Enhanced tumor retention of a radiohalogen label for site-specific modification of antibodies

Article abstract of DOI:10.1021/jm401365h

A known limitation of iodine radionuclides for labeling and biological tracking of receptor targeted proteins is the tendency of iodotyrosine to rapidly diffuse from cells following endocytosis and lysosomal degradation. In contrast, radiometal-chelate complexes such as indium-111-1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (In-111-DOTA) accumulate within target cells due to the residualizing properties of the polar, charged metal-chelate-amino acid adduct. Iodine radionuclides boast a diversity of nuclear properties and chemical means for incorporation, prompting efforts to covalently link radioiodine with residualizing molecules. Herein, we describe the Ugi-assisted synthesis of [I-125]HIP-DOTA, a 4-hydroxy-3-iodophenyl (HIP) derivative of DOTA, and demonstration of its residualizing properties in a murine xenograft model. Overall, this study displays the power of multicomponent synthesis to yield a versatile radioactive probe for antibodies across multiple therapeutic areas with potential applications in both preclinical biodistribution studies and clinical radioimmunotherapies.

Full text of DOI:10.1021/jm401365h

Article
pubs.acs.org/jmc
Enhanced Tumor Retention of a Radiohalogen Label for Site-Specific
Modification of Antibodies
C. Andrew Boswell,* Jan Marik, Michael J. Elowson, Noe A. Reyes, Sheila Ulufatu, Daniela Bumbaca,
Victor Yip, Eduardo E. Mundo, Nicholas Majidy, Marjie Van Hoy, Saritha N. Goriparthi, Anthony Trias,
Herman S. Gill, Simon P. Williams, Jagath R. Junutula, Paul J. Fielder, and Leslie A. Khawli
Genentech Research and Early Development, 1 DNA Way MS 463A, South San Francisco 94080, United States
S
* Supporting Information
ABSTRACT: A known limitation of iodine radionuclides for
labeling and biological tracking of receptor targeted proteins is the
tendency of iodotyrosine to rapidly diffuse from cells following
endocytosis and lysosomal degradation. In contrast, radiometal−
chelate complexes such as indium-111−1,4,7,10-tetraazacyclodode-
cane-1,4,7,10-tetraacetic acid (In-111-DOTA) accumulate within
target cells due to the residualizing properties of the polar, charged
metal-chelate-amino acid adduct. Iodine radionuclides boast a
diversity of nuclear properties and chemical means for incorporation,
prompting efforts to covalently link radioiodine with residualizing molecules. Herein, we describe the Ugi-assisted synthesis of [I-
125]HIP-DOTA, a 4-hydroxy-3-iodophenyl (HIP) derivative of DOTA, and demonstration of its residualizing properties in a
murine xenograft model. Overall, this study displays the power of multicomponent synthesis to yield a versatile radioactive probe
for antibodies across multiple therapeutic areas with potential applications in both preclinical biodistribution studies and clinical
radioimmunotherapies.
emission computed tomography (SPECT) imaging may be
performed with 123I, 131I, and 125I, the latter being limited to
preclinical small animal cameras. Positron emission tomography
(PET) with 124I is also feasible,9 although the highly energetic
emissions limit the image quality.
Labeling of antibodies with radiometals results in a different
cellular retention of radioactivity relative to iodotyrosine-based
labeling.10 For both labeling methods, antibodies undergo
receptor-mediated endocytosis and lysosomal degradation.
However, a third critical step, cellular efflux of the radiolabel
with its covalently associated amino acid, does not readily occur
for radiometal-labeled antibodies (Figure 2).11 Unlike [125I]-
iodotyrosine, which diffuses out of the cell (intact or as free
iodide) following proteolysis, 111In-DOTA−lysine cannot easily
cross the plasma membrane due to its charge and polarity.12
Because of its tendency to become intracellularly trapped, 111In-
DOTA is referred to as a residualizing label.
Significant effort has been made to derive strategies for
labeling antibodies with radioiodine such that residualization
occurs in a similar manner as for radiometals. This reflects, in
part, the wide availability of iodine radionuclides with diverse
nuclear properties, in terms of both decay half-lives and
emission energies (Supporting Information, Table S1), and an
abundant knowledge of halogen radiochemistry.8 For instance,
the decay half-lives of 111In and 125I are 2.8 and 60 days,
respectively, making the latter radionuclide far better suited for
INTRODUCTION
■
The antigen specificity of monoclonal antibodies is a powerful
attribute that allows the specific in vivo delivery of payloads,
including chemotherapeutic drugs and radionuclides.1 To date,
only two radioimmunotherapeutic agents have been approved
for clinical use, and both feature murine monoclonal antibodies
targeting the CD20 receptor for treatment of lymphoma.2
Tositumomab is administered with the β-emitting iodine
radionuclide, 131I, attached via tyrosine residues as shown in
Figure 1A. Ibritumomab tiuxetan is administered with the β-
emitting yttrium radionuclide, 90Y, attached using tiuxetan, an
isothiocyanate analogue of diethylenetriamine pentaacetic acid
(DTPA), through lysine residues via thiourea bonds.3 This
labeling strategy is somewhat analogous to the complexation of
the indium radionuclide, 111In, by 1,4,7,10-tetraazacyclodode-
cane-1,4,7,10-tetraacetic acid (DOTA), whose succinimidyl
ester can form amide bonds via lysine as shown in Figure 1B.
Beyond their clinical utility, radioimmunoconjugates are also
useful as tools in preclinical and translational research for
studying conventional, nonradioactive antibody therapeutics.7
Noninvasive small animal imaging modalities, whole-body
autoradiography, and invasive biodistribution studies can assist
drug development by providing valuable mechanistic informa-
tion including confirmation of target localization, screening for
off-target uptake, and assessing receptor occupancy. In addition
to 131I, 125I is commonly used for such studies, with the latter
having the advantages of a roughly 10-fold lower γ energy, the
absence of a β particle emission, and a much longer decay half-
life (Supporting Information, Table S1).8 Single photon
Received: September 6, 2013
© XXXX American Chemical Society
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emissions often associated with superior autoradiographic
image quality and lower radiation exposure to workers. A
residualizing iodine probe would combine the long decay half-
life and low energy of 125I with the superior tumor accretion of
radiometals while providing a facile translational route to
clinical imaging (via 123I or 124I) or radioimmunotherapy (via
131I)13 (Supporting Information, Table S1).
To date, strategies used to derive residualizing iodine probes
include the use of (i) nonmetabolizable carbohydrates, (ii)
nonmetabolizable peptide adducts, and/or (iii) highly charged
moieties. The carbohydrate derivative dilactitol-125I-tyramine is
a member of the first class of residualizing radioiodine probes.14
However, the use of carbohydrates poses a potential risk, as
pendant sugar groups are important for binding of antibodies to
Fc receptors and other critical functions.15 Representing the
second class is the residualizing peptide, IMP-R4 (MCC-
Lys(MCC)-Lys(X)-D-Tyr-D-Lys(X)-OH, where MCC is 4-(N-
maleimidomethyl)-cyclohexane-1-carbonyl and X is 1-((4-
thiocarbonylamino)benzyl)-DTPA).16 This approach relies on
a synthetic peptide that is conjugated with the chelate DTPA,
whose charge imparts residualizing properties.17 Selected
examples of the third class are shown in Figure 1C, many of
which require the use of organostannane intermediates in their
syntheses.4−6 Overall, we sought to design a simple
residualizing iodine probe that is accessible to laboratories
with basic organic synthesis capabilities while avoiding the use
of bulky peptides, carbohydrates, organostannanes, and lengthy
synthetic routes.
Figure 1. Selected nonresidualizing (A) and residualizing (B−D)
methods of radiolabeling antibodies including (A) oxidative tyrosine
radioiodination, (B) lysine modification with radiometal chelate, (C)
lysine modification with charged iodinated groups,4−6 and (D) thiol
modification with the novel compound 6, designated [125I]HIP-
DOTA, that can be attached to either the Fc region or the heavy (HC)
or light (LC) chains of an antibody containing engineered cysteine
residues.
Herein, we report the Ugi multicomponent synthesis,18,19
radiolabeling, and biological evaluation of a residualizing
radiohalogen probe, [125I]HIP-DOTA, (Figure 1D). The
probe was site-specifically conjugated to an engineered
THIOMAB20 (i.e., a full-length antibody having engineered
cysteine residues) and demonstrated to exhibit superior tumor
uptake relative to a conventional tyrosine radioiodinated
control antibody. This novel method for introducing radio-
iodine labels into antibodies is a useful preclinical tool for
studying the biodistribution, metabolism, and excretion of
antibody therapeutics. Furthermore, antibodies labeled with
residualizing radionuclides offer unique advantages as both
diagnostic and radioimmunotherapeutic agents because they
may provide a more sustained retention of radioactivity inside
tumor cells.21 Assuming that efficacy is related to tumor
radiation exposure, the use of residualizing radioimmunother-
apeutic agents may encourage high target radiation exposure
and favorable clinical responses.22
RESULTS
■
In choosing a synthetic strategy, we considered that our
molecule should consist of three critical functional compo-
nents: an iodotyrosine-like moiety, an activated group for
antibody conjugation, and a residualizing anchor. The success
of residualizing peptides, which contain iodotyrosine, suggested
that a hydroxyphenyl residue is a sufficient scaffold on which to
introduce radioiodine.23 This avoided the necessity of tin
precursors, which are necessary to produce the N-succinimidyl
m-iodobenzoate derivatives whose corresponding antibody
conjugates are depicted in Figure 1C. The choice of the
macrocyclic polyaminopolycarboxylic acid DOTA was based on
both its known ability to residualize10 and on its commercial
availability with a wide array of functional groups.
Figure 2. Schematic depicting the cellular fates of nonresidualizing and
residualizing labels following antibody binding to an internalizing cell-
surface antigen. Antibodies labeled by both methods undergo
receptor-mediated endocytosis and lysosomal degradation. Diffusion
of radioactivity out of the lysosome and/or cell occurs readily for
iodotyrosine but is resisted for radiometal−chelate complexes and
other residualizing labels.
long-term preclinical studies. This is especially true for
antibodies, whose pharmacokinetic half-lives are often on the
order of 1−2 weeks. Another consideration is that the γ energy
of 125I is nearly 10-fold lower relative to 111In, with lower energy
We sought a chemical reaction with the ability to rapidly
combine multiple chemical components in high yields and with
B
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Figure 3. Synthetic route to the shelf compound, maleimide 4, and its radiolabeling and antibody conjugation to LC-V205C thio-trastuzumab.
ample flexibility in reactant structure. These requirements led
us to the Ugi multicomponent reaction involving a ketone or
aldehyde, an amine, an isocyanide, and a carboxylic acid to form
a bisamide.18,19 Indeed, there is one previous report of the Ugi
reaction applied to synthesis of ditopic chelating agents.24
We used formaldehyde, a primary amine-functionalized
DOTA derivative (compound 1), 4-hydroxyphenylacetic acid,
and ethyl isocyanoacetate as our four commercially available
Ugi components (Figure 3). The ethyl ester 2 was converted to
the acid 3 by saponification using lithium hydroxide. All
attempts to synthesize the succinimidyl ester of this acid, our
original desired molecule, were unsuccessful, with the
formation of dimers (phenolic esters) evident by mass
spectrometry. This problem was averted by changing from a
lysine- to a cysteine-based conjugation strategy, taking
advantage of recent developments in antibody engineering to
introduce site-specific cysteines.20 Our previous work demon-
strated that the maleimide functionality is compatible with the
oxidative conditions of radioiodination.25 This guided our
synthetic route to introduce a maleimide group by coupling the
acid to N-(2-aminoethyl)maleimide using 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC), yielding the shelf
compound 4 in only three steps (Figure 3) as compared to the
six-step synthesis of the protected tin precursor of SIB-DOTA
(SIB = N-succinimidyl-3-iodobenzoate).6 Compounds 2, 3, and
4 eluted on a RP-HPLC gradient at 14.8, 13.8, and 13.7 min,
respectively (Supporting Information, Figure S1).
S3−S4). Compound 5 could be purified from free iodide and
oxidant using reverse-phase solid-phase extraction cartridges,
but this step was not necessary for the Iodogen method. Acid
deprotection yielded the triacid [125I]6, with completeness of
reaction monitored by reverse-phase radio-HPLC (Supporting
Information, Figure S5). This 4-hydroxy-3-iodophenyl deriva-
tive of DOTA, compound [125I]6, was designated [125I]HIP-
DOTA. After exhaustive removal of acid by repeated addition
and evaporation of toluene, the thiol-containing antibody could
be introduced for thiol-maleimide coupling. Any remaining free
thiols were reacted with iodoacetic acid to avoid dimerization
or formation of adducts with thiol-containing endogenous
proteins. All radioimmunoconjugates were analyzed for purity
by size-exclusion HPLC and compared to the profile of
unlabeled trastuzumab (Supporting Information, Figure S6−
S7).
The site-specificity of our labeling strategy was confirmed by
SDS-PAGE analysis including both protein staining and
phosphorimaging (Figure 4). Digestion with the endoprotease
Lys-C was able to distinguish radiolabeled Fc from Fab.
Similarly, reduction by dithiothreitol was able to resolve
radiolabeled light chain (LC) from radiolabeled HC/Fc
regions.
On the basis of our previous experience with THIOMAB
technology, we anticipated differences in stability between the
heavy chain (HC-A114C), light chain (LC-V205C), and
crystallizable fragment (Fc-S396C) radioimmunoconjugate
variants (Kabat numbering is used).26 This hypothesis was
confirmed using an in vitro plasma stability assay in which each
of the [125I]6-labeled trastuzumab derivatives was incubated in
mouse plasma at 37 °C. Consistent with the previously
reported stabilities of antibody−drug conjugates,26 the rank
order of plasma stability for our site-specific conjugates was LC
> HC > Fc (Supporting Information, Figure S8−S10). These
site specific differences in stability likely result from the local
electrostatic environment (i.e., charged residues) causing
variations in the rate of succinimide ring hydrolysis, which
prevents further maleimide exchange with reactive thiols and
Our radiochemical strategy initially involved use of the mild
oxidant, N-chlorosuccinimide, to achieve iodination in chloro-
form on the hydroxyphenyl group. However, we subsequently
discovered that maleimide 4 is sufficiently water-soluble to
allow a more efficient labeling in aqueous 0.1% acetic acid in a
test tube that is precoated with the water insoluble oxidizing
agent, 1,3,4,6-tetrachloro-3α,6α-diphenylglucoluril (i.e., “Iodo-
gen”) (Figure 3). The intermediate [125I]5 had a retention time
of 15.5 min on reversed-phase HPLC (Supporting Information,
Figure S2) and could be detected in terms of both [127I] and
[
125I] by mass spectrometry (Supporting Information, Figure
C
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Figure 4. SDS-PAGE analysis by protein staining (top) and
phosphorimaging (bottom) of trastuzumab labeled site specifically
with [125I]6 through its heavy chain (HC-A114C), light chain (LC-
V205C), and Fc (Fc-S396C) region. Digestion of antibodies with the
endoprotease, Lys-C, results in cleavage between the Fab and Fc
regions. In contrast, dithiothreitol (DTT) reduction separates the
heavy (still attached to Fc) and light chains. Differential exposure of
radioactivity in selected bands is evident despite equal loading of
proteins, demonstrating the site-specificity of labeling.
enhances stability.26 The loss of the main (intact) peak at 18
min was accompanied by the appearance of two new peaks. An
earlier peak at 16.5 min was attributed to protein aggregation
and/or dimerization, while a later peak at 19 min was consistent
with the retention time of albumin, an abundant plasma protein
that is known to possess a reactive thiol.
To evaluate the ability of compound 6, HIP-DOTA, to
residualize, a biodistribution study was performed in the
previously validated KPL-4 xenograft mouse model27 of human
epidermal growth factor receptor 2 (HER2)-expressing breast
cancer as previously reported.28 The radioimmunoconjugates
Figure 5. Plasma pharmacokinetics (A) and biodistribution at 3 days
(B) of trastuzumab radiolabeled by various methods in KPL-4
xenograft-bearing mice. Trastuzumab was labeled with 125I by
traditional tyrosine modification (black), by site-specific (HC-
A114C/LC-V205C/Fc-S396C) modification with [125I]6 (blue,
green, and red, respectively), or by lysine modification with 111In-
DOTA (gray). Uptake is expressed as percentage of injected dose per
g of tissue (%ID/g).
residualized to a greater extent than [125I]iodotyrosine. Apart
from tumor, the radioactivity levels in tissues obtained by
trastuzumab labeled by the five different methods were largely
similar. A higher renal uptake of radioactivity was observed for
[
125I]6-[HC]−, [125I]6-[LC]−, and [125I]6-[Fc]−trastuzumab
were directly compared with tyrosine-labeled 125I-trastuzumab
prepared by the Chizzonite (i.e., indirect Iodogen) method.
Trastuzumab labeled with 111In-DOTA, a known residualizing
probe, was coadministered with each radioiodinated antibody
to serve as an internal control. Simultaneous measurement of
both 125I and 111In in a single sample is feasible due to the
distinct γ energies of these two radionuclides.
[
125I]6−trastuzumab, particularly the Fc variant, relative to
125I−trastuzumab. Renal levels of radioactivity following
injection of 125I-, 125I-6- (HC), 125I-6- (LC), 125I-6- (Fc), and
111In-DOTA-labeled trastuzumab were roughly 3, 10, 8, 20 and
6%ID/g, respectively. In addition, both splenic and hepatic
uptake of 111In were higher than for any of the [125I]-labeled
molecules.
The plasma clearance profiles for all three radioimmuno-
conjugates were overlapping (Figure 5A), indicating that the
tumor uptakes of radioactivity for 111In-DOTA-, [125I]6-, and
125I-labeled trastuzumab were not influenced by systemic
exposure and that modification of the antibody with [125I]6
did not deleteriously affect its pharmacokinetics. The lack of
difference among [125I]6−trastuzumab variants, despite differ-
ences in plasma stability, reflects the inability of the radiometric
pharmacokinetic assay to distinguish between intact antibody
and [125I]6−albumin, a likely product of maleimide exchange
with reactive thiols.26
At 1 week, the trends in tumor and tissue uptake were
somewhat similar to those observed in the 3-day data, although
the residualization of 111In-DOTA appears to be more sustained
than [125I]6 (Supporting Information, Figure S11). Mean
tumor uptakes were 6.9 3.7 (HC-A114C), 8.2 3.8 (LC-
V205C), 5.9 0.7 (Fc-S396C), 1.7 0.3 (tyrosine-modified),
and 21.2 5.6(111In-DOTA)%ID/g, respectively. The levels of
111In uptake in %ID/g for liver (7.2) and spleen (13) were
considerably higher than that for any of the 125I-labeled
analogues.
Noninvasive single photon emission computed tomography
(SPECT) imaging was performed in live, anesthetized KPL-4
tumor-bearing mice in order to complement the biodistribution
study arm (Figure 6, upper). X-ray computed tomography
(CT) was performed prior to SPECT without movement or
bed adjustment to allow anatomical coregistration of radio-
activity with tissue structures. Seventy-two hour SPECT-CT
At 3 days, tumor uptake for each of the three [125I]6−
trastuzumab variants was more than triple that of 125I−
trastuzumab with values of 24.6 4.2 (HC-A114C), 27.7 1.4
(LC-V205C), 21.2 1.8 (Fc-S396C), and 6.1 0.3 (tyrosine-
modified) percentage of injected dose per gram (%ID/g),
respectively (Figure 5B). By comparison, 3-day tumor uptake of
111In-DOTA−trastuzumab was even higher at 48.7 3.8%ID/
g. These data suggest that [125I]6-associated catabolites are
D
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the reconstructed field-of-view, which is smaller for SPECT
than for CT on our system. In contrast, mostly blood pool
uptake was visible in the midline plane for the other four
variants (Supporting Information, Figure S12). The image
quality and resolution for 111In-DOTA was noticeably inferior
to the 125I-labeled variants, as evident from the degree of
pixelation and bleed-over (Figure 6). This observation may be
attributed to the roughly 10-fold γ energy of 111In relative to
125I.
DISCUSSION
■
The HC-A114C, LC-V205C, and Fc-S396C variants of [125I]
6−trastuzumab yielded more than triple the tumor radioactivity
relative to traditional tyrosine radioiodination at 3 days
postinjection but still fell short (roughly 50%) of 111In-DOTA
(Figure 5B). The presence of the metal in the latter may be the
reason for this discrepancy; however, previous studies showed
that the presence of In3+ did not affect the level of
residualization in antibodies labeled with DTPA-appended
radioiodinated peptides.17 Furthermore, as others have noted,6
chelates have an ability to scavenge iron or other adventitious
metals in tissue culture media or in vivo. Importantly, DOTA
forms complexes with transition metals, alkaline earth metals
(e.g., calcium), and several lanthanides.29 The stabilities (log
Kd, defined as dissociation constant) for the transition metal
complexes of DOTA range from 24 to 29 for Fe(III) to 19−21
for Zn(II), with a less stable value of 16 for the Ca(II)
complex.29 The promiscuous complexation chemistry of DOTA
makes it possible that a range of metal ions could be bound in
the chelator cavity within a biological matrix, but iron is of
particular relevance because of its presence in circulating
plasma proteins such as transferrin.
Figure 6. SPECT-CT imaging (upper) at 24 and 72 h and whole-body
autoradiographic imaging (lower) at 72 h indicate relative degrees of
tracer residualization in KPL-4 xenograft-bearing mice following
intravenous administration of trastuzumab radiolabeled by five
different methods. In the three-dimensional volume renderings from
a coronal perspective (upper), the skeletal images are derived from the
X-ray (anatomical) CT data while the relative levels of radioactivity
from the SPECT data are indicated in a false-color scale. Tumor-to-
blood (T:B) ratios of radioactive uptake are shown. Post-mortem
cryosection images from a sagittal perspective (lower) were acquired
from the same mice as in the upper panel. False-colored
phosphorimages (left) and digital photographs (right) are shown for
each mouse in the tumoral plane. Tissues are labeled as tumor (T),
liver (L), and kidney (K).
Alternatively, the level of tumor residualization of [125I]6-
associated catabolites may be inferior to that of 111In-DOTA−
lysine, potentially placing HIP-DOTA at a disadvantage with
respect to radiometal-based labels. However, size exclusion
radiochromatographic analysis of kidney homogenates from a
follow-up study in nontumor-bearing mice revealed higher
levels of nonintact, low molecular weight radiocatabolites for
images of mice receiving a single intravenous injection of
radiolabeled trastuzumab qualitatively revealed tumor-to-blood
ratios in the following rank order: 111In-DOTA > 125I-6 (LC-
V205C) > 125I-6 (HC-A114C) > 125I-6 (Fc-S396C) > 125I
(tyrosine-modified). The better agreement between 111In-
DOTA and [125I]6 by SPECT-CT in Figure 6, relative to
Figure 5, may be explained by higher receptor occupancy and/
or altered internalization rates due to the higher doses of
radiolabeled antibody necessary for image acquisition.
Whole-body localization of radioactivity in KPL-4 tumor-
bearing mice was determined by autoradiography (Figure 6,
lower). Digital photographs of the sagittal cryosections allow
anatomical coregistration. The same rank order of relative
tumor uptake (upper panel) was qualitatively observed: 111In-
DOTA > [125]I6 (LC-V205C) > [125]I6 (HC-A114C) > [125]I6
(Fc-S396C) > 125I (tyrosine-modified). Elevated renal uptake,
especially for the Fc variant of 125I-6−trastuzumab, was evident
by both SPECT and autoradiography and recapitulated the data
obtained by organ harvest in Figure 5. In the midline plane
(lower panel), a strikingly high uptake of radioactivity in the
thyroid gland was the dominant feature in the 125I−trastuzumab
(tyrosine-modified) autoradiograph (Supporting Information,
Figure S12) despite the NaI blocking doses administered at
both 24 and 1 h prior to dosing. The absence of this feature in
the SPECT images could be explained by image artifacts near
the top/bottom of the images or by the thyroid lying outside of
[
125I]6-[HC]−trastuzumab relative to 125I-trastuzumab (65 and
5% of renal radioactivity, respectively) (Supporting Informa-
tion, Figure S13). Importantly, these data demonstrate that
renal accumulation of [125I]6-associated radioactivity occurs
independently of tumor uptake and are further supported by in
vitro evidence of partial deconjugation in plasma (Supporting
Information, Figure S8−S10). Another possibility is that
dehalogenation, cleavage of the halogen−carbon bond, is
affecting the probe’s tumor retention (see below).
Enzymatic dehalogenation also contributes to the underlying
discrepancy in tumor uptake between targeted molecules
labeled with radiohalogens and radiometals. Although mostly
concentrated in the thyroid, dehalogenases are also present
elsewhere including the liver and kidneys.30,31 The use of
organostannane precursors to derive charged metaiodoben-
zoate derivatives as residualizing iodine probes (see Figure 1C)
is intended to avert this problem by eliminating the presence of
a phenol group, thus escaping specific molecular recognition by
these enzymes.4−6 Indeed, similar strategies have yielded
nonresidualizing iodine probes that are more resistant to
dehalogenation.25,32,33 However, both the present and previous
work17,34 demonstrate that even 2-iodophenol derivatives may
be successfully incorporated into residualizing probes. Con-
sequently, the efflux of iodotyrosine and/or its catabolites
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following lysosomal proteolysis may be a more vexing problem
than enzymatic dehalogenation. However, it is important to
note that dehalogenases are specific for mono- or diiodinated L-
tyrosine, therefore the D-tyrosine-containing IMP-R4 and the
Ugi-derived [125I]6 might not even be recognized by these
enzymes.
DOTA chelate forms very kinetically stable complexes with
larger +3 metal cations including the γ emitter 111In, the low
energy β/negatron (β−) emitter lutetium-177 (177Lu), the high
energy β-emitter yttrium-90 (90Y), the positron (β+) emitter
yttrium-86 (86Y),9 and nonradioactive gadolinium (natGd) as a
magnetic resonance spectroscopy contrast agent.3
We have developed a facile, tin-free, three-step synthetic
route to a residualizing probe amenable to oxidative radio-
iodination and antibody labeling. In particular, the multi-
component Ugi reaction proved to be an efficient means for
covalently linking our three desired components: a charged
residualizing anchor, a phenol for iodine incorporation, and an
activated linker for antibody conjugation. When conjugated to
trastuzumab, a marketed anti-HER2 antibody, this novel probe
demonstrated a 3−4-fold increase, relative to traditional
tyrosine radioiodination, in tumor uptake at 3 days post-
injection. Overall, our synthetic route to [125I]6, a novel
residualizing radioiodine probe, is potentially useful for targeted
radioimmunotherapy of cancer and may also benefit preclinical
and translational research efforts for antibodies across multiple
therapeutic areas.
Higher renal uptake of the HC-A114C, LC-V205C, and Fc-
S396C analogues of [125I]6 thio-trastuzumab, relative to 125I-
trastuzumab, was expected based on our previous experience
with residualizing labels.28 In particular, the Fc variant
demonstrated high renal and lower tumor uptakes (Figure
5B), consistent with its poor plasma stability (Supporting
Information, Figure S10). Administration of a basic amino acid
such as lysine is a potential future strategy to lower the renal
uptake associated with [125I]6, as it has been previously
demonstrated to reduce renal tubular reabsorption of radio-
metal-labeled antibody fragments by neutralization of negative
charges within the luminal tubular cell surface.35 Despite the
differences in both in vitro plasma stability (Supporting
Information, Figure S8−S10) and in vivo radioactivity levels
in tumor and kidneys (Figure 5B), no differences in plasma
pharmacokinetics were observed among the three analogues of
[
125I]6−trastuzumab (Figure 5B). Since albumin is the most
EXPERIMENTAL SECTION
■
abundant free thiol-containing plasma protein, maleimide
exchange from trastuzumab to albumin is a likely scenario
that is further supported by the appearance of radiolabeled
albumin in the in vitro plasma stability study (Supporting
Information, Figure S10). The similar clearances of the HC,
LC, and Fc analogues may reflect the fact that both
immunoglobulins and albumin are protected from lysosomal
degradation by the recycling receptor, the neonatal Fc receptor
(FcRn), thereby promoting long serum half-lives in both cases.
Although the present study utilized a THIOMAB version of
trastuzumab with engineered cysteines,26 the maleimide
derivative [125I]6 has the potential to be conjugated to any
thio-containing protein. Although THIOMAB labeling confers
advantages such as excellent batch-to-batch reproducibility, it
does require the production, time, and expense of locating an
appropriate site to introduce the unpaired cysteine into an
antibody. For those who prefer to work with conventional
antibodies, established methods allow conjugation to hinge-
region thiols made available through mild reduction. Alter-
natively, thiols may be introduced to any antibody through the
ε-amino groups of lysine residues by use of a heterobifunctional
linker.25 Although the current approach offers several
advantages over traditional radioiodination, further refinements
could offer significant improvements. For instance, a future
generation derivative of 6 allowing direct conjugation to lysines
could additionally circumvent the problematic stability of
thioether bonds which are highly dependent on the local
electrostatic environment. This could improve label stability in
vivo by eliminating losses to reverse Michael reactions and
reducing overall renal uptake.
General Synthetic Methods. Unless otherwise noted, all
reactions were run under an argon atmosphere in oven-dried
glassware. Reactions were stirred using Teflon-coated magnetic stirrer
bars. Reactions were monitored using thin layer silica gel
chromatography (TLC) using 0.25 mm silica gel 60F plates with
fluorescent indicator from EMD Chemicals. Plates were visualized
under UV light. Products were purified via preparative reverse phase
chromatography with UV detection at 254 nm using a gradient of 5−
50% water/ACN (0.1% formic acid) at 70 mL/min in 10 min; the
column was a Phenomenex Gemini-NX 10 μ C18 110 Å, 100 mm ×
30.00 mm.
Acetonitrile (CH3CN) and trifluoroacetic acid (TFA) were
purchased from EMD Chemicals. Acetic acid (CH3COOH), ethyl
isocyanoacetate, deuterated chloroform (CDCl3), and N-chlorosucci-
nimide (NCS) were purchased from Alpha Aesar. Chloroform
(CHCl3) was purchased from Mallinckrodt. Methanol (MeOH) was
purchased from J. T. Baker. 4-Hydroxyphenylacetic acid was purchased
from Sigma-Aldrich. Lithium hydroxide monohydrate (LiOH·H2O)
was purchased from Acros. Paraformaldehyde was purchased from
TCI America. 2-Maleimidoethylamine hydrochloride was purchased
from Oakwood Products. 2-Aminoethyl-monoamide-DOTA-tris(t-
butyl ester) hydrobromide was purchased from Macrocyclics, Inc.
NMR spectra were acquired on either a Bruker 300 UltraShield (1H
at 300 MHz, 13C at 75 MHz) or a Bruker Avance II 400 (1H at 400
MHz, 13C at 100 MHz) magnetic resonance spectrometer. 1H
chemical shifts are reported relative to the residual solvent peak
(chloroform = 7.26 ppm) as follows: chemical shift (δ), (multiplicity,
integration). 13C chemical shifts are reported relative to the residual
deuterated solvent 13C signals (CDCl3 = 77.16 ppm). High resolution
LC-MS was performed using an Agilent 1200 series LC with PLRP-S,
1000 Å, column (50 mm × 2.1 mm, Varian Inc.) coupled to an Agilent
6220 Accurate-Mass TOF LC/MS mass spectrometer.
Chromatography. Reversed-phase radiochromatography was
performed using an Agilent 1200 HPLC system coupled with a γ-
RAM model 4 radioactive detector (LabLogic, formerly IN/US)
running Laura version 4 software. A flow rate of 1 mL/min and a 30
min gradient of 5% A, 95% B to 95% A, 5% B were utilized where A =
0.1% trifluoroacetic acid (aq) and B = acetonitrile.
Size-exclusion radiochromatography was performed using an
Agilent 1100 HPLC system coupled with a Raytest Gabi Star
radioactive flow monitor running ChemStation software. The colum
was a BioSep SEC S-3000, 300 mm × 7.8 mm, 5 μm (Phenomenex).
The mobile phase was PBS, and the flow rate was 0.5 mL/min for 32
min (isocratic). The ChemStation analogue to digital converter was set
The ability to conjugate a residualizing probe to thiols may
be useful as a quantitative means to estimate cumulative drug
delivery of antibody−drug conjugates in which chemother-
apeutic drugs are conjugated using similar chemical method-
ologies.1 In addition, a residualizing halogen probe could
benefit the development of radioimmunotherapeutic agents
labeled with the β-emitter, 131I (Supporting Information, Table
S1).13 Moreover, aside from its residualizing properties, the
presence of DOTA in [125I]6 lends the possibility of
incorporating a metal to yield a multimodal probe. The
F
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Journal of Medicinal Chemistry
Article
to 25000 units/mV, peak width 2 s, slit 4 nM (Agilent Technologies).
Radioactivity was detected with a raytest Gabi Star radiodetector in
line with a standard Agilent 1100 HPLC module system. The data
were collected using ChemStation for LC 3D Systems (revision
B.01.03[204]).
Chemistry. Purities of nonradioactive compounds were assessed by
UV monitoring during analytical RP-HPLC and found to be 100, 96.3,
and 93.1% for 2, 3, and 4, respectively (see Supporting Information,
Figure S1).
addition of 3 μL (1 mCi) of Na125I in 0.1 N NaOH (aq) (Perkin-
Elmer). The mixture was briefly vortexed and transferred to a glass test
tube (Pierce) that is precoated with 1,3,4,6-tetrachloro-3α,6α-
diphenylglucoluril (i.e., Iodogen). The radioiodination reaction was
allowed to proceed without heating for 5 min with gentle intermittent
shaking every 30 s on an Eppendorf thermomixer. The solution was
transferred to a 5 mL glass vial and characterized by reversed-phase
HPLC without purification. After solvent (i.e., water) removal by
rotary evaporation, a 0.5 mL quantity of 95% trifluoroacetic acid/5%
water was added followed by magnetic stirring for 2 h. Complete
removal of acid by rotary evaporation was facilitated by successive
additions of toluene in 250 μL aliquots followed by an overnight
vacuum. To the residue, still in a 5 mL glass vial, was added 50 μL of
phosphate-buffered saline, pH 7.4 (PBS). After a brief vortex, a pH
strip was used to ensure that the pH was in the range of 6.5−7.5. This
aqueous solution was quickly transferred to a freshly thawed 500 μg
aliquot of deblocked ThioMab (thio-trastuzumab−HC-A114C (9.7
mg/mL), thio-trastuzumab−LC-V205C (15.1 mg/mL), or thio-
trastuzumab−Fc-S396C (5 mg/mL) (Kabat numbering); Genentech,
Inc.), followed by an additional 150 μL of PBS. These ThioMabs were
engineered such that site-specific amino acid residues were mutated to
cysteine.20 If necessary, the final pH was carefully adjusted to 7.5 by
addition of 50 mM borate buffer pH 8.5 in 10 μL increments. The
reaction mixture was constantly mixed at 350 rpm for 1 h, followed by
addition of a 2-fold molar excess of iodoacetic acid to quench, for an
additional 10 min, any remaining free thiols. The amount of
radioactivity in these reactions ranged from 500 to 527 μCi, with
the remainder of the initial 1 mCi presumably lost by sticking to vials
and/or volatility during vacuum. The desired radioimmunoconjugates
were purified using PBS-equilibrated NAP5 desalting columns (GE
Healthcare) and analyzed by size exclusion chromatography. Final
product activities were 255, 298, and 224 μCi for the HC-A114C, LC-
V205C, and Fc-S396C variants, respectively. These corresponded to
conjugation yields of 62, 55, and 50%.
Trastuzumab was radiolabeled by traditional means, through its
tyrosine residues, with 125I by a modified indirect Chizzonite method
as previously described (840 μCi, 88% radiochemical yield).36
Trastuzumab was conjugated to DOTA and radiolabeled with 111In
as previously described (1.19 mCi, 71% radiochemical yield).37
SDS-PAGE. Samples of [125I]6-labeled thio-trastuzumab (LC-
V205C, HC-A114C, and Fc-S396C) in phosphate buffered saline
were analyzed by sodium-dodecyl-sulfate polyacrylamide gel electro-
phoresis (SDS-PAGE). For the endoproteinase lys-C digestion, 2.5 μg
of each antibody (5 × 106 cpm) was combined with 1 μL of
reconstituted (0.5 mg/mL) lys-C (lysyl endopeptidase, Wako, cat.
129-02541) and incubated at pH 8 for 1 h at 37 °C. Following the
incubation, the antibodies were combined with NuPAGE 4× LDS
sample buffer (pH 8.4) (cat. NP0007) and water. For the
dithiothreitol (DTT) reductions, 2.5 μg of each antibody was
combined directly with sample buffer, DTT, and water. Antibodies
from both preparations were incubated at 70 °C for 10 min, then
applied to a NuPAGE 4−12% Bis-Tris gel (cat. NP0321BOX) with
NuPAGE 1× MOPS SDS running buffer (cat. NP0001). The gel was
stained with Coomassie Blue R250 dye. All NuPAGE reagents were
obtained from Invitrogen, Corp. The gel was exposed for 5 min at
room temperature to phosphor-imaging plates (YBIP 2025MS; Fuji
Film Medical Systems, Inc.) and scanned using a Fuji Film BAS-5000
scanner (Fuji Film Medical Systems, Inc.) to obtain digital images of
the radioactivity in the gel.
Tri-tert-butyl-2,2′,2″-(10-(6-(2-(4-hydroxy-phenyl)acetyl)-2,8,11-
trioxo-12-oxa-3,6,9-triaza-tetradecyl)-1,4,7,10-tetraazacyclodode-
cane-1,4,7-triyl)triacetate (2). To a 50 mL round-bottom flask were
added the following: tri-tert-butyl-2,2′,2″-(10-(2-((2-aminoethyl)-
amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)-
triacetate (0.5025 g, 0.724 mmol), paraformaldehyde (0.022 g, 0.724
mmol, 1 equiv), 4-hydroxyphenylacetic acid (0.110 g, 0.724 mmol, 1
equiv), and ethyl isocyanoacetate (0.081 g, 0.724 mmol, 1 equiv). The
mixture was refluxed in 10 mL of MeOH under argon at 70 °C for 5 h.
After solvent removal in vacuo, purification was achieved by
preparative reverse-phase HPLC (gradient from 100% water
containing 0.1% formic acid to 50% water with 0.1% formic acid/
50% CH3CN). Lyophilization yielded a fluffy white hygroscopic solid;
1
58.3%. H NMR (300 MHz, CDCl3) δ 8.87 (m, 1H), 8.48 (m, 1H),
7.03 (m, 2H), 6.81 (m, 2H), 6.34 (br s, 1H), 4.20−2.80 (m, 36H),
1.45 (s, 27H), 1.25 (t, J = 7.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) δ
173.66, 173.23, 170.18, 169.99, 169.77, 169.65, 167.67, 156.60, 156.40,
130.36, 130.04, 125.58, 116.03, 115.81, 82.11, 82.02, 81.98, 81.93,
61.34, 61.24, 56.61, 55.81, 55.48, 52.81, 52.54, 52.06, 50.79, 50.58,
49.99, 49.74, 49.25, 48.77, 47.08, 41.43, 39.59, 37.97, 28.13, 14.16.
HRMS (m/z): [M]+ for C44H73N7O12 892.5395, found 892.5368.
2-(2-(2-(4-Hydroxyphenyl)-N-(2-(2-(4,7,10-tris(2-(tert-butoxy)-2-
oxoethyl)-1,4,7,10-tetra-azacyclododecan-1-yl)acetamido)ethyl)-
acetamido) acetamido)acetic Acid (3). To a 50 mL round-bottom
flask loaded with 2 (0.260 g, 0.291 mmol) was added LiOH·H2O
(0.018 g, 0.437 mmol, 1.5 equiv), 6 mL of EtOH, and 2 mL of H2O.
The reaction was stirred at room temperature for 12 h. After solvent
removal in vacuo, purification was achieved by preparative reverse-
phase HPLC (gradient from 100% 0.1% formic acid in water to 50%
0.1% formic acid in water/50% CH3CN). Lyophilization yielded a
1
fluffy white hygroscopic solid; 65.5%. H NMR (300 MHz, CDCl3) δ
9.34 (m, 1H), 8.57 (s, 1H), 7.02 (m, 2H), 6.86 (m, 2H), 5.82 (br s,
1H), 4.10−2.80 (m, 34H), 1.43 (s, 27H). 13C NMR (75 MHz, CDCl3)
δ 174.37, 173.76, 173.47, 170.14, 169.72, 169.24, 166.96, 156.90,
156.73, 130.30, 129.92, 124.58, 116.17, 115.92, 81.91, 81.86, 81.73,
56.55, 55.74, 52.98, 52.61, 52.25, 51.88, 51.84, 50.55, 49.16, 49.06,
47.69, 43.65, 43.63, 43.21, 39.51, 38.29, 37.84, 28.15. HRMS (m/z):
[M]+ for C42H69N7O12 864.5082, found 864.5082.
Tri-tert-butyl-2,2′,2″-(10-(14-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)-6-(2-(4-hydroxyphenyl)-acetyl)-2,8,11-trioxo-3,6,9,12-tetraazate-
tradecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (4).
To a 50 mL round-bottom flask loaded with 3 (0.113 g, 0.131
mmol) were added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(0.038 g, 0.198 mmol, 1.5 equiv) and 2-maleimidoethylamine·HCl
(0.035 g, 0.198 mmol, 1.5 equiv). The mixture was dissolved in 10 mL
of CH3CN and stirred for 12 h at room temperature. After solvent
removal in vacuo, purification was achieved by preparative reverse-
phase HPLC (gradient from 100% 0.1% formic acid in water to 50%
0.1% formic acid in water/50% CH3CN). Lyophilization yielded a
1
fluffy white hygroscopic solid; 41.4%. H NMR (300 MHz, CDCl3) δ
9.02 (m, 1H), 8.07 (m, 1H), 7.01 (m, 2H), 6.79 (m, 2H), 6.65 (s, 1H),
4.23−2.80 (m, 34H), 1.45 (s, 27H). 13C NMR (75 MHz, CDCl3) δ
173.67, 173.43, 171.04, 170.33, 170.26, 170.08, 169.94, 169.86, 168.64,
156.55, 156.47, 134.16, 130.33, 130.06, 125.57, 125.09, 115.95, 115.84,
81.93, 81.87, 81.81, 81.70, 56.63, 56.08, 55.90, 53.12, 52.01, 50.95,
49.79, 49.41, 49.39, 47.22, 43.44, 43.07, 39.58, 39.15, 38.25, 38.10,
37.74, 37.34, 28.17. HRMS (m/z): [M]+ for C48H75N9O13 986.5562,
found 986.5579. Purity was determined to be 93.1% by RP-HPLC.
Radiochemistry. A 5 μL aliquot of a 1 mg/mL solution of 4 in
chloroform was evaporated to dryness by N2 blowdown. The residue
was dissolved in 100 μL of 0.1% CH3COOH (aq) followed by the
In Vitro Plasma Stability. The LC-V205C, HC-A114C, and Fc-
S396C (Kabat numbering) versions of [125I]6−trastuzumab were
added to CD-1 mouse plasma (Bioreclamation, LLC) at 2 × 107
counts per min (cpm)/mL. The mixture was incubated at 37 °C with
gentle rotation for 0, 6, 24, and 96 h. The samples were transferred to
dry ice and stored at −70 °C until analysis by size-exclusion HPLC.
Biodistribution and Pharmacokinetics. All animal studies were
conducted in accordance with the guidelines of the American
Association for Accreditation of Laboratory Animal Care and the
Genentech Institutional Animal Care and Use Committee. The HER2-
expressing (3+) human breast cancer cell line KPL-4, obtained in 2006
G
dx.doi.org/10.1021/jm401365h | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
from Dr. J. Kurebayashi,27 was used in most studies. The cells were
cultured in RPMI 1640 media plus 1% L-glutamine with 10% FBS. The
KPL-4 cell line was authenticated by short tandem repeat profiling
(AMEL, X; CSF1PO, 11,13; D13S317, 12; D16S539, 12; D5S818,
11,13; D7S820, 9,10; TH01, 9; TPOX, 8; vWA, 14,19) and by single-
nucleotide polymorphism genotyping. Both methods indicate that
KPL-4 cells are unique when compared with a database of more than
1000 human cancer cell lines. C.B-17 Icr SCID (severe combined
immunodeficient; Inbred) female mice (Charles River Laboratories),
weighing between 20 and 25 g were inoculated in the right mammary
fat pad with approximately 3 million KPL-4 cells in a 50:50 suspension
of Hanks’ Buffered Salt Solution (Invitrogen) and Matrigel (BD
Biosciences) in at most 0.2 mL/mouse. When mean tumor volume
reached at least 250 mm3, mice received a single bolus intravenous
injection via the tail vein containing 111In-trastuzumab (5 μCi)
together with either 125I-trastuzumab (5 μCi) or 125I-6-trastuzumab (5
μCi). The total protein doses associated with these radiolabeled
trastuzumab tracers were 0.4 mg/kg for [125I]6 (HC-A114C), 0.5 mg/
kg for [125I]6 (LC-V205C), 0.3 mg/kg for [125I]6 (Fc-S396C), and 0.2
mg/kg for 125I (tyrosine-labeled). To minimize thyroid sequestration
of 125I, 100 μL of 30 mg/mL of sodium iodide was intraperitoneally
administered 1 and 24 h before dosing. Blood samples were collected
at 5 min, 2 h, 1, 3, and 7 days postinjection via retroorbital bleed, with
terminal tissue harvests performed at 3 and 7 days postinjection.
Terminally collected samples included liver, spleen, kidneys, lungs,
intestine (ileum), muscle (gastrocnemius), blood, and tumor. Tissues
were counted for radioactivity using a 2480 Wizard2 automatic gamma
counter (Perkin-Elmer). Counts per minute values were used to
calculate the percent of injected dose per gram of tissue (%ID/g) as
previously described.36
SPECT-CT Imaging. In vivo distribution was obtained by single
photon emission computed tomography/X-ray computed tomography
(SPECT-CT) using modification of previously reported methods.38
Radiolabeling procedures were identical as for the biodistribution
study. Doses of radiolabeled trastuzumab were 202 μCi for [125I]6
(HC-A114C), 156 μCi for [125I]6 (LC-V205C), 164 μCi for [125I]6
(Fc-S396C), 414 μCi for 125I (tyrosine-labeled), and 514 μCi for 111In-
DOTA. These doses were calculated such that each mouse would
receive a protein dose of 10 mg/kg without adjustment in specific
activity. Immediately after CT acquisition, SPECT images were
acquired in a window centered at 35.5 keV for 125I or on two 20%
windows centered at the 173- and 247-keV photopeaks of 111In using a
high-resolution 5-pinhole collimator and a 5.5 cm radius of rotation.
Mice were subsequently euthanized under sedation and tissues
collected for whole-body autoradiography. SPECT quantitation was
accomplished using Amira software (TGS).
(trastuzumab) is marketed in the U.S. by Genentech and
internationally by Roche.
ACKNOWLEDGMENTS
■
We thank Helga Raab, Rachana Ohri, Emily Chan, Gillian
Smelick, Joe Ware, Rami Hannoush, Michelle Schweiger, Jose
Imperio, Kirsten Messick, Nicole Valle, Cynthia Young, Nina
Ljumanovic, Bernadette Johnstone, and Shannon Stainton.
ABBREVIATIONS USED
■
CD20, cluster of differentiation 20; DOTA, 1,4,7,10-tetraaza-
cyclododecane-1,4,7,10-tetraacetic acid; DTPA, diethylenetria-
m i n e p e n t a a c e t i c a c i d ; E D C , 1 - e t h y l - 3 - ( 3 -
dimethylaminopropyl)carbodiimide; Fc, fragment crystallizable;
HC, heavy chain; HER2, human epidermal growth factor
receptor 2; HIP, 4-hydroxy-3-iodophenyl; %ID/g, percentage
of injected dose per g; keV, kiloelectron volt; LC, light chain;
MCC, N-maleimidomethyl)-cyclohexane-1-carbonyl; SCID,
severe combined immunodeficient; SGMIB, N-succinimidyl 4-
guanidinomethyl-3-iodobenzoate; SIB, N-succinimidyl-3-iodo-
benzoate; SIPMB, N-succinimidyl-3-[131I]iodo-4-phosphono-
methylbenzoate
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ASSOCIATED CONTENT
* Supporting Information
■
S
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Notes
The authors declare the following competing financial
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