A traceless vascular-targeting antibody-drug conjugate for cancer therapy

Article abstract of DOI:10.1002/anie.201106527

Right on target: A chemically defined vascular-targeting antibody-drug conjugate (ADC) that offers comprehensive tumor coverage has been developed. When injected intravenously, this ADC potently inhibited tumor growth in a syngeneic immunocompetent model of murine cancer which cannot be cured by conventional cytotoxic agents. Copyright

Full text of DOI:10.1002/anie.201106527

Angewandte
Chemie
DOI: 10.1002/anie.201106527
Cancer Therapy
A Traceless Vascular-Targeting Antibody–Drug Conjugate for Cancer
Therapy**
GonÅalo J. L. Bernardes, Giulio Casi, Sabrina Trꢀssel, Isabelle Hartmann, Kathrin Schwager,
Jçrg Scheuermann, and Dario Neri*
Monoclonal antibodies have demonstrated considerable util-
ity in the clinical treatment of cancer,[1] but unmodified
immunoglobulins are rarely curative, especially when used as
single agents. Thus, there is considerable interest in arming
antibodies with bioactive payloads (e.g., drugs, radionuclides,
cytokines), to improve their potency and selectivity, thus
increasing activity at the tumor site while sparing normal
tissues.[2,3]
Significant progress has been made in the past few years in
the area of antibody–drug conjugates (ADCs) for the
selective delivery of cytotoxic drugs to tumors.[4,5] As a
result of these investigations, new agents with pronounced
clinical activities have been developed, including SGN-35 (an
ADC directed against CD30-positive hematological malig-
nancies)[6] and trastuzumab-DM1 (which has shown activity in
metastatic breast cancer).[7] As conventional drug conjugation
strategies yield heterogeneous ADC preparations, intense
efforts are being devoted to the development of methods for
site-selective modification of therapeutic antibodies, thus
leading to products with improved performance and batch-to-
batch reproducibility.[8] Furthermore, comparative evalua-
tions of intact immunoglobulins in IgG format and other
recombinant antibody formats for ADC development have
been conducted.[9,10]
negative cells.[11] However, monoclonal antibodies specific
to tumor cell antigens often exhibit limited diffusion into the
solid tumor mass by several mechanisms, including slow
extravasation and antibody trapping by perivascular tumor
cells (the so-called antigen barrier).[12] In view of the fact that
the formation of new blood vessels (angiogenesis) is a rare
process in a healthy adult but a characteristic feature of
virtually all types of aggressive cancers, it would be reason-
able to develop vascular-targeting ADCs.[3,13,14] Unlike the
use of cell-specific markers, vascular targeting offers com-
prehensive tumor coverage, as the majority of cancers express
splice isoforms of tenascin-C and of oncofetal fibronectin.[15]
In addition, vascular targeting helps address the issue of
heterogeneity of antigen expression within the tumor mass
(i.e., tumor cells which are positive or negative for the
antigen). Despite the remarkable potency of cytotoxic com-
pounds targeting the tumor vasculature and the strong
dependence of growing neoplastic masses on florid angio-
genesis, only limited efforts were directed in the past towards
the investigation of ADCs that target tumor vascular
antigens.[16] We have recently shown that the antibody-based
delivery of photosensitizers to tumor blood vessels and
irradiation may induce complete and long-lasting cancer
eradication, in a process that also involves the action of
natural killer cells.[17] Thus, there appears to be a strong
rationale for the targeted delivery of cytotoxic agents to the
tumor neovasculature for cancer therapy.
Given that antibodies are large molecules compared to
cytotoxic agents, potent drugs need to be used to generate
ADCs that can be administered at reasonably low doses and
that are compatible with industrial development activities at
acceptable cost of goods.[4] Herein, we aimed at generating a
novel class of chemically defined vascular-targeting ADCs
that release cytotoxic drugs with a mechanism that does not
require antibody internalization. We reasoned that ADCs
based on linkerless antibody modification with a potent thiol-
containing drug would allow the formation of homogeneous
products by the formation of a mixed disulfide. These agents
could release the cytotoxic payload in the extracellular space,
when tumor cell death is initiated and releases high concen-
trations of reducing agents (e.g., cysteine, glutathione) to the
surrounding environment. Provided that a sufficiently large
amount of ADC can be delivered to the subendothelial
extracellular matrix, the drug release process would be
amplified as tumor cell death progresses.
It is generally assumed that ADCs may need to be
internalized by the tumor cells for the active release of
cytotoxic drugs.[4,5] Once ADCs are internalized and the drug
is released in the intracellular compartments, a cross-fire
effect (corresponding to the migration to neighboring cells)
may occur, as has been reported for the treatment of tumors
consisting of a mixture of antigen-positive and antigen-
[*] Dr. G. J. L. Bernardes, Dr. S. Trꢀssel, I. Hartmann,
Dr. J. Scheuermann, Prof. D. Neri
Department of Chemistry and Applied Biosciences
Swiss Federal Institute of Technology (ETH Zꢀrich)
Wolfgang-Pauli Str. 10, 8093 Zꢀrich (Switzerland)
E-mail: dario.neri@pharma.ethz.ch
Dr. G. Casi, Dr. K. Schwager
Philochem AG, Libernstrasse 3, 8112 Otelfingen (Switzerland)
[**] G.J.L.B. is an EMBO and Novartis Foundation Research Fellow. We
thank Katrin Gutbrodt for her help in histology and immunofluor-
escence experiments, Dr. Kathrin Zuberbꢀhler and Nadine Pasche
for their help during therapy experiments, and Dr. Eveline Traschel
for treating tumor-bearing mice with IgG(F8). Financial contribu-
tion from ETH Zꢀrich, Swiss National Science Foundation, Swiss-
Bridge/Stammbach Stiftung, Kommission fꢀr Technologie und
Innovation (KTI) and Philochem AG is gratefully acknowledged.
Dolastatins are a group of small peptides isolated from the
Indian ocean hare Dolabella auricularia[18,19] that bind to
tubulin subunits and inhibit new microtubule assembly and
depolymerize existing microtubules, thus blocking cell cycle
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201106527.
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progression during mitosis.[20] Initial clinical investigations of
dolastatin 10, a linear pentapeptide, and dolastatin 15, a seven
unit depsipeptide, showed no significant benefit in patients
with advanced solid malignancies, possibly as a result of poor
cellular uptake and inadequate pharmacokinetics.[20] Ana-
logues of dolastatins were thus synthesized, in an attempt to
improve in vivo performance. For example, auristatin PE,
cemadotin, and tasidotin were synthesized by variation of the
amide moiety at the C terminus of the molecule and have
been studied in clinical trials in patients with cancer.[20]
Cemadotin (Figure 1a, R = H), a water-soluble analogue of
dolastatin 15 with a terminal benzylamine moiety instead of
cytotoxic activity in the low nanomolar concentration range
comparable to cemadotin (Figure 1b).
As a tumor-targeting moiety we used the human antibody
F8, which is specific to the EDA domain of fibronectin, a
marker of tumor angiogenesis. The antibody was used in a
small immune protein (SIP) format,[24,25] which displays
superior tumor-targeting properties compared to IgGs and
smaller antibody fragments, as evidenced by quantitative
biodistribution studies[26] and by nuclear medicine investiga-
tions.[27] The antibody SIP(F8) can be produced in high yields
in mammalian cells (see the Supporting Information) and
contains a cysteine residue at the C-terminal position of each
eCH4 domain, which can be chemically modified without loss
of immunoreactivity or tumor-targeting performance.[28]
Based on its relative ease of access and reversibility under
mild reducing conditions, we decided to pursue a disulfide
strategy for site-selective antibody modification.[22,23] The C-
terminal cysteine residues involved in the interchain covalent
disulfide bond, may be reduced under mild conditions for
further chemical manipulation without affecting the intra-
domain disulfide bridges and antibody binding, because the
C-terminal cysteine residues are remote from the binding site.
In addition, the resulting noncovalent homodimer remains
kinetically stable after chemical modification (see below).
Our general conjugation strategy (Figure 2a) involved mild
reduction of the covalent disulfide bond using tris(2-carboxy-
ethyl)phosphine hydrochloride (TCEP·HCl) followed by
activation of the resulting cysteine residues with 5,5’-dithio-
bis(2-nitrobenzoic acid) (DTNB; Ellmanꢀs reagent). Subse-
quent incubation with as little as 10 equivalents of CemCH2-
SH for 10 minutes at room temperature in phosphate-
buffered saline (PBS) pH 7.4 with purified SIP(F8)–Ellmanꢀs
yielded a homogeneous mixed disulfide, which we denote
SIP(F8)-SS-CH2Cem. The low pKa of the aromatic thiol
accelerated formation of the mixed disulfide at neutral pH
and prevented competing formation of a symmetric disulfide.
This conjugation method proceeded with complete conver-
sion (> 95%) within 10 minutes (see the Supporting Infor-
mation), thus enabling the preparation of ADCs with
excellent overall yields. The resulting chemically defined,
linkerless SIP(F8)-SS-CH2Cem conjugate displayed excellent
purity and antigen-binding properties, as confirmed by
sodium dodecylsulfate–polyacrylamide gel electrophoresis
(SDS-PAGE) analysis (Figure 2b), ESI-MS analysis (Fig-
ure 2c), size-exclusion chromatography (Figure 2d), and
Biacore studies (Figure 2e). The conjugate was demonstrated
to be stable under storage conditions (À808C and 48C) and
could be detected in mouse plasma even after 72 hours
incubation at 378C as evidenced by ESI-MS analysis (see the
Supporting Information). In addition, SIP(F8)-SS-CH2Cem
readily reacted in the presence of as little as 0.3 mm reduced
glutathione (see the Supporting Information). This observa-
tion is of particular importance since we expect the presence
of small amounts of physiological reducing agents (e.g.,
cysteine, glutathione) to trigger disulfide bond reduction and
the consequent release of the cytotoxic payload from the
antibody.
Figure 1. CemCH2-SH, a potent cytotoxic analogue of cemadotin.
a) Chemical structure of cemadotin (Cem; R=H) and the synthetic
analogue CemCH2-SH (R=CH2SH) used in this study. The analogue
CemCH2-SH was synthesized using peptide coupling chemistry start-
ing from S-4-(aminomethyl)benzyl ethanethioate and the pentapeptide
P5, which was assembled by step-wise solid-phase peptide synthesis.
Hydrolysis of the thioester gave the desired thiol drug. See the
Supporting Information for detailed synthetic procedures and charac-
terization. b) Bioactivity assay of Cem and its analogue CemCH2-SH.
The biological activity of the two drugs was determined by their ability
to inhibit proliferation of cells.
the original pyrrolidone, is one of the most potent cytotoxic
agents described so far.[21] This potent cytotoxic activity
together with its simple chemical structure (when compared
to other known anticancer drugs) makes cemadotin an ideal
candidate for use in our vascular-targeting ADC strategy. We
reasoned that cemadotin could be synthetically manipulated,
by introduction of a thiol reactive tag for disulfide site-
selective conjugation to an antibody,[22,23] without compro-
mising its activity. Thus, a thiol-containing cemadotin ana-
logue (CemCH2-SH, R = CH2SH; Figure 1a) was designed
and synthesized using peptide coupling chemistry and starting
from S-4-(aminomethyl)benzyl ethanethioate and the penta-
peptide P5, which was assembled by step-wise solid-phase
peptide synthesis. A final hydrolysis of the thioester gave the
desired thiol drug (see the Supporting Information for
synthetic details). The in vitro potency of CemCH2-SH was
evaluated in different cell lines and demonstrated to retain
Having established a robust method for the site-selective
modification of SIP(F8) with a potent cytotoxic analogue of
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Figure 2. Construction of a traceless, chemically defined tumor-vascu-
lature-targeting antibody–drug conjugate (ADC). a) Schematic illustra-
tion for the generation of a chemically defined tumor-vasculature-
targeting ADC. The F8 antibody is specific to the EDA domain of
human and murine fibronectin. b) SDS-PAGE analysis of site-selective
conjugation of CemCH2-SH to SIP(F8): M=molecular marker; 1) unre-
duced SIP(F8); 2) SIP(F8) reduced with TCEP; 3) purified SIP(F8)–
Ellman’s; 4) purified SIP(F8)-SS-CH2Cem. c) ESI-MS spectrum of puri-
fied SIP(F8)-SS-CH2Cem. Ion count (main spectrum)=6.07ꢁ105, ion
count (inset)=6.10ꢁ105. d) Gel-filtration analysis of purified SIP(F8)-
SS-CH2Cem. The peak eluting at a retention volume of 15.3 mL
corresponds to the noncovalent homodimeric form of SIP(F8)-SS-
CH2Cem. Arrows indicate standard proteins (11 mL: Ferritin 440 kDa;
14.1 mL: BSA 67 kDa; 15.4 mL: b-lactoglobulin 35 kDa). e) Biacore
analysis of purified SIP(F8)-SS-CH2Cem towards recombinant 11A12
fibronectin.
Figure 3. Therapeutic efficacy of SIP(F8)-SS-CH2Cem in a syngeneic
murine tumor model. a) Immunocompetent 14-week-old female
129SvEv mice bearing subcutaneous F9 teratocarcinomas were treated
intravenously with 960 mg of SIP(F8)-SS-CH2Cem (targeting EDA),
960 mg of SIP(KSF)-SS-CH2Cem (specific to an irrelevant antigen),
18 mg of unconjugated CemCH2-SH or saline (5 mice per group).
Treatment was performed daily for a period of 7 days (arrows). Therapy
was initiated when tumors reached a size of 125 mm3. Data represents
mean tumor volumes (Æstandard error). Tumor growth curves were
stopped when tumors reached a size of 1800 mm3. b) Body weight
variations of the mice during and after therapy. No detectable weight
loss was observed. c) Survival curves of treatment and control groups;
substantial prolongation for mice which received SIP(F8)-SS-CH2Cem.
d) Photos of mice bearing subcutaneous F9 teratocarcinomas on
day 10 after tumor implantation, after having received five daily treat-
ments. e) Fluorescence microscopy analysis of F9 tumor sections, co-
stained for EDA and endothelial cells with SIP(F8) (green) and rat anti-
mouse CD31 antibody (red), respectively. SIP(KSF) was used as
negative control (see the Supporting Information). Scale bar, 100 mm.
f) Effect of a single injection of SIP(F8)-SS-CH2Cem on tumor histol-
ogy. Sections of F9 tumors were excised 24 h after a single intravenous
injection of 960 mg SIP(F8)-SS-CH2Cem, stained with haematoxylin/
eosin and analyzed by light microscope. Left (saline group), Right
(treated tumor). Scale bar, 2 mm.
cemadotin, we evaluated the anticancer activity of this
vascular tumor-targeting ADC in immunocompetent
Sv129Ev mice, bearing subcutaneously grafted F9 tumors.
As negative controls, groups of mice (n = 5) received injec-
tions of saline, unconjugated CemCH2-SH, or SIP(KSF)-SS-
CH2Cem, an ADC which recognizes an antigen of irrelevant
specificity in the mouse (hen egg lysozyme; see the Support-
ing Information).[29] At 8 mgkgÀ1 doses of SIP(F8)-SS-
CH2Cem, around 50% tumor growth retardation was
observed (see the Supporting Information, Figure S1a and
b). The use of the antibody in SIP format was shown to be
preferable when compared to the recombinant diabody(Db)-
Cys format (see the Supporting Information, Figure S2 and
S1c and d). Encouraged by these results, a new therapy
experiment was performed using 43 mgkgÀ1 doses of the
conjugate (corresponding to 18 mg of CemCH2-SH per
mouse). Under these conditions, a very potent tumor
growth retardation was observed for SIP(F8)-SS-CH2Cem,
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In conclusion, a novel approach for the delivery of
cytotoxic drugs using ADCs has been developed. Current
ADCs target tumor cell surface markers and rely on the ADC
being internalized in the cells for drug delivery. Instead, our
approach relies on targeting the tumor neovasculature offer-
ing comprehensive tumoral coverage. The site-selective
chemistry used gives defined products conjugated directly at
cysteine by a disulfide bridge. Unlike current strategies for
ADC preparation, our strategy does not require the use of
elaborate linkers that complicate chemical synthesis and may
be immunogenic. Additionally, our approach leads to a
traceless ADC from which only two species will result upon
cleavage of a disulfide bridge, the native antibody and free
drug.
To our knowledge, this is the first experimental demon-
stration that a noninternalizing vascular-targeting ADC can
be used to mediate a strong antitumor activity in vivo. The
traceless ADC presented here allows the progressive am-
plification of drug release, as tumor cell lysis mediates
glutathione and cysteine release to the surrounding tissue. It
is likely that ADCs that target the tumor vasculature instead
of cell surface markers may offer additional opportunities for
cancer therapy, as angiogenesis is a common feature of
virtually all types of aggressive cancers.
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Received: September 14, 2011
Published online: December 15, 2011
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[30] The naked F8 antibody in scFv or SIP format does not inhibit a
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where the Fc portion could theoretically mediate antibody-
dependent cell cytotoxicity (ADCC). See Figure S3 in the
Supporting Information.
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Keywords: angiogenesis · antibody–drug conjugates · cancer ·
cemadotin · drug delivery
.
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