ORIGINAL PAPER

177Lu–DO3A–HSA–ZEGFR:1907: characterization as a potentialradiopharmaceutical for radionuclide therapyof EGFR-expressing head and neck carcinomas

Susan Hoppmann • Shibo Qi • Zheng Miao •

Hongguang Liu • Han Jiang • Cathy S. Cutler •

Ande Bao • Zhen Cheng

Received: 18 December 2011 / Accepted: 7 March 2012 / Published online: 16 March 2012

� SBIC 2012

Abstract Epidermal growth factor receptor 1 (EGFR) is an

attractive target for radionuclide therapy of head and neck

carcinomas. Affibody molecules against EGFR (ZEGFR)

show excellent tumor localizations in imaging studies.

However, one major drawback is that radiometal-labeled

Affibody molecules display extremely high uptakes in the

radiosensitive kidneys which may impact their use as radi-

otherapeutic agents. The purpose of this study is to further

explore whether radiometal-labeled human serum albumin

(HSA)–ZEFGR bioconjugates display desirable profiles for

the use in radionuclide therapy of EGFR-positive head and

neck carcinomas. The ZEFGR analog, Ac–Cys–ZEGFR:1907,

was site-specifically conjugated with HSA. The resulting

bioconjugate 1,4,7,10-tetraazacyclododecane-1,4,7-triace-

tic acid (DO3A)–HSA–ZEGFR:1907 was then radiolabeled

with either 64Cu or 177Lu and subjected to in vitro cell uptake

and internalization studies using the human oral squamous

carcinoma cell line SAS. Positron emission tomography

(PET), single photon emission computed tomography

(SPECT), and biodistribution studies were conducted using

SAS-tumor-bearing mice. Cell studies revealed a high

(8.43 ± 0.55 % at 4 h) and specific (0.95 ± 0.09 % at 4 h)

uptake of 177Lu–DO3A–HSA–ZEGFR:1907 as determined by

blocking with nonradioactive ZEGFR:1907. The internaliza-

tion of 177Lu–DO3A–HSA–ZEGFR:1907 was verified in vitro

and found to be significantly higher than that of 177Lu-

labeled ZEFGR at 2–24 h of incubation. PET and SPECT

studies showed good tumor imaging contrasts. The biodis-

tribution of 177Lu–DO3A–HSA–ZEGFR:1907 in SAS-tumor-

bearing mice displayed high tumor uptake (5.1 ±

0.44 % ID/g) and liver uptake (31.5 ± 7.66 % ID/g) and

moderate kidney uptake (8.5 ± 1.08 % ID/g) at 72 h after

injection. 177Lu–DO3A–HSA–ZEGFR:1907 shows promising

in vivo profiles and may be a potential radiopharmaceutical

for radionuclide therapy of EGFR-expressing head and neck

carcinomas.

Keywords 177Lu � Affibody � Epidermal growth factor

receptor � Human serum albumin � Radionuclide therapy

Introduction

Epidermal growth factor receptor 1 (EGFR) is an approx-

imately 180-kDa transmembrane glycoprotein consisting of

an extracellular-ligand-binding domain, a transmembrane

domain, and an intracellular domain with tyrosine kinase

activity for signal transduction. As a member of the erbB

S. Hoppmann � S. Qi � Z. Miao � H. Liu � H. Jiang �Z. Cheng (&)

Molecular Imaging Program at Stanford (MIPS),

Department of Radiology and Bio-X Program,

Canary Center at Stanford for Cancer Early Detection,

Stanford University,

1201 Welch Road,

Lucas Expansion, P095,

Stanford, CA 94305, USA

e-mail: zcheng@stanford.edu

C. S. Cutler

Research Reactor Center (MURR),

Radiopharmaceutical Sciences Institute,

Nuclear Engineering and Sciences Institute,

Nuclear Engineering,

University of Missouri,

Columbia, MO 65211, USA

A. Bao

Departments of Radiology and Otolaryngology—Head

and Neck Surgery,

University of Texas Health Science Center at San Antonio,

San Antonio, TX 78229, USA

123

J Biol Inorg Chem (2012) 17:709–718

DOI 10.1007/s00775-012-0890-3

receptor family, EGFR is widely expressed in most epi-

thelial cells from several normal tissues, including skin,

liver, and basal cells of the prostate epithelium [1–3].

Dysregulation of EGFR disrupts normal cellular patterns

by increasing cell proliferation and angiogenic potential

and inhibiting apoptosis [4]. Thus, overexpression of

EGFR has been frequently detected in a wide range of

human tumors, including small cell carcinoma of the head

and neck [1–3, 5–8]. Moreover, recent studies have dem-

onstrated a correlation between EGFR overexpression and

metastasis formation, therapy resistance, poor prognosis,

and short survival for some cancer types [1, 3, 5–9].

Head and neck squamous cell carcinoma (HNSCC),

including cancers of the oral cavity, oropharynx, hypo-

pharynx, and larynx, is one of the most commonly diag-

nosed cancers worldwide, with over 600,000 new cases

every year [10]. Although there have been slightly

decreased incidence and mortality rates during recent

years, 260 new cases and 11,480 deaths of HNSCC

occurred in the USA alone in 2010 [11]. HNSCC has been

characterized by high levels of EGFR expression in

approximately 90 % of tumors [12], correlating with poor

clinical outcome [13], decreased response to radiotherapy,

and increased locoregional recurrence following definitive

radiotherapy [14].

EGFR has become an attractive target for treatment of

HNSCC. Inhibition of tumor growth can be obtained by

either blocking the extracellular-ligand-binding domain of

EGFR or by inhibiting intracellular tyrosine kinase activity

[15]. Owing to the intrinsic radiosensitivity of this tumor

type, targeted radionuclide therapy offers a realistic

approach to improve the treatment of HNSCC [16].

Affibody proteins have been shown to be a promising

platform for the development of imaging or therapeutic

agents for different molecular targets [17, 18]. Affibody

molecules are engineered small proteins with 58 amino acid

residues (6–7 kDa) and a three-a-helical bundle structure,

as derived from one of the IgG-binding domains of staph-

ylococcal protein A [19]. Several anti-EGFR Affibody

proteins (ZEFGR) with high affinities in the nanomolar range

have been reported recently [20, 21]. Particularly

ZEGFR:1907 was demonstrated to show excellent EGFR-

tumor-targeting abilities, which was confirmed in our

research using either 64Cu or Cy5.5 fluorescent dye labeled

ZEGFR:1907 for small-animal positron emission tomography

(PET) and optical imaging studies [22, 23]. However,

besides the high and specific tumor imaging contrast, che-

lator modified and radiometal-labeled Affibody proteins

typically exhibit extremely high renal uptake (more than

100 % of the injected dose per gram of tissue, ID/g). This

could result in very high radiation doses to the radiation-

sensitive kidneys and thus represents a critical concern

when using Affibody molecules for radionuclide therapy.

We have recently demonstrated that conjugation of human

serum albumin (HSA) with an anti-human epidermal growth

factor receptor 2 (HER2) Affibody (ZHER2) can produce

HSA–ZHER2 bioconjugates which exhibit improved phar-

macokinetics in terms of high tumor uptake, but dramatically

reduced kidney uptake [24]. These promising results have

encouraged us to further explore whether radiometal-labeled

HSA–ZEFGR bioconjugates (Fig. 1) could display desirable in

vivo profiles and be suitable for radionuclide therapy of

EGFR-positive HNSCC.

The EGFR-binding Affibody molecule, ZEGFR:1907, and

the metal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,

10-tetraacetic acid (DOTA) were chemically conjugated to

HSA to produce the Affibody–HSA bioconjugate DO3A–

HSA–ZEGFR:1907. To study its in vitro cell uptake and inter-

nalization as well as its in vivo pharmacokinetics, biodistri-

bution, and potential radiotherapeutic applications, DO3A–

HSA–ZEGFR:1907 was radiolabeled with the PET radionuclide64Cu [t1/2 = 12.7 h, Ebþ = 278.2 keV (17.4 %), Eb� =

190.4 keV (39.0 %)] and the therapeutic and single photon

emission computed tomography (SPECT) radionuclide 177Lu

[t1/2 = 6.647 days, Eb� = 149.1 keV (78.6 %), Ec =

208.4 keV (11.0 %)], respectively. The use of 64Cu as a

radionuclide provides the possibility to obtain the in vivo

behaviors, e.g., tumor-targeting ability and clearance of

DO3A–HSA–ZEGFR:1907 through quantitative PET analysis.

Furthermore, the resulting 64Cu–DO3A–HSA–ZEGFR:1907

may also be a promising agent for PET EGFR imaging.

The biologic profiles of the resulting radiopharmaceuticals

(64Cu–DO3A–HSA–ZEGFR:1907 and 177Lu–DO3A–HSA–

ZEGFR:1907) were evaluated in human oral squamous carci-

noma cell line SAS and in nude mice bearing subcutaneous

SAS tumors expressing high levels of EGFR [25].

Materials and methods

General

The Affibody molecule Ac–Cys–ZEGFR:1907 (Ac-CVDNK

FNKEMWAAWEEIRNLPNLNGWQMTAFIASLVDDPS

QSANLLAEAKKLNDAQAPK-NH2) was synthesized

and analyzed as previously described [22]. The 1,4,7,

10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-

N-hydroxysuccinimide ester (DOTA-NHS ester) was

obtained from Macrocyclics. Sulfosuccinimidyl 4-[N-male-

imidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) was

purchased from Thermo Fisher Scientific. HSA and all other

standard reagents were purchased from Sigma-Aldrich.64CuCl2 was provided by the Department of Medical Physics,

University of Wisconsin at Madison. 177LuCl3 was provided

by the University of Missouri (Research Reactor Center) with

710 J Biol Inorg Chem (2012) 17:709–718

123

a specific activity of 25 Ci/mg. Human oral squamous car-

cinoma cell line SAS was obtained from the American Type

Culture Collection. The cells were maintained in high-glu-

cose Dulbecco’s modified Eagle’s medium (DMEM) sup-

plemented with 10 % fetal bovine serum, 100 U penicillin

per milliliter, and 100 lg streptomycin per milliliter (Invit-

rogen). Female athymic nude mice (nu/nu) were purchased

from Charles River Laboratories. All other instruments,

including the radioactive dose calibrator, were the same as

those previously reported [26].

Bioconjugation of HSA with DOTA

and Ac–Cys–ZEGFR:1907

The conjugation of DOTA-NHS ester and Ac–Cys–

ZEGFR:1907 was performed in the same fashion as described

previously for preparation of HSA–ZHER2 bioconjugates

[24]. Briefly, the HSA was conjugated to DOTA-NHS ester

in 200 lL borate buffer (50 mM, pH 8.5) in a molar ratio

of 1:100. The resulting product is referred to as DO3A–

HSA to indicate it is no longer a full-fledged DOTA che-

lator but is rather a derivative of DO3A. The HSA–DO3A

conjugate was then reacted with 1 mg sulfo-SMCC in a

molar ratio of 1:5. Finally, Ac–Cys–ZEGFR:1907 was site-

specifically conjugated to the DO3A- and Sulfo-SMCC-

modified HSA via the cysteine residue. The reaction was

performed in a molar ratio of 1:5 using 200 lg DO3A–

HSA–sulfo-SMCC and 150 lg Ac–Cys–ZEGFR:1907. The

final bioconjugate, DO3A–HSA–ZEGFR:1907, was then

concentrated through a microcentrifuge tube (Millipore,

30 kDa, 0.5 mL) to a final volume of 20 lL. After each

step, the protein concentration was measured by the bicinch-

oninic acid assay (Pierce), and the samples were analyzed via

matrix-assisted laser desorption/ionization time of flight mass

spectrometry. The 1,4,7,10-tetraazacyclododecane-1,4,7-tria-

cetic acid-10-maleimidoethylacetamide-conjugated Ac–Cys–

ZEGFR:1907, DO3A–ZEGFR:1907, which was reported by us

before, was also used in our studies for comparison [22].

Radiolabeling of DO3A–HSA–ZEGFR:1907

Approximately 200 lg DO3A–HSA–ZEGFR:1907 was radi-

olabeled with either 64Cu or 177Lu by addition of

37–74 MBq (1–2 mCi) of 64CuCl2 or 177LuCl3 in 0.1 N

sodium acetate buffer of pH 6.0 or pH 5.0, respectively.

After an incubation time of 1 h at 39 �C, the radiolabeled

complexes were purified by a PD-10 column (GE Health-

care), eluted with phosphate-buffered saline (PBS; pH 7.4),

and passed through a 0.22-lm Millipore filter for both

in vitro cell uptake studies and animal experiments. To

prevent radiolysis, 1 % ascorbic acid was added to the177Lu-labeled bioconjugate. For comparison, radiolabeling

of DO3A–ZEGFR:1907 with 177Lu was performed in the

same way as for DO3A–HSA–ZEGFR:1907.

In vitro stability

The stability of 177Lu–DO3A–HSA–ZEGFR:1907 was asses-

sed in vitro by adding 50 lL tracer to 1 mL mouse serum

(final activity concentration 2.59 MBq/mL; 70 lCi/mL).

The samples were incubated at 37 �C. The stability of the

tracer was assessed by size-exclusion chromatography on

Fig. 1 A 1,4,7,10-

tetraazacyclododecane-1,4,7-

triacetic acid (DO3A)–human

serum albumin (HSA)–anti-

epidermal growth factor

receptor 1 (EGFR) Affibody

(ZEGFR:1907) bioconjugate. The

red regions indicate the lysine

residues of HSA suitable for

conjugation with 1,4,7,10-

tetraazacyclododecane-1,4,7,10-

tetraacetic acid (DOTA) and

ZEFGR molecules (bluestructures). The radiolabeling

occurs via complexation of

either 177Lu or 64Cu with the

DO3A chelator of the

bioconjugates

J Biol Inorg Chem (2012) 17:709–718 711

123

NAP-5 columns (GE Healthcare) at incubation times of 4,

24, 48, 72, and 168 h. The column was equilibrated with

PBS and the contents were eluted at room temperature;

0.35-mL fractions were collected. The radioactivity of all

fractions was counted using a PerkinElmer 1470 automatic

c-counter, and the stability was expressed as the percentage

of the radioactivity in the original tube.

In vitro cell uptake and internalization assays

In vitro cell uptake assays of both 64Cu–DO3A–HSA–

ZEGFR:1907 and 177Lu–DO3A–HSA–ZEGFR:1907 were per-

formed as previously described [24]. Briefly, SAS cells

(2 9 105 per well) were seeded in 12-well tissue culture

plates and allowed to attach overnight. The cells were

washed twice with serum-free DMEM and incubated with

the probes [1.5 lCi per well, final concentration approxi-

mately 6 nM (0.2 lg)] in 400 lL serum-free DMEM at 4

and 37 �C. The nonspecific binding of the probes with SAS

cells was determined by co-incubation with nonradioactive

Ac–Cys–ZEGFR:1907 (6 lg per well, final concentration

2.14 lM). After 0.5, 1, 2, and 4 h (177Lu–DO3A–HSA–

ZEGFR:1907) and 2 h (64Cu–DO3A–HSA–ZEGFR:1907) the

cells were washed three times with cold PBS and lysed

with the addition of 200 lL of 0.2 M NaOH. The radio-

activity of all fractions was counted using a PerkinElmer

1470 automatic c-counter. The uptake (counts per minute)

was expressed as the percentage of added radioactivity.

For the cell internalization assay, SAS cells (5 9 105 per

well) were seeded in six-well tissue culture plates and

allowed to attach overnight. The cells were washed twice

with serum-free DMEM medium and incubated with 177Lu–

DO3A–HSA–ZEGFR:1907 and 177Lu–DO3A–ZEGFR:1907 [each

5 lCi per well, final concentrations approximately 10 nM

(0.66 lg) and approximately 10 nM (0.1 lg), respectively]

in 800 lL serum-free DMEM at 37 �C. After 1 min, 30 min,

1 h, 2 h, 4 h, 6 h, 12 h, and 24 h the medium was collected

and the cells were washed twice with cold PBS. Then, the

cells were treated with an acid washing buffer (0.2 M glycine/

HCl buffer with 4 M urea, pH 2.0) for 5 min at 4 �C and

additionally rinsed with the same buffer. The solution col-

lected was considered as the membrane-bound fraction. The

cells were lysed in 500 lL of 0.2 M NaOH and the solution

collected was considered as the internalized fraction. The

radioactivity of all fractions was counted using a PerkinElmer

1470 automatic c-counter.

Small-animal PET

All animal studies were performed in compliance with

federal and local institutional rules for animal experimen-

tation. Protocols were approved by the Stanford Adminis-

trative Panel on Laboratory Animal Care (APLAC 18086).

Approximately 3 9 106 SAS cells suspended in PBS were

subcutaneously implanted in the right upper shoulder of

female athymic nu/nu mice. Tumors were allowed to grow

to 0.5–0.7 cm in diameter (3–4 weeks). The tumor-bearing

mice were subjected to in vivo biodistribution and imaging

studies. Small-animal PET of tumor-bearing mice (n = 4

for each group) was performed using a micro-PET R4

rodent-model scanner (Siemens Medical Solutions USA).

A 180–210-lCi dose (6.7–7.8 MBq, 36–42 lg) of 64Cu–

DO3A–HSA–ZEGFR:1907 was injected via the tail vein into

mice bearing SAS tumors . At various times after injection

(1, 4, 24, 48, and 72 h) the mice were anesthetized with

2 % isoflurane and placed in the prone position near the

center of the field of view of the scanner. Static scans (for

1, 4, and 24 h, 3-min scans; for 48 and 72 h, 5-min scans)

were obtained, and the images were reconstructed by a

two-dimensional ordered subsets expectation maximum

algorithm. The method for quantification analysis of small-

animal PET images was the same as previously reported

[26].

Small-animal SPECT/X-ray computed tomography

For small-animal SPECT and X-ray computed tomography

(CT), a 200–300-lCi dose (7.4–11.1 MBq, 26.7–40.5 lg)

of 177Lu–DO3A–HSA–ZEGFR:1907 was injected via the tail

vein into SAS-tumor-bearing mice (n = 3). After 4, 24, 72,

and 96 h, the mice were anesthetized with 2 % isoflurane

and placed in the prone position near the center of the field

of view of the scanner. Nuclear imaging and CT were

performed with a combined SPECT/CT scanner for small

animals (X-SPECT; Gamma Medica).

For micro-CT image acquisition, the 512 images (170-lm

slice thickness) were acquired in 5 min at 0.4 mA and

80 kVp. SPECT was performed using a 1-mm multipinhole

collimator (single head, 360� of rotation, 64 projections, 30 s

per projection, and a 5-cm field of view). The SPECT and

CT images were then reconstructed as described previously

[24]. All data were imported into Amira (Mercury Com-

puting Systems, Chelmsford, UK) for processing and

visualization.

Biodistribution studies

For biodistribution studies, a 20–25-lCi dose (740–925 kBq;

2.7–3.3 lg) of 177Lu–DO3A–HSA–ZEGFR:1907 was injected

through the tail vein into mice bearing SAS xenografts (n = 4

for each group), and they were killed at different times after

injection (1, 4, 24, 48, 72, 120, and 240 h). Tumor and normal

tissues were excised and weighed, and their radioactivity

measured using a c-counter. The radioactivity uptake in the

tumor and normal tissues was expressed as the percentage of

injected radioactivity per gram of tissue.

712 J Biol Inorg Chem (2012) 17:709–718

123

Statistical analysis

Statistical analysis was performed using Student’s two-

tailed t test for unpaired data. A 95 % confidence level was

chosen to determine the significance between groups, with

P \ 0.05 being significantly different.

Results

Synthesis, conjugation, radiochemistry, and stability

Ac–Cys–ZEGFR:1907 with an N-terminal cysteine residue

and DOTA were successfully conjugated with HSA. Three

DO3A molecules were conjugated on average to one HSA

molecule as determined by matrix-assisted laser desorp-

tion/ionization time of flight mass spectrometry. The

average molecular masses of DO3A–HSA–ZEGFR:1907

bioconjugates were 75.3, 81.8, 89.6, 95.0 and 101.9 kDa,

representing different numbers of Affibody molecules

conjugated per HSA protein (one to five Affibody mole-

cules on each HSA molecule). Owing to the more complex

nature of the final agent and the conjugation method,

several bioconjugates (e.g., bioconjugates with a different

number of DO3A molecules) may be hidden under one

average molecular mass signal. DO3A–HSA–ZEGFR:1907

was then successfully radiolabeled with either 64Cu or177Lu. Purification of the radioactive reaction mixtures

using a PD-10 column resulted in 64Cu–DO3A–HSA-

ZEGFR:1907 and 177Lu–DO3A–HSA–ZEGFR:1907 with decay-

corrected yields of more than 70 %. High specific activities

of 64Cu–DO3A–HSA–ZEGFR:1907 or 177Lu–DO3A–HSA–

ZEGFR:1907 (14–36 MBq/nmol, 5.0–12.9 mCi/mg) were

obtained at the end of the synthesis.

The serum stability showed that 177Lu–DO3A–HSA–

ZEGFR:1907 has good resistance to dissociation of 177Lu

from the tracer, with more than 95 % of the probe intact at

an incubation time of 4 h and more than 80 % of the probe

intact at incubation times of 24, 48, 72, and 168 h in mouse

serum.

In vitro assay and small-animal PET of 64Cu–DO3A–

HSA–ZEGFR:1907

The uptake of 64Cu–DO3A–HSA–ZEGFR:1907 in SAS cells

over an incubation period of 2 h at 37 �C is shown in

Fig. 2a. The probe showed a high and specific uptake

(26.84 ± 2.5 vs. 9.64 ± 1.51 % for the control and the

blocking group, respectively). Decay-corrected coronal and

transaxial microPET images of a mouse bearing SAS

tumor at 1, 4, 24, and 48 h after tail vein injection of 64Cu–

DO3A–HSA–ZEGFR:1907 are shown in Fig. 2b. The SAS

tumor was visible but with a low tumor-to-background

contrast at 1 and 4 h after injection, and excellent tumor-to-

background contrasts were observed at 24, 48, and 72 h

after injection. Quantification analysis revealed that the

SAS tumor uptake values increased with time, and they

were 3.34 ± 0.21, 5.19 ± 0.35, 7.01 ± 1.13, 7.72 ± 1.14,

and 7.48 ± 0.96 % ID/g at 1, 4, 24, 48 and 72 h, respec-

tively (Fig. 2c). In addition to the tumor, moderate activity

accumulation was observed in the liver (e.g., 11.34 ±

1.08 % ID/g at 24 h), but much lower activity accumula-

tion was found in the kidneys (e.g., 2.45 ± 1.72 % ID/g at

24 h). Relatively low radioactivity concentrations were

found in the blood (region of interest over the heart):

4.73 ± 0.49 and 3.51 ± 0.31 % ID/g at 1 and 4 h after

injection., respectively (Fig. 2c). Although the stability of

this conjugate was not tested in this study, previous studies

using a 64Cu–DOTA–Affibody conjugate showed that the

complex is stable in mouse plasma in vitro. A low (below

5 %) decomposition was found at an incubation time of 4 h

in mouse serum in vitro [22].

In vitro assays of 177Lu–DO3A–HSA–ZEGFR:1907

Cell uptake of 177Lu–DO3A–HSA–ZEGFR:1907 at 37 and 4 �C

over an incubation period of 0.5–4 h is shown in Fig. 3a.177Lu–DO3A–HSA–ZEGFR:1907 accumulated slowly in the

SAS cells and reached 1.97 ± 0.08 % of the applied activity

at 0.5 h at 37 �C. The uptake increased to 8.43 ± 0.55 % at

4 h at 37 �C. An approximate fourfold to fivefold lower

accumulation of 177Lu–DO3A–HSA–ZEGFR:1907 in the cell

lysates was observed at all time points at 4 �C compared with

37 �C, indicating internalization that occurs at physiologic

temperature. Finally, cell surface receptor binding and

internalization of the radiopharmaceutical were shown to be

inhibited by the presence of a large molar excess of unlabeled

ZEGFR:1907 (P \ 0.05) at all incubation time points (e.g.,

0.95 ± 0.09 % at 4 h). Cell association of 177Lu–DO3A–

HSA–ZEGFR:1907 at 4 h was inhibited by 80 and 65 % at 37

and 4 �C, respectively, indicating that the probe was specif-

ically targeting EFGR.

The internalization data for 177Lu–DO3A–HSA–

ZEGFR:1907 and 177Lu–DO3A–ZEGFR:1907 are shown in

Fig. 3b and c, respectively. For both radiopharmaceuticals,

the percentage of the internalized tracers was much higher

than that of the cell-surface-bound fraction. 177Lu–DO3A–

HSA–ZEGFR:1907 showed a relatively slow internalization,

with 1.45 ± 0.1 and 7.9 ± 0.5 % internalization of the

receptor-bound tracer at incubation times 1 and 30 min,

respectively, whereas 38.74 ± 1.4 % of the tracer was inter-

nalized at 24 h. In comparison, 177Lu–DO3A–ZEGFR:1907

internalized faster, with 2.27 ± 0.2 and 9.6 ± 0.8 % at 2 and

30 min, respectively, whereas 28.9 ± 2.2 % of the tracer was

internalized at 24 h. A significantly higher percentage of

added 177Lu–DO3A–HSA–ZEGFR:1907 compared with

J Biol Inorg Chem (2012) 17:709–718 713

123

177Lu–DO3A–ZEGFR:1907 was internalized into SAS cells at

24 h (P \ 0.05). This suggests that the presence of multiple

Affibody molecules on HSA may help to promote internali-

zation of the tracer.

In vivo studies of 177Lu–DO3A–HSA–ZEGFR:1907

The in vivo biodistribution of 177Lu–DO3A–HSA–

ZEGFR:1907 was examined in an SAS-tumor-bearing mouse

model. The biodistribution of 177Lu–DO3A–HSA–

ZEGFR:1907 at 1, 4, 24, 48, 72, 120, and 240 h is shown in

Fig. 4a. Slow but relatively high levels of radioactivity

accumulation in the SAS tumors were observed. The tumor

uptake in SAS tumors was 2.00 ± 0.80 % ID/g at 1 h and

continually increased to 3.13 ± 0.95 % ID/g at 4 h and

4.57 ± 0.23 % ID/g at 24 h. The tumor uptake reached a

plateau of 5.24 ± 0.67 and 5.31 ± 0.44 % ID/g at 48 and

72 h, respectively. Finally, the tumor uptake of 177Lu–

DO3A–HSA–ZEGFR:1907 decreased to 3.72 ± 0.64 and

0.66 ± 0.2 % ID/g at 120 and 240 h after injection, respec-

tively. 177Lu–DO3A–HSA–ZEGFR:1907 also displayed a rel-

atively slow blood clearance. Blood values of 31.87 ± 0.95

and 19.07 ± 1.05 % ID/g were observed at 1 and 4 h after

injection, respectively. The blood clearance of 177Lu–

DO3A–HSA–ZEGFR:1907 fits the second-order exponential

decay model (R2 = 0.9987). With the administered dose used

for this study, 177Lu–DO3A–HSA–ZEGFR:1907 exhibited a

two-phase clearance pattern, in which 64.3 % of the injected

dose cleared fast with1.99-h half clearance time and 35.7 %

cleared more slowly with 25.50-h half clearance time. The

tracer showed high liver uptake at all time points (e.g., 4 h

after injection; 54.93 ± 4.05 % ID/g). The kidneys, how-

ever, showed a moderate radioactivity accumulation (e.g.,

11.14 ± 1.0 and 7.65 ± 1.68 % ID/g at 4 and 48 h after

injection, respectively). These data indicate the tracer was

cleared predominantly through the hepatobiliary system and

to a minor extent through the renal system. Figure 4b shows

the tumor-to-organ ratios of 177Lu–DO3A–HSA–ZEGFR:1907.

The tumor-to-muscle ratio increased slowly from 3.3 at 1 h

and reached a plateau of approximately 7.9 at 72–240 h.

However, the tumor-to-blood ratio increased tenfold from 1.7

at 48 h to 16.7 at 240 h.

The SPECT images correlate with the results obtained

from the biodistribution studies. Images acquired at 4, 24,

48, and 72 h after injection of 177Lu–DO3A–HSA–

ZEGFR:1907 demonstrated significant liver accumulation and

good tumor localization, and high tumor-to-background

contrasts were observed at the later times after injection

(Fig. 5).

Discussion

Affibody molecules have shown excellent tumor-targeting

properties in molecular imaging studies of tumors

expressing EGFR [22, 27–30]. However, the use of

radiometal-labeled, chelator-modified Affibody molecules

for radiotherapeutic applications is questionable, mainly

Fig. 2 a Cell uptake of 64Cu–

DO3A–HSA–ZEGFR:1907 in SAS

cells in the presence or absence

of nonradioactive Ac–Cys–

ZEGFR:1907 after 120 min

incubation. Data are shown as

the mean ± the standard

deviation (SD) percentage of

applied radioactivity (n = 4).

b Representative decay-

corrected coronal (top) and

transaxial (bottom) positron

emission tomography (PET)

images at 1, 4, 24, 48, and 72 h

after tail vein injection of 64Cu–

DO3A–HSA–ZEGFR:1907.

Arrows indicate the location of

the tumors. c Uptake levels

(percentage of injected dose per

gram of tissue) of tumor,

muscle, liver, and kidney

derived from multiple time

point small-animal PET images

after tail vein injection of 64Cu–

DO3A–HSA–ZEGFR:1907. Data

are shown as the mean ± SD of

four measurements

714 J Biol Inorg Chem (2012) 17:709–718

123

because of their extremely high kidney uptakes (generally

more than 100 % ID/g for radiometal-labeled Affibody

proteins) [31–33]. The high kidney retention is caused by

reabsorption of radiolabeled Affibody molecules in the

proximal tubules via luminal endocytosis after glomerular

filtration. Furthermore, it is known that peptides

conjugated with residualizing radiolabels are trapped in

the tubular cell lysosomes and therefore deliver high

radiation doses to the radiation-sensitive kidneys [34]. As

shown in earlier studies, the fusion of an Affibody protein

to a small albumin-binding domain (ABD) peptide results

in reduced kidney uptake [35, 36]. In our previous study

we developed an anti-HER2 HSA–Affibody conjugate, a

multimeric ligand with a size much smaller than that of

conventional antibodies (below 100 kDa), which exhibits

high tumor targeting and reduced kidney uptake in

SKOV3 xenograft mouse models [24]. That work also

revealed that Affibody–albumin conjugates display several

distinct advantages for imaging or therapy applications.

For example, (a) a quick and efficient covalent conjuga-

tion and labeling technique, (b) improved pharmacoki-

netics in terms of a lower kidney accumulation, and more

importantly, (c) a possible multiple and simultaneous

binding to HER2 owing to the attachment of several Af-

fibody molecules.

In the present study, HSA was further used to modify a

novel EGFR binder, Ac–Cys–ZEGFR:1907, and the radiola-

beled bioconjugate was evaluated for its potential in

radionuclide therapy of head and neck cancer. Targeted

radionuclide therapy has great potential for treatment of

HNSCC, because this type of cancer has a decreased

response to radiotherapy and increased locoregional

recurrence following definitive radiotherapy [14]. More-

over, the radiopharmaceutical developed could also be used

for radionuclide therapy for many other types of EGFR-

positive cancer. This fact leads to a broader application

range for using HSA–ZEFGR than for HSA–ZHER2. The

series of experiments performed confirmed our hypothesis

that anti-EGFR Affibody–HSA conjugates display good

tumor-targeting abilities and preferred pharmacokinetics

for potential radiotherapeutic applications in treatment of

HNSCC.

The human oral squamous carcinoma cell line SAS used

in this study exhibits high expression of EGFR in vitro and

in xenotransplants in vivo [25]. In vitro cell uptake studies

demonstrate the specificity and ability of the radiolabeled

conjugates to target EGFR. Furthermore 177Lu–DO3A–

HSA–ZEGFR:1907 can be internalized into SAS cells after

binding EGFR at the membrane. Interestingly, in vitro cell

internalization studies of both 177Lu–DO3A–HSA–

ZEGFR:1907 and 177Lu–DO3A–ZEGFR:1907 demonstrate that

the radiolabeled HSA–Affibody bioconjugate displays

significantly higher internalization rates than the radiola-

beled Affibody molecule alone at later time points. This is

another important advantage for HSA–Affibody conjugates

for potential use in radionuclide therapy. The higher

internalization rate might be caused by albumin-mediated

endocytosis of the radiopharmaceutical in addition to the

EGFR-mediated internalization. The supportive effect of

Fig. 3 In vitro cell studies of 177Lu–DO3A–HSA–ZEGFR:1907. a In

vitro cell uptake of 177Lu–DO3A–HSA–ZEGFR:1907 in SAS cells over

time at 4 and 37 �C in the presence or absence of nonradioactive

Ac–Cys–ZEGFR:1907. b, c Cell-associated radioactivity as a function of

time after incubation of SAS cells with 177Lu–DO3A–HSA–

ZEGFR:1907 (b) or 177Lu–DO3A–ZEGFR:1907 (c). The radioactivity that

was removed from the cells by treatment with acidic washing buffer

(0.2 M glycine buffer containing 4 M urea, pH 2.0) was considered as

the membrane-bound fraction. The radioactivity measured in the cell

lysates was considered as the internalized fraction. All data are

presented as means ± SD (n = 4)

J Biol Inorg Chem (2012) 17:709–718 715

123

albumin contributing to the internalization of drugs in

tumor cells was also shown by others [37].

Quantitative PET, SPECT, and biodistribution data con-

firm the good tumor targeting properties of both 64Cu- and177Lu-labeled DO3A–HSA–ZEGFR:1907. Importantly, the

retention of the radioactivity measured in the tumor is very

long. The retention of radiopharmaceuticals in the tumor is a

critical factor in radionuclide therapy applications. In contrast,

the short peptide EGFR is rapidly degraded in lysosomes after

binding with and internalization in EGFR-positive tumors

[38]. This disadvantageous property leads to a short intra-

cellular retention of the radionuclide and decreases the effi-

ciency of the therapy and increases unwanted side effects,

such as uptake in normal tissues. Compared with the uptake

of 64Cu–DO3A–ZEGFR:1907 in A431 tumors determined by

PET quantification [22], the uptake of 64Cu–DO3A–HSA–

ZEGFR:1907 in SAS tumors was found to be half as much at 4 h

after injection. In contrast, the tumor uptake of 111In–DOTA–

ZEGFR:1907 in A431-tumor-bearing mice is only half as much

as for 64Cu–DO3A–HSA–ZEGFR:1907 (2.4 ± 0.3 % ID/g

Fig. 4 Biodistribution results

(a) and tumor-to-organ ratios

(b) for 177Lu–DO3A–HSA–

ZEGFR:1907 in nude mice bearing

subcutaneously xenografted

SAS human head and neck

cancer. The biodistribution data

are expressed as the percentage

of the injected dose per gram of

tissue after intravenous injection

of the probe at 1, 4, 24, 48, 120

,and 240 h (n = 4)

Fig. 5 Small-animal single

photon emission computed

tomography/X-ray computed

tomography of a mouse bearing

SAS tumor xenograft at 4, 24,

48, and 72 h after

administration of 177Lu–DO3A–

HSA–ZEGFR:1907. Arrowsindicate the location of tumors.

White indicates the highest

radioactivity accumulation

716 J Biol Inorg Chem (2012) 17:709–718

123

vs. 5.19 ± 0.35 at 4 h after injection) [39]. These differences

can be explained by the size and different pharmacokinetic

properties of the tracers, by the different radiometals used,

and also by the lower expression of EGFR in SAS tumors

compared with A431 tumors.

The differences in tumor uptake between 64Cu–DO3A–

HSA–ZEGFR:1907 calculated from PET studies and 177Lu–

DO3A–HSA–ZEGFR:1907 obtained from biodistribution

studies, e.g., 7.72 ± 1.14 versus 5.24 ± 0.67 % ID/g at

72 h, could be caused by the different amounts of injected

radiotracer (approximately 40 lg for the 64Cu tracer and

approximately 3 lg for the 177Lu tracer). Several authors

have shown an influence of the injected dose of EGFR-

targeting proteins on the organ distribution [22, 28, 39–40].

In previous studies, we showed that the tumor uptake of64Cu–DO3A–ZEGFR:1907 is higher when there is blocking

with a small amount (50 lg) of cold tracer [22]. Further-

more, Tolmachev et al. [28] identified a maximum uptake

of 111In–DOTA–ZEGFR:2377 in A431 tumors between 30

and 50 lg of injected protein. The liver uptake measured

from the PET studies was shown to be one third as high as

that determined via biodistribution experiments. It was

observed that an increase of the injected dose of the

radiotracer resulted in a partial blocking with unlabeled

compound and thus a decrease in the uptake in EGFR-

expressing organs, e.g., the liver but also in the blood and

muscle. On the other hand, the differences between the64Cu- and the 177Lu-labeled bioconjugates could also be

caused by the different stabilities of the DO3A–metal

complex.

Compared with many radiometal-labeled Affibody pro-

teins (64Cu, 111In), the 64Cu- and 177Lu-labeled DO3A–

HSA–ZEGFR:1907 conjugates show much lower uptake in

the kidneys as determined in PET, SPECT, and biodistri-

bution studies. In comparison with the biodistribution

studies we performed recently using 64Cu–DO3A–

ZEGFR:1907, the kidney uptake for 177Lu–DO3A–HSA–

ZEGFR:1907 is eight times lower at 4 h after injection

(11.14 ± 1.0 vs. 88.45 ± 14.42 % ID/g). However, the

liver uptake of 177Lu–DO3A–HSA–ZEGFR:1907 was three

times higher than that of 64Cu–DO3A–ZEGFR:1907 at 4 h

after injection (54.93 ± 4.05 vs. 18.99 ± 2.88 % ID/g).

This indicates clearly that the conjugation of HSA signif-

icantly reduces the kidney uptake of the protein but

increases the liver uptake. However, increased liver uptake

does not make the agent questionable for therapeutic

use. The predicted dose of radiation absorbed by liver

for 177Lu–DO3A–HSA–ZEGFR:1907 was calculated be

2.6 mGy/MBq. If a radiation-treatment absorbed dose of

30–50 Gy is assumed for the tumor, the corresponding

dose for the liver will be 8.1–13.5 Gy, which is far below

the dose limit for liver in humans [41]. The high liver

uptake of the conjugate can be explained by several facts,

e.g., the high EGFR expression in the liver tissue [22], a

nonspecific clearance of the probe through the hepatobil-

iary system, and a presumable transchelation of 64Cu in the

liver [42]. Furthermore, the high accumulation in liver

tissues can be caused by the metabolic fate of the HSA

component of the radiotracer.

In conclusion, HSA–ZEFGR conjugates were prepared

and labeled successfully with both 64Cu and 177Lu. 177Lu–

DO3A–HSA–ZEGFR:1907 provides high specificity, high

sensitivity, and displays good tumor contrasts as shown in

SPECT studies. The in vivo properties and pharmacoki-

netics of the 177Lu–DO3A–HSA–ZEGFR:1907 conjugate

make it a potential radiopharmaceutical for treatment of

head and neck cancer.

Acknowledgments This work was supported, in part, by National

Cancer Institute (NCI) grant 5R01 CA119053 and NCI In Vivo

Cellular Molecular Imaging Center grant P50 CA114747. The pro-

duction of 177Lu was supported by Department of Energy grant

84900-001-10.

Conflict of interest The authors declare that they have no conflict

of interest.

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