminum overload for many

years, its application as a chelate in clinical immuno-PET

studies is possible without the need of performing large

toxicity/safety studies in advance.

DFO consists of three hydroxamate groups for the com-

plexation of zirconium as well as of iron, gallium and nio-

bium and results in 6-coordination. Recently, Holland et al.

did density functional theory (DFT) calculations on the com-

plexation of DFO with zirconium [12]. The calculations pro-

vide an explanation for the observed high in vivo stability of89Zr-DFO-labeled mAbs and suggest that in aqueous solution

the coordination sphere of zirconium by DFO is expanded by

two water molecules resulting in thermodynamically stable

8-coordinated zirconium [12].

http://www.cyclotron.nl/

http://www.cyclotron.nl/

http://www.iba-molecular.com/

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Vol. 8, No. 2–4 2011 Drug Discovery Today: Technologies | Imaging techniques

89Zr can be transchelated from oxalic acid to DFO at neutral

pH (optimum: 6.8–7.2). To this end a strong base, like sodium

carbonate, is added to the 89Zr in 1 M oxalic acid solution,

followed by 4-(2-hydroxyethyl)-1-piperazineethanesulfonic

acid (Hepes) buffer to obtain a final pH of 6.8–7.2. Hepes is

an excellent buffer at neutral pH and outperforms phosphate

buffer in radiolabeling efficiency: while with the latter label-

ing yields of 30% were achieved, with Hepes buffer >95%

yield was obtained. Recently Holland et al. [9] reported on the

transchelation of 89Zr-oxalate to 89Zr-chloride, with the aim

to get rid of potentially toxic oxalic acid. However, in our

hands using this 89Zr-chloride did not result in optimal

radioimmunoconjugates.

To prevent deterioration of 89Zr-labeled mAbs due to radi-

olytically formed species in the solution, gentisic acid (2,5-

dihydroxybenzoic acid) proved to be the best preservative.

Ascorbic acid is not recommended, because Zr4+ will be

reduced to Zr2+ and zirconium will dissociate from DFO.

Quality specifications

For clinical application it is important that 89Zr-labeled

mAbs are produced under current good manufacturing

practice (cGMP). After radiolabeling and purification it is

important to assess (i) appearance: a clear and colorless

solution should be observed; (ii) chelate-to-mAb ratio: in

general one to two DFO groups per mAb molecule do not

change the immunoreactivity and pharmacokinetics and

allow patient dose preparations; (iii) radiochemical purity:

this should be ideally 100%. Aggregate/dimer formation

caused by modification and radiolabeling and free 89Zr

and 89Zr-DFO should be <10%. High pressure liquid chro-

matography (HPLC) analysis can be used to evaluate both

criteria, while instant thin layer chromatography (iTLC) can

only be used to determine the percentage of free 89Zr and89Zr-DFO. It is thus important to select a HPLC column that

is able to distinguish dimers/aggregates from the native

protein and to use iTLC strips and an eluent that indeed

separates 89Zr–mAb from free 89Zr and 89Zr-DFO; (iv) immu-

noreactivity: this should not be affected by modification and

radiolabeling and can be examined by a Lindmo assay [13]

or enzyme-linked immunosorbent assay (ELISA); (v) apyr-

ogenicity: the product should be endotoxin free. This can be

assessed by, for example, an endosafe PTS reader; (vi) steri-

lity: the product should be filtered after preparation and the

filter integrity should be tested by, for example, a bubble

point test. Sterility tests have to be performed to ensure that

the product is sterile. Sterility determinations cannot be

done before injection, because of the time needed to per-

form this test. When no facilities are available for micro-

biological analysis of radioactive samples, medium can be

filtered and analyzed to monitor the filtration process.

Finally, during validation experiments it is important to

evaluate the stability of the 89Zr-labeled mAbs to ensure that

during storage the radiochemical purity and immunoreac-

tivity are preserved.

Comparison of DFO coupling strategies

There are five different DFO–mAb complexes (Scheme 1)

described in literature. Three DFO–mAb complexes use the

lysine groups of the mAb (Scheme 1, routes A–C) and two

DFO–mAb complexes use the thiol groups of specially engi-

neered ThiomAbs for coupling DFO (Scheme 1, routes D–E).

Modification of lysine groups is applicable for every mAb,

mAb fragment or peptide that contains lysine groups, while

the use of the modification via ThiomAbs is restricted to these

specially prepared mAbs.

89Zr–DFO–thioether–mAbs (A)

The synthesis of this thioether–DFO chelate and subse-

quently coupling to proteins has been described by Meijs

et al. in 1996 [14]. A S-acetylthioacetate (SATA) molecule is

coupled to DFO, a succinimidyl 4-(N-maleimidomethyl)

cyclohexane-1-carboxylate (SMCC) group to the mAb and

the resulting products are reacted with each other giving the

DFO–thioether–mAb products (Scheme 1, path A). Although

the first in vivo results looked promising for 89Zr–DFO–

thioether labeled mAbs A323/E3 and E48, showing biodis-

tribution comparable to the 99mTc and 123I-labeled analogs

[15], Verel et al. showed in 2003 [8] that 89Zr–DFO–thioether–

mAbs are not stable. Using mAb U36 they showed lower

tumor uptake, faster blood kinetics and lower uptake in all

non-target tissues of the 89Zr–DFO–thioether–U36 compared

to the hereafter described 89Zr–N-suc-DFO–U36. In vitro

experiments with 89Zr–DFO–thioether–U36 in serum showed

that the radioactivity transferred to serum albumin, while

also aggregates and 89Zr-labeled low molecular weight pro-

ducts were formed. The hypothesis for this instability is that

upon opening of the succinimide ring the chelate can break

off at either side of the sulfur atom. Once the S-containing89Zr–DFO fragment is formed, coupling with human serum

albumin (HSA) can occur, while cleavage at the other side of

the S-bond can result in the reactive S-atom at the mAb side,

making it susceptible to aggregation [8]. Therefore, this con-

jugation method is not suitable for stable coupling of DFO to

mAbs.

89Zr–N-suc-DFO–mAb (B)

In 2003 Verel et al. described the synthesis of iron-N-succinyl-

desferrioxamine-tetrafluorophenol ester (Fe–N-suc-DFO–TFP

ester) [8]. Till 2010 this was the chelate of choice for the

formation of stable 89Zr-labeled mAb conjugates (Scheme 1,

path B). In the preparation of Fe–N-suc-DFO–TFP ester, DFO is

first reacted with succinic anhydride resulting in N-succinyl-

DFO (N-suc-DFO), then the chelate DFO is temporarily

blocked with iron, and finally the acid group becomes acti-

vated to its Fe–N-suc-DFO–TFP-ester. The Fe–N-suc-DFO–TFP

www.drugdiscoverytoday.com e55


O

O

O

OO

O

O

O

O

O

O

O

O

OH

OH

DFONH2

NH

HON

N

N

HN OO

O

OH S

N

NCS

NCS

SCN

H

F

F F

FS

S

H

HN

HN

H

pH 6.5

pH 8.3

N

N

DFO

DFO

DFO

DFODFODFO

HS HSSH SH

HS SH

pH 9

S S

S S

DFO-Ac-ThiomAb

DFO-Chx-Mal-ThiomAb

pH 9

pH 7.5

DFO

DFO DFO

DFO

DFODFO

DFO

Fe-DFO

H

N-suc-DFO-mAb

H2N

H2N

H2N

1. pH 9.5

DFO-Bz-NCS-MAbDFO-thioether-MAb

1. FeCl32. TFP-OH

pyridine

pH 9 A

D1D2

E

BC

pH~9

2. removal of Fe, pH 4.2-4.5, EDTA

(b)

(a) (c)

(d)

(e)

N

O

S NHN

O

O

O

O

O O

O

O

OO

O

O

OO

O

N N

O

OO

O

O

O

OO

O

O

O

N

N

SHN

SHN I

I

BrBr

Br

S

SATA

NHS

S

OO

SHN

SMCC

N

O

ONO


Scheme 1. Summary of the different approaches to DFO–mAbs. Routes A–C are based on lysine modifications of mAbs and routes D1, D2 and E are

based on using specifically engineered Thiomabs having a cysteine group available for conjugation of DFO. In route A the synthesis of DFO–thioether–mAb

is depicted, in route B the synthesis of N-suc-DFO–mAb, in route C the synthesis of DFO–Bz–NCS–mAb, in route D the two synthesis possibilities of

DFO–Ac–ThiomAb and in route E the synthesis of DFO–CHx–Mal–Thiomab.



Table 1. Summary of preclinical data obtained with 89Zr–N-suc-DFO–mAb conjugates

mAb Target antigen Study results Refs

U36 CD44v6 Tumors as small as 19 mg containing 17 kBq 89Zr can be detected with PET. [16,17]89Zr–DFO–U36 can be used as scouting procedure for radioimmunotherapy with 90Y–DOTA–U36.

Slightly higher uptake of 89Zr in kidney, bone, liver and sternum.

cG250 G250 Comparable in vivo behavior of 89Zr–cG250 and 111In–DTPA–cG250 except for spleen, kidney

and liver, which showed a slightly higher percentage of 89Zr.

[18]

89Zr–DFO–cG250 PET images are superior to 111In–DTPA–cG250 SPECT images with respect

to tumor delineation.

Cetuximab EGFR 89Zr can predict the biodistribution of the residualizing labels 177Lu and 90Y, except for

the bone marrow uptake, which was significantly higher for 89Zr.

[19,24]

EGFR tumor expression levels do not correlate with the relative signal obtained with

PET. Possible causes: additional pharmacokinetic and pharmacodynamic mechanisms influence

tumor uptake of cetuximab.

Bevacizumab VEGF-A

binding

89Zr–DFO–bevacizumab can be used as specific VEGF tracer showing excellent

in vivo quantitative measurements.

[20,26]

89Zr–DFO–bevacizumab can be used for response monitoring upon treatment with

HSP90 inhibitor NVP-AUY922.

DN30 c-Met 89Zr–DFO–DN30 can be used as specific c-Met tracer and outperforms 124I–DN30 [21]

Zevalin CD20 89Zr–DFO–Zevalin and 88Y–DTPA–Zevalin show comparable biodistribution in tumor

bearing mice, except for the liver and bone accumulation, which was significantly higher for 89Zr.

[22]

Trastuzumab HER2 89Zr–DFO–trastuzumab can be used for imaging the HER2/neu status of the tumor. [23,25,29]89Zr–DFO–trastuzumab can be used for monitoring of HER2 downregulation by the

HSP90 inhibitor NVP–AUY922 and PU–H71

R1507 IGF-1R IGF-1R expression can be imaged with 89Zr–R1507 in triple-negative xenografts [27]

cG250–F(ab’)2 CAIX Hypoxia can be imaged with 89Zr–DFO–cG250–F(ab’)2 [28]

J591 PSMA 89Zr–DFO–J591 can be used for imaging and quantification of PSMA expression in prostate tumors [12]

Ranibizumab VEGF Alteration of VEGF expression during sunitinib treatment can be assessed

with 89Zr–DFO–ranibizumab.

[30]

ester can be stored in acetonitrile at �808C for at least a year

without hydrolysis of the TFP-ester. When the protection of

the DFO group with iron was omitted, intractable results were

obtained. In that case the TFP ester most probably reacted

with one of the hydroxamate groups and thus no conjugation

to proteins was possible anymore. When N-suc-DFO was first

radiolabeled with 89Zr, then no TFP-ester could be formed.

This prelabeling strategy is normally applied for other radio-

nuclide–chelate combinations like for 99mTc-MAG3 and this

would circumvent the protection and deprotection step with

iron. We hypothesize that zirconium is not fully saturated by

the coordination of the three hydroxamate groups of N-suc-

DFO and uses additional groups for coordination like the acid

group of N-suc-DFO, which is then not available anymore for

activation to its TFP-ester. Although the synthesis of Fe–TFP–

N-suc-DFO-ester is time consuming, the advantage of this

chelate is that the chelate-to-mAb molar ratio can easily be

determined by the analysis of the reaction mixture by size

exclusion chromatography HPLC using UV analysis at

430 nm for the assessment of the amount of Fe–N-suc-DFO

that is attached to a mAb. For obtaining optimal radioimmu-

noconjugates it is important that not too many DFO groups

are coupled per mAb molecule, because otherwise immunor-

eactivity, pharmacokinetics and pharmacodynamics of the

mAb can change. This is normally achieved when on average

less than four DFO molecules are coupled per mAb molecule,

and in practice mostly just one DFO molecule is coupled [8].

After coupling of Fe–N-suc-DFO to a mAb, iron must be

removed. It has been proven that a 100-fold molar excess of

ethylenediaminetetraacetic acid (EDTA) at pH 4.3–4.5 effi-

ciently transchelates iron to EDTA and that the integrity of

the mAb is not harmed under these conditions. Via this

method several commercial available and new therapeutic

mAbs have been labeled and their in vivo behavior assessed in

preclinical and clinical studies. An overview of the achieve-

ments with 89Zr–N-suc-DFO–mAbs in a preclinical setting is

provided in Table 1. The first studies described the compar-

able in vivo behavior of 89Zr-labeled mAbs with their 90Y, 111In

and 177Lu-labeled mAb counterparts [16–20], while later the

potential of 89Zr-immuno-PET was shown in response mon-

itoring and validation of new antibodies [12,21–30] (Table 1).

The first clinical study ever with a 89Zr-labeled mAb was

reported in 2006 by Borjesson et al. [31]. This feasibility study

with 20 patients showed that 89Zr–N-suc-DFO–U36 can be



(a)

(b)

0.28 g 0.76 g

Max

Min

A B C


Figure 2. (a) PET images of FaDu xenograft bearing nude mouse obtained at 72 hours post injection of 89Zr–N-suc-DFO–cmAb U36. Coronal slices from

ventral (left) to dorsal (right). Tumor is indicated with arrows [35]; (b) PET images of a head and neck cancer patient with a tumor in the left tonsil (thick

arrow) and lymph node metastases (thin arrows) obtained at 72 hours post injection of 89Zr–N-suc-DFO–cmAb U36 (A: sagittal image; B: axial image and C:

coronal image) [31].

safely applied in patients and that it is a promising method

for imaging of primary head and neck tumors as well as

metastases in the neck. All primary tumors (n = 17) were

detected (Fig. 2). Furthermore lymph node metastases in

18 of 25 positive levels and in 11 of 15 positive sides were

detected. The missed tumor involved lymph nodes were

relatively small and contained just a small proportion of

tumor tissue and were also missed in most cases by computed

tomography (CT) and/or magnetic resonance imaging (MRI).

The sensitivity of immuno-PET for the detection of lymph

node metastases was at least as good as of CT/MRI: 72% versus

60% [31].

In aforementioned study with 89Zr-U36, also biodistribu-

tion, radiation dose and quantification potential of immuno-

PET were assessed [32]. PET quantification of blood-pool

activity in the left ventricle was in good agreement with

sampled blood activity (difference equals 0.2 � 16.9%),

except for heavy-weight patients (>100 kg). The same

accounts for the uptake in tumor tissue, where a good agree-

ment was observed between the PET-derived data and biopsy

data (mean deviation: �8.4 � 34.5%). This suggests that

patients with high and low uptake can be differentiated

and therefore it is possible to select patients who may or

may not benefit from mAb therapy. The mean radiation dose

for patients in this study receiving 74 mBq 89Zr was 40 mSv,

which is high and will limit repeated application of


89Zr–mAbs. However with the next generation PET/CT scan-

ners a lower 89Zr radioactivity dose could be used while the

image quality is preserved.

High quality images at lower radioactivity dose have been

shown by Dijkers et al. in 2010 [33]. In this study 37 MBq 89Zr-

trastuzumab showed excellent quality images at an effective

dose of 20 mSv. In this feasibility study with 14 patients three

different dose cohorts were evaluated: 10 or 50 mg for tras-

tuzumab-naıve patients and 10 mg for patients on trastuzu-

mab treatment. It was proven that the latter two performed

equally. Although this study was not aiming for the compar-

ison with conventional staging modalities or for assessing

specificity and sensitivity, lesions with 89Zr-trastuzumab

uptake were generally in good agreement with CT, MRI

and bone scans. PET images showed a high spatial resolution,

and a good signal-to-noise ratio, which resulted in an image

quality unapproachable by previous 111In-trastuzumab single

photon emission computed tomography (SPECT) scans.

Excellent visualization of mAb uptake in HER2-positive

lesions as well as in metastatic liver, lung, bone and even

brain HER2-positive lesions was observed. The best moment

to assess 89Zr-trastuzumab tumor uptake was four to five days

post injection. 89Zr-trastuzumab PET allowed quantification

of conjugate uptake in HER2-positive lesions, and it became

clear that for some patients with extensive tumor load no

HER2 saturation occurred during trastuzumab therapy.


Although this N-suc-DFO–TFP method for labeling of 89Zr

showed great potential in clinical immuno-PET studies, there

are also some drawbacks of this method. The lengthy proce-

dure which is relatively complicated and time consuming is

challenging with respect to good manufacturing practice

(GMP) compliancy. Furthermore the low pH (pH 4.3–4.5)

as used during the removal of iron from the chelate by EDTA

can be harmful for the mAb.

89Zr–DFO–Bz–NCS–mAb (C)

The disadvantages of the above described method of coupling

DFO to mAbs have been overcome by the introduction of a

new DFO chelate, p-isothiocyanatobenzyl-desferrioxamine

(DFO–Bz–NCS), that has been published in 2010 [34,35].

DFO–Bz–NCS has been prepared by the reaction of DFO with

1,4-phenylendiisothiocyanate and is commercially available,

also at cGMP grade, from Macrocyclics (Dallas, TX, USA). The

isothiocyanate group is frequently used as reactive group for

conjugation to mAbs or other biologicals and reacts with

lysine groups of mAb resulting in thiourea bonds (Scheme 1,

path C). Because the isothiocyanate group is not reactive

toward hydroxamate groups, DFO does not need to be tem-

porarily blocked with iron and thus making this strategy

faster, easier and more widely applicable. With this method

precautions should be taken to prevent aggregation of the

protein during coupling of the chelate to the mAb. DFO–Bz–

NCS in dimethylsulfoxide (DMSO) should be added stepwise

while shaking to prevent high local concentrations of DMSO

resulting in excessive local reaction of DFO to mAb and thus

in aggregate formation [34].

This method has been applied for the coupling of 89Zr to

mAb U36, rituximab and cetuximab by Perk et al. [35] and the

performance of this new chelate was comparable with that of

the previously discussed N-suc-DFO chelate with respect to in

vitro stability and in vivo biodistribution. In vitro stability of89Zr–DFO–Bz–NCS–mAbs, however, appeared to be depen-

dent on the storage buffer. The best storage conditions proved

to be 48C and sodium acetate buffer in the presence of the

anti-oxidant gentisic acid. The presence of Cl� should be

avoided, because this results in impaired integrity. Most

Table 2. Comparison of technologies for the conjugation of DF

Technology DFO–thioether–

mAbs (A)

N-suc-DFO–

mAbs (B)

Modificationa 3 steps 5 steps

Reaction conditions pH 6.5–9 pH 4.3–9.5

Stability Poor Good

Generally applicable Yes Yes

Clinical application No Yes, current stan

a Starting from DFO.

probably due to radiation induced formation of OCl� ions,

which react with the SH group of the enolizable thiourea

bond and thus cause detachment of the chelate-89Zr [35].

DFO–Ac–Thiomab (D) and DFO–Chx–Mal–ThiomAb (E)

In 2010 another new DFO-based conjugation method to

MAbs has been reported using site-specifically modified

mAbs. These so-called thiomAbs contain two engineered

cysteine residues in the protein, which can be used for

coupling of DFO [36]. These ThiomAbs could provide tracers

with unaltered binding affinity and scaffold stability. To this

end DFO is reacted with (i) bromoacetyl bromide resulting in

bromoacetyl–DFO (DFO–Bac), (ii) N-hydroxysuccinimidyl

iodoacetate resulting in iodoacetyl–DFO (DFO–Iac) or (iii)

SMCC resulting in maleimidocyclohexyl–DFO (DFO–Chx–

Mal). Then thio-trastuzumab is reacted with either of these

DFO-constructs resulting in DFO–Ac–thio-trastuzumab (D) or

DFO–Chx–Mal-thio-trastuzumab (E) (Scheme 1, routes D and

E). Tinianow et al. showed that these 89Zr–DFO–thio-trastu-

zumab constructs have comparable in vitro and in vivo char-

acteristics as the randomly modified lysine conjugates 89Zr–

N-suc-DFO–trastuzumab (B) and 89Zr–DFO–Bz–NCS–trastuzu-

mab (C) [36]. Therefore, side-specific conjugation of DFO to

ThiomAbs did not show an advantage over random lysine

modification of mAbs in the model employed.

Conclusion

Five different approaches to 89Zr–DFO–mAbs are reported in

literature, of which three in 2010, which exemplifies the

growing interest in 89Zr-immuno-PET. This is also supported

by the increasing number of publications in recent years:

while between 2003 and 2009 on average one or two papers

were published per year, in 2010 more than ten research

papers have been published on the application of 89Zr-labeled

compounds.

The most important characteristics of the five different

DFO–mAbs have been summarized in Table 2. Because all

chelates consist of a DFO moiety for the chelation of 89Zr the

linker determines in general the stability of the 89Zr-labeled

mAbs. Except for 89Zr–DFO–thioether–mAb (A) all other four

O to mAbs

DFO–Bz–NCS–

mAb (C)

DFO–ThiomAbs

(D/E)

2 steps 2 steps

pH 7–9 pH 7.5–9

Good Good

Yes No, specially engineered

mAbs

dard First trials will start soon –



89Zr–DFO–mAbs (B–E) show good stability and comparable in

vivo behavior. Comparison of 89Zr-labeled mAbs with other

radiometal–mAb complexes like 177Lu–mAbs in preclinical

studies revealed that bone uptake was higher for the 89Zr–

mAb constructs. This can either be explained by release of89Zr from DFO in the blood or via catabolism in tumor and for

example, liver and redistribution in the blood and subse-

quent uptake in the bone. However, up to now no conclusion

can be drawn from the available preclinical studies on what

the actual cause is of the slightly increased bone uptake seen

in some cases. Differences between the lysine modified 89Zr–

DFO–mAbs B and C and the cysteine modified 89Zr–DFO–

mAbs D and E are not the stability but the ease of synthesis

and application. While the lysine modifications are general

applicable, the cysteine modification is limited to specially

engineered antibodies, which limits the application of this

strategy. The difference between these cysteine modified

DFO–mAbs is small, the DFO–Chx–Mal–ThiomAb (E) is

slightly faster synthesized than DFO–Ac–Thiomab (D), but

their in vitro and in vivo behavior is comparable. There are

three differences between the lysine modified mAbs: (i) TFP–

N-suc-DFO needs to be synthesized from DFO, while DFO–Bz–

NCS is commercially available; (ii) preparation of 89Zr-labeled

mAbs using TFP–N-suc-DFO requires an extra de-iron step at

relatively low pH after coupling to mAbs compared to the

DFO–Bz–NCS (Table 2) and (iii) the DFO–Bz–NCS in DMSO

requires more careful handling, exemplified by the faster

aggregate formation with this DFO-chelate. In conclusion,

stable 89Zr-labeled mAbs can be prepared for clinical applica-

tion and we envision that in the near future DFO–Bz–NCS will

take over TFP–N-suc-DFO as DFO-chelate.

Acknowledgements

Funding support was provided by CTMM, the Center for

Translational Molecular Medicine (http://www.ctmm.nl,

project AIRFORCE number 030-103); and the European Com-

munity Seventh Framework Programme (FP7/2007-2012

under grant agreement no. Health-F2-2008-201342: ADA-

MANT).

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