A small-molecule drug conjugate for the treatment of carbonic anhydrase IX expressing tumors

Article abstract of DOI:10.1002/anie.201310709

Antibody-drug conjugates are a very promising class of new anticancer agents, but the use of small-molecule ligands for the targeted delivery of cytotoxic drugs into solid tumors is less well established. Here, we describe the first small-molecule drug co

Full text of DOI:10.1002/anie.201310709

Angewandte
Chemie
DOI: 10.1002/anie.201310709
Cancer Therapy
A Small-Molecule Drug Conjugate for the Treatment of Carbonic
Anhydrase IX Expressing Tumors**
Nikolaus Krall, Francesca Pretto, Willy Decurtins, GonÅalo J. L. Bernardes, Claudiu T. Supuran,
and Dario Neri*
Abstract: Antibody–drug conjugates are a very promising
class of new anticancer agents, but the use of small-molecule
ligands for the targeted delivery of cytotoxic drugs into solid
tumors is less well established. Here, we describe the first small-
molecule drug conjugates for the treatment of carbonic
anhydrase IX expressing solid tumors. Using ligand–dye
conjugates we demonstrate that such molecules can preferen-
tially accumulate inside antigen-positive lesions, have fast
targeting kinetics and good tumor-penetrating properties, and
are easily accessible by total synthesis. A disulfide-linked drug
conjugate with the maytansinoid DM1 as the cytotoxic payload
and a derivative of acetazolamide as the targeting ligand
exhibited a potent antitumor effect in SKRC52 renal cell
carcinoma in vivo. It was furthermore superior to sunitinib and
sorafenib, both small-molecule standard-of-care drugs for the
treatment of kidney cancer.
effects. To date, monoclonal antibodies have been considered
as the ligands of choice[1,2] and, indeed, research in the field of
antibody–drug conjugates (ADCs) has led to the recent
approval of two ADCs for applications in oncology: brentux-
imab vedotin and trastuzumab emtansine.[3]
In spite of these very encouraging developments, the
limitations of using antibodies for drug-delivery applications
are becoming increasingly clear.[4] Antibodies are large
macromolecules and thus often have difficulties penetrating
deeply into solid tumors.[5] In addition, they can be immuno-
genic[6] and typically long circulation times[7] can lead to
premature drug release and undesired side effects. Moreover,
the production of ADCs is expensive, reflecting the need for
the clinical-grade manufacturing of antibodies, drugs, and the
resulting conjugates.[4]
The use of smaller ligands as delivery vehicles such as
peptides or small drug-like molecules could potentially
overcome some of the above-mentioned problems. Their
reduced size should aid tissue penetration, and they should be
nonimmunogenic and amenable to classic organic synthesis
thus reducing manufacturing costs.[4,8] Indeed, the favorable
properties of drug conjugates that use folic acid[9] and those
with ligands against prostate-specific membrane antigen
(PSMA)[10] as delivery vehicles have been demonstrated and
a folate conjugate has recently entered Phase III clinical
studies.[11] In spite of growing interest from both academia and
industry, the field is still in its infancy as few ligands have been
T
he targeted delivery of highly potent cytotoxic agents into
diseased tissues has emerged as a promising strategy for the
treatment of cancer and other serious conditions. By attaching
a therapeutic effector through a cleavable linker to a ligand
specific to a marker of disease, the effector preferentially
accumulates and acts at the intended site of action, thus
increasing the effectively applied dose while reducing side
[*] N. Krall, F. Pretto, W. Decurtins, Dr. G. J. L. Bernardes, Prof. D. Neri
Institute of Pharmaceutical Sciences, ETH Zurich
HCI, G391.4, Vladimir-Prelog-Weg 1-5/10
8093 Zurich (Switzerland)
E-mail: dario.neri@pharma.ethz.ch
Dr. G. J. L. Bernardes
Current address: Department of Chemistry
University of Cambridge
Lensfield Road, CB2 1EW, Cambridge (UK)
and
Instituto de Medicina Molecular
Faculdade de Medicina da Universidade de Lisboa
Av. Prof. Egas Moniz, 1649-028 Lisboa (Portugal)
Prof. C. T. Supuran
University of Florence, Neurofarba Department
Via Ugo Schiff 6, Polo Scientifico
50019 - Sesto Fiorentino (Firenze) (Italy)
[**] The research conducted by the Neri group on small-molecule-based
tumor targeting is funded by ETH Zurich, the Swiss National
Science Foundation, and Oncosuisse. Prof. Dario Neri is a share-
holder of Philochem AG, which has licensed small-molecule drug-
delivery technology from ETH Zurich. We thank EMBO for a Long-
Term Fellowship to G.J.L.B. and Dr. Raphael Franzini and Dr. Giulio
Casi for critically reading the manuscript.
Scheme 1. Chemical structures of ligand–linker–dye conjugates synthe-
sized for in vitro binding and in vivo targeting studies. FITC=fluor-
escein isothiocyanate.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201310709.
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studied systematically and only a small number of targets
have been tested successfully.
(Figures 2 and 3 (SI)). In contrast to previous reports that
suggested receptor-based internalization of CAIX-specific
ligands,[22–25] acetazolamide-based fluorophore conjugates
were found to preferentially bind to the cell membrane of
kidney cancer cells without efficient internalization (Fig-
ure 1c, Figure 5 (SI)), while the same cells were not stained by
fluorophores lacking the tumor-homing moiety (Figure 1d).
These results collectively suggest that fluorescent probes 1a–c
derived from the approved antiglaucoma drug acetazolamide
(AAZ) were high-affinity CAIX binders (KD = 12.6 nm for
1a) and, thus, potentially suitable for pharmacodelivery
applications.
Here, we report the design and characterization of
a family of novel small-molecule drug conjugates (SMDCs)
targeting carbonic anhydrase IX (CAIX) expressing solid
tumors. CAIX is over-expressed in many different forms of
cancer such as glioblastoma,[12] colorectal,[13] and breast
cancer[14] as a marker of hypoxia, and thus represents a very
attractive antitumor target.[15] In renal cell carcinoma it is
often constitutively expressed and is among the best-charac-
terized cell-surface markers of this disease.[16] Other carbonic
anhydrase isoforms fulfill diverse physiological roles in the
body. Whilst many are intracellular (e.g., the abundant CAII)
some are also extracellularly located (e.g., CAXII).[17,18]
Based on known structure–activity relationship data[19–21]
a series of high-affinity CAIX ligand-linker-dye conjugates
1a–5c (Scheme 1) was prepared. The dissociation constants
of 1a–3a and 5a towards recombinant CAIX were deter-
mined by fluorescence polarization (FP, Figure 1a). Flow
cytometry experiments indicated ligand-dependent binding of
Alexa546 and IRDye750 conjugates 1b and 1c–5c to CAIX-
positive cells (Figure 1b, Figure 1 in the Supporting Informa-
tion (SI)), but not to control cell lines lacking CAIX
We thus investigated the potential of 1b and 1c to to reach
tumors in vivo (Figure 2). An intravenous dose of 3 nmol
Figure 2. Evaluation of tumor-homing performance of targeted conju-
gates 1b and 1c compared to that of untargeted controls 6b and 6c.
a) Whole-animal near-infrared fluorescence images of a representative
mouse 1, 2, and 4 h after intravenous administration of 3 nmol
targeted ligand–IRDye750 conjugate 1c or 6c. A preferential accumu-
lation of construct 1c in the tumor can be observed. Without the
targeting ligand the dye conjugate 6c can only be seen in the kidneys
as a major route of excretion. b) Quantification of targeted conjugate
1c or 6c in homogenized samples of tumors extracted 1, 2, and 4 h
after intravenous administration of 3 nmol dye conjugate. Error bars
indicate standard deviations. Averages of three experiments are
shown. c) 10 mm section of tumor 30 min and 1 h after intravenous
administration of 50 nmol targeted ligand–Alexa546 conjugate 1b or
untargeted 6b. Penetration of the fluorescent dye conjugate (green)
into the tumor increases over time. Areas in the immediate surround-
ing of blood vessels were stained by a 5 min perfusion of the animal
with Hoechst33342 (blue) prior to organ extraction. Scale bar:
200 mm. The untargeted conjugate does not reach the tumor in
detectable levels.
Figure 1. In vitro characterization of ligand–linker–dye conjugates
a) Determination of the KD values of 1a–3a, 5a, and 6a towards
recombinant CAIX extracellular domain in vitro by fluorescence polar-
ization. The KD value of the diazo ligand 4a could not be determined
due to its strong absorption at 495 nm. Data points are given as
averages of three experiments. Error bars indicate standard deviations.
KD values are given in brackets in nm Æstandard errors of fit. b) Flow
cytometric analysis of the binding of Alexa546 conjugates 1b and 6b
to CAIX-expressing SKRC52 cells. Only conjugate 1b containing the
CAIX ligand binds to the cells, whereas nonbinding conjugate 6b
produces a histogram superimposable to that of untreated cells.
c) Confocal microscopy image of CAIX-expressing SKRC52 cells after
exposure to targeted dye conjugate 1b (30 nm) for 1 h. The conjugate
is mainly bound to the cell surface. d) Confocal microscopy image of
CAIX-expressing SKRC52 cells exposed to untargeted dye conjugate 6b
(30 nm) for 1 h. No cell surface binding can be detected. Scale bar:
35 mm.
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AAZ-based conjugate 1c preferentially accumulated in
subcutaneous CAIX-expressing SKRC52 tumors[26] in nude
mice (Figure 2a, Figures 7 and 8 (SI)). Intravenous admin-
istration of 3 nmol of 2c resulted in moderate visible tumor
accumulation after 1 h (Figure 8 (SI)), but the tumor-to-
background ratio was only poor. By contrast, conjugates 3c–
5c and untargeted dye conjugate 6c did not exhibit a prefer-
ential tumor homing (Figure 2a, Figure 8 (SI)). Since 1a has
the lowest KD value followed by 2a, these results suggest that
binding affinity contributes to efficient tumor targeting. As
the best cancer localization results were obtained with 1c, we
decided to quantitatively evaluate the biodistribution of this
compound, comparing it to the tissue distribution properties
of the untargeted dye 6c.
Accumulation of 1c in the tumor was rapid and efficient
with (13.4 Æ 3.0)% of injected dose per gram of tissue
(%IDgÀ1) after only 1 h (Figure 2b, Figures 10 and 11 (SI)).
This result compares favorably with previous work on the
antibody-based targeting of CAIX-expressing tumors, where
only markedly lower tumor uptake values (a maximum of
(2.4 Æ 0.2)%IDgÀ1) could be detected.[27] In our case, the dye
conjugate 1c, however, progressively dissociated from the
tumor (residence half-life t1/2 ꢀ 1 h), suggesting that an
improvement of CAIX binding affinity may further contrib-
ute to efficient tumor targeting performance.
tumors, 1b and 6b were intravenously injected into SKRC52
tumor-bearing mice. Already after 30 min, 1b (Figure 2c) had
diffused outside the immediate surrounding of the blood
vessels stained by perfusion of the animal with Hoechst33342
shortly before tumor extraction. Nontargeted dye conjugate
6b did not reach the tumor in detectable levels (Figure 2c).
As a further indication of targeting selectivity, fluorescence
was restricted to CAIX-positive tissues and excretory organs
(Figure 13 (SI)).
The promising tumor-targeting performance of AAZ
derivatives motivated us to investigate the therapeutic
activity of conjugates comprising a linker cleavable in the
extracellular space and a potent cytotoxic payload. We
focused on conjugates 7a,b and 8a,b with a duocarmycin
derivative as the payload and on the DM1 conjugates 9a,b,
since these cytotoxic drugs are actively being used for the
development of anticancer antibody–drug conjugates.[28,29]
Since CAIX did not internalize efficiently in our hands
(Figure 1c), we hypothesized that the disulfide bond would be
cleaved by reducing agents in the tumor extracellular space
(e.g., glutathione liberated by dying tumor cells). Subse-
quently, the drug would diffuse into the neighboring neo-
plastic cells. A similar process has been proposed for non-
internalizing antibody–drug conjugates.[30]
The syntheses of the drug conjugates 7a–9a are outlined
in Scheme 2a. For all conjugates, a charged peptide spacer
related to a previously described linker was used in order to
improve water solubility.[11] The targeting ligand was attached
A tumor-to-blood ratio of 13.8:1 was observed 1 h after
intravenous injection of 1c (Table 2 (SI)) and further
improved to 79.2:1 after 4 h. Tumor-to-organ ratios for
excretory organs ranged between 0.2:1
for liver and 1.4:1 for kidneys after 1 h
(Tables 1 and 2 (SI)) but a high level of
selectivity was observed for other
organs (e.g., 27.6:1 for tumor to heart
after 1 h, Table 2 (SI)). AAZ is a CA
ligand with broad isoform selectivity,[19]
but the generation of derivatives con-
taining multiple charges may limit their
tissue distribution to the extracellular
space and thus restrict binding to extra-
cellular membrane-associated CAs.
Importantly, tumor targeting was
clearly dependent on the CAIX-binding
moiety, as revealed by strikingly differ-
ent areas under the curve of tumor
uptake over time (Figure 2b) and by the
fact that the AAZ-based targeted dye
conjugate 1c had a 22-fold higher tumor
accumulation at 1 h than the nontar-
geted dye 6c (Table 1 (SI)). Assuming
that 6c is a good model for the tissue
distribution of “naked” (i.e., untar-
Scheme 2. Structure, synthesis, and stability of drug conjugates. a) A CuI-catalyzed azide–alkyne
cycloaddition was used to couple the targeting ligand 10 to the charged linker, which was
prepared by fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis (SPPS). Therapeu-
tic payloads were installed by disulfide exchange. b) Hydrolytic stability of drug conjugates in
PBS pH 7.4 at 378C as determined by mass spectrometry (7a and 8a) and high-performance
liquid chromatography (9a). The carbamate derivative of duocarmycin conjugate 8a (t1/2 >24 h)
was found to be more stable than the carbonate (t1/2 =15 h). DM1 conjugate 9a was found to
be hydrolytically stable. TBTA=tris(benzyltriazolylmethyl)amine, TFA=trifluoroacetic acid,
TIS=triisopropylsilane.
geted) anticancer agents, this compar-
ison highlights the potential impact of
the ligand-based drug delivery of ther-
apeutically relevant doses of drugs into
neoplastic masses.
To gain an understanding of the
microscopic distribution of small ligand-
based targeted dye conjugates inside
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using an CuI-catalyzed alkyne–azide cycloaddition since
direct peptide coupling of the AAZ succinic acid derivative
as in 1a consistently failed. For the duocarmycin conjugates
7a,b and 8a,b, the residual carbonate or carbamate linker was
designed to self-immolate upon disulfide cleavage, yielding
the active drug.[11,31,32] Conjugates 7b, 8b, and 9b lacked AAZ
as the targeting ligand and were used as negative controls.
Drug conjugates bound CAIX in vitro with low nano-
molar affinities whilst control compounds only interacted
weakly (KD > 1 mm), as tested by competitive fluorescence
polarization against 1a (Figure 14 (SI)).[33] As expected, the
carbonate 7a (t1/2 = 15 h) was less stable in PBS at 378C than
the carbamate 8a (t1/2 > 24 h). No decomposition was
observed for the DM1 conjugate 9a under the same
conditions (Scheme 2b). The stability of 7a and 8a was
reduced in mouse serum in vitro[34] (t1/2 = 43 and 61 min
respectively, Figure 15a (SI)), but occurred in a time range
compatible with the preferential accumulation of the AAZ
conjugates at the tumor site (Figure 2b). The DM1 conjugate
9a was significantly more stable (t1/2 = 20 h, Figure 15b (SI)).
The conjugates were cytotoxic when tested in an in vitro assay
(Figures 16 and 17 (SI)).
The therapeutic activity of the conjugates 7–9a,b was
tested in mice bearing subcutaneous SKRC52 tumors. Drugs
were administered at a recommended dose, which was
experimentally determined by dose escalation studies (Fig-
ures 18 and 19 (SI)). Equimolar amounts of untargeted
conjugates 7b, 8b, and 9b together with AAZ were used as
negative controls.
The therapeutic results obtained with the duocarmycin–
AAZ conjugates only indicated a modest tumor growth
inhibition effect (Figure 3a). Nevertheless, targeted carbon-
ate 7a gave rise to statistically significant tumor growth
retardation compared with that observed in mice that only
received vehicle (p < 0.0001) and mice receiving untargeted
conjugate 7b plus equimolar amounts of AAZ (p < 0.05). The
carbamate-based constructs 8a and 8b did not lead to any
retardation in tumor growth. It seems reasonable that the low
affinity of 8a towards the antigen (KD = (40.3 Æ 2.6) nm versus
KD = (7.3 Æ 0.5) nm for 7a) and inefficient extracellular acti-
vation may have been partly responsible for this effect. The
treatment could be performed with a weight loss lower than
15% of body weight (Figure 3b).
For the DM1 conjugate 9a, a potent antitumor effect was
observed at doses that gave only minimal toxicity (i.e., no
detectable body weight loss when 7 ꢀ 70 nmol of DM1
conjugate 9a was given on 7 consecutive days). During the
treatment period tumors shrank and continued to reduce in
volume for 7 additional days. Only 20 days after the start of
treatment did tumors start to grow again, as a consequence of
the mice not receiving additional drug treatment. Impor-
tantly, neither sorafenib nor sunitinib, which represent the
most commonly used chemotherapeutic agents for the treat-
ment of kidney cancer,[35] exhibited any detectable antitumor
effect, in line with previous reports in different models of
kidney cancer.[36] These findings suggest that the targeted
delivery of potent cytotoxic agents may provide a therapeutic
advantage over the current standard of care. DM1 may be
a particularly suitable payload for the development of
Figure 3. Therapy experiments a) Growth of SKRC52 xenografts in
balb/c nu/nu mice treated 5 times on 5 consecutive days (arrows)
with 4 nmol of duocarmycin derivative-based drug conjugates 7–8a,b,
or vehicle (5% DMSO in PBS pH 7.4). Untargeted conjugates 7b and
8b were administered with equimolar amounts of AAZ. Error bars give
standard errors. b) Change of average weight of animals treated with
compounds from (a); error bars give standard deviations. c) Growth of
SKRC52 xenografts in balb/c nu/nu mice treated 7 times on 7
consecutive days (arrows) with 70 nmol of DM1-based drug conju-
gates 9a and 9b, or vehicle (5% DMSO in PBS pH 7.4). Untargeted
conjugate 9b was administered with equimolar amounts of AAZ. The
targeted conjugate 9a has a significantly stronger antitumor effect
than the untargeted conjugate 9b and leads to prolonged suppression
of tumor growth. After 20 days, tumors started regrowing because the
mice had not received any additional drug treatment. Clinically used
kinase inhibitors sorafenib and sunitinib were given as further controls
at a standard dose of 30 mgkgÀ1 but did not exhibit any activity; error
bars give standard errors. d) Change of average weight of animals
treated with compounds from (c); error bars give standard deviations.
* indicates p<0.05; **** indicates p<0.0001. Groups were stopped
when the tumor of one animal in the group reached >2000 mm3 in
volume or weight dropped by >15% with the exception of 9a where
all animals were still alive after 40 days. Each group consisted of five
or six animals.
targeted cytotoxics, since the presence of, for example, an
ester moiety in its structure may facilitate its detoxification by
esterases in clearance-related organs, thus sparing healthy
tissues.[37,38]
To the best of our knowledge, this is the first report of
a therapeutic effect of small-molecule drug conjugates
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[14] S. A. Hussain, R. Ganesan, G. Reynolds, L. Gross, A. Stevens, J.
directed against CAIX, a validated and accessible marker of
renal cell carcinoma.[16] Our work demonstrates that targeting
noninternalizing antigens with small-molecule conjugates can
lead to a preferential product uptake at the tumor site and
release of cytotoxic payloads, with a potent antitumor activity.
A judicious choice of linker–payload combinations contrib-
utes to therapeutic performance, as evidenced by the different
results obtained with conjugates of DM1 and duocarmycin
derivatives, two widely used cytotoxic drugs for ADC
development.[28,29]
Our quantitative biodistribution studies revealed the
contribution of the CAIX-binding moiety to the preferential
accumulation of payloads at the tumor site. By improving the
affinity and isoform selectivity of CAIX-targeting ligands we
expect that therapeutic performance may further be
improved in the future. Importantly, similar studies could be
performed using nuclear medicine techniques in cancer
patients, thus bridging the translational gap between rodent
models of cancer and man. We believe that our findings may
facilitate the development of targeted drugs for the treatment
of CAIX-expressing tumors. Some of the findings presented
here may have a broader applicability for the developments of
targeted cytotoxics directed against other tumor-associated
antigens.
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Published online: && &&, &&&&
Keywords: cancer therapy · carbonic anhydrase IX ·
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Angew. Chem. Int. Ed. 2014, 53, 1 – 6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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These are not the final page numbers!

.
Angewandte
Communications
Communications
Cancer Therapy
Size advantage: Antibodies are currently
the most widely used vehicles for drug-
delivery applications. Small-molecule
ligands, however, may have improved
pharmacokinetics and better tissue pen-
etration. Targeted small-molecule drug
conjugates directed against the tumor
marker carbonic anhydrase IX were syn-
thesized, characterized in vitro, and
tested in vivo.
N. Krall, F. Pretto, W. Decurtins,
G. J. L. Bernardes, C. T. Supuran,
D. Neri*
&&&& — &&&&
A Small-Molecule Drug Conjugate for the
Treatment of Carbonic Anhydrase IX
Expressing Tumors
6
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
These are not the final page numbers!