Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery

Article abstract of DOI:10.1021/ja211589m

Aldehyde drugs are gaining increasing research interest, considering that aldehyde dehydrogenases overexpression is characteristic of cancer stem cells. Here, we describe the traceless site-specific coupling of a novel potent drug, containing an aldehyde

Full text of DOI:10.1021/ja211589m

Article
pubs.acs.org/JACS
Site-Specific Traceless Coupling of Potent Cytotoxic Drugs
to Recombinant Antibodies for Pharmacodelivery
Giulio Casi,§ Nicolas Huguenin-Dezot,‡ Kathrin Zuberbuhler,‡ Jorg Scheuermann,‡ and Dario Neri*,‡
̈
̈
§Philochem AG, Libernstrasse 3, CH-8112 Otelfingen, Switzerland
‡Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zurich), Wolfgang-Pauli-Strasse 10,
̈
CH-8093 Zurich, Switzerland
̈
S
* Supporting Information
ABSTRACT: Aldehyde drugs are gaining increasing research interest,
considering that aldehyde dehydrogenases overexpression is characteristic
of cancer stem cells. Here, we describe the traceless site-specific coupling
of a novel potent drug, containing an aldehyde moiety, to recombinant
antibodies, which were engineered to display a cysteine residue at their
N-terminus, or a 1,2-aminothiol at their C-terminus. The resulting
chemically defined antibody-drug conjugates represent the first example in
which a thiazolidine linkage is used for the targeted delivery and release of
cytotoxic agents.
INTRODUCTION
■
The selective chemical modification of proteins represents an
important avenue for the study of protein function and for the
development of novel biopharmaceuticals.1,2 In particular, bio-
orthogonal reactions, which modify proteins at specific sites
using cleavable linkers, are essential for the development of
antibody-drug conjugates (ADCs).3−5 This emerging class of
biopharmaceuticals consists of armed versions of therapeutic
antibodies, which mediate the selective delivery and release of
potent cytotoxic agents at sites of disease, sparing healthy
tissues. In spite of the large variety of bio-orthogonal reactions
available, most of the current ADCs manufacturing processes
still exploit the chemical modification of the primary amino
group of lysine or of the thiol group of cysteine residues,6,7
thus, yielding heterogeneous mixtures, with possible potency
loss and batch-to-batch reproducibility problems.
Ketones and aldehydes have received particular attention for
the implementation of innovative bio-orthogonal protein modi-
fication strategies. These electrophiles readily condense with
weak amine nucleophiles whose reactivity is enhanced by the
α-effect,8 while being poorly reactive in the presence of other
biological nucleophiles (e.g., amines, thiols, and alcohols). Recent
elegant studies reported the genetic encoding of an aldehyde tag
into proteins, allowing their site selective modification with
hydroxylamines9 or the formation of hydrazones for the selective
capture and release of proteins from complex biological mix-
tures.10 The condensation between carbonyls and 1,2- amino-
thiols at pH 4−5 to yield thiazolidine heterocycles (Figure 1a)
represents a bio-orthogonal reaction8 that has been used in
convergent protein synthesis and covalent capture techniques,11
but not with biosynthetically produced proteins and for ADC
development. In proteins, a cysteine residue in N-terminal posi-
tion represents a natural source of 1,2-aminothiol functionality;
however, protein biosynthesis incorporates methionine as first
Figure 1. (a) Thiazolidine heterocycle forming reaction. (b) An
aldehyde drug undergoes thiazolidine formation by reaction with a 1,2-
aminothiol functional group introduced in a protein either by
engineering of the N-terminus or by the use of a suitable cross-linker.
Relevant antibody formats are also depicted, together with a protein
modified using a maleimido-based bifunctional linker.
amino acid. Methods for producing recombinant proteins with
an N-terminal cysteine residue all rely on cleavage of an
appropriate upstream amino acid sequence.12,13 Recently, 1,2-
aminothiols have been genetically encoded into recombinant
Received: December 12, 2011
Published: March 6, 2012
© 2012 American Chemical Society
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proteins and used for site-specific protein labeling.14 We
reasoned that introducing a cysteine immediately after the leader
peptide sequence in mammalian cell production would afford an
N-terminal cysteine upon protein secretion. The newly introduced
1,2-aminothiol would be compatible with highly selective protein
modification strategies, such as native chemical ligation,15 cyano-
benzothiazole condensation,14 or the coupling and release of po-
tent aldehyde drugs. Indeed, thiazolidine formation would repre-
sent a general approach for the synthesis of a new generation of
ADCs, which would release both the aldehyde drug and the
original antibody in a scarless manner (Figure 1).
METHODS
■
Mammalian Cell Culture and Antibody Production. Antibod-
ies were expressed using transient gene expression (TGE).16 Cells
were handled under sterile conditions in a laminar air flow hood using
disposable sterile plastic pipettes (TPP), sterile pipet tips, and tissue
culture flasks of different sizes (TPP). Incubation of cells occurred either
in a humidified incubator (series II Water Jacketed CO2 Incubator,
ThermoForma) with a 5% CO2/95% air atmosphere or in a shaking
incubator (Innova 4000 or 4430) at 180 rpm (only suspension cells).
The temperature for protein production was 31 °C and for cell growth,
37 °C. The plasmids containing the correct construct were used to
transfect Chinese Hamster Ovary cells (CHO-cells): 1.25 μg of DNA
was used to transfect 1 mllion cells. Polyethyleneimine (PEI) was used
to help DNA in the phagocytosis. A total of 50 μg/mL DNA and
250 μg/mL PEI were mixed and incubated at room temperature for
10 min. Subsequently, the DNA−PEI complex was added to the cells,
previously resuspended in proCHO-4 medium at a concentration of
2 million cells/mL, and gently mixed. The transfected cultures were then
incubated at 37 °C for 4 h in a shaking incubator (160 rpm), diluted
with the same volume of powerCHO-2 CD medium, and then incuba-
ted for 6 days at 31 °C in a shaking incubator (140 rpm). The anti-
bodies were subsequently purified from the cell culture by protein A
affinity chromatography (GE Healthcare, n Protein A, sepharose 4 fast
flow, 17-5280-04), after an initial passage on a sepharose resin (PD10
column, GE Healthcare 17-0851-01) to remove unspecific resin binders
and cellular debris. The protein A column was washed with phosphate-
buffered saline (PBS) pH 7.4, then with 0.1 M NaCl in PBS pH 7.4,
containing 0.1% Tween and finally again with PBS pH 7.4. Proteins were
eluted with 0.1 M glycine, pH 3, adjusting the pH immediately after
elution by addition of 1/10 of the volume of 1 M Tris-HCl, pH = 7. The
optical density (OD) of the different fractions was measured at 280 nm
(Cary300 UV−visible spectrophotometer, Varian) and all fractions with an
OD280 higher than 0.1 were pooled. The combined fractions were dialyzed
at 4 °C against PBS using SpectraPor dialysis membranes (12−14 kDa
MWCO). Typical nonoptimized antibody yields were 5 mg/L of culture.
N-Terminal Cysteine Modified Antibody Pretreatment. After
purification, antibodies were first reduced in PBS with 1,4-dithiothreitol
(DTT) (10 mM, 1 h) and subsequently treated with 4 M methoxy-
amine hydrochloride (MeONH2·HCl) (final concentration 400 mM,
pH 3−4, 16 h). The mixture was purified by fast protein
liquid chromatography (FPLC) (Amersham Pharmacia) using two
HiTrap desalting columns in series (GE Healthcare) of 5 mL volume
equilibrated with potassium acetate buffer (AcOK, 100 mM, pH 4.5,
for thiazolidines condensation) or with PBS (for native chemical
ligation experiments, see Supporting Information (SI)) at a flow rate
of 1 mL/min. The fractions containing the antibody were collected,
concentrated, and analyzed using liquid chromatography−mass
spectrometry (LC−MS) and sodium dodecylsulfate−polyacrylamide
gel electrophoresis (SDS−PAGE). ESI−MS: Cys-Db(F8) (calcd
24890.0 Da, found 24890.0 Da, Figure 2e); Cys-Gly-Db(F8) (calcd
24947.0 Da, found 24946.0 Da, Figure 2h).
Figure 2. (a) DNA structure and amino acid sequence of an engine-
ered F8 antibody mutant, carrying a Cys residue (C) immediately after
the last residue of the leader peptide sequence used for protein
secretion (S). The exact position of the cleavage site is shown. Three
different mutants were cloned, containing 0, 1, or 4 Gly residues
after the Cys residue. (b) Schematic representation of a recom-
binant antibody in diabody format (Cys-Db), carrying a Cys residue
at the N-terminus of the VH domain (gray), after cleavage of the
leader peptide. The diabody format consists of a VH domain
sequentially linked to the VL domain (white) of the same antibody by
means of a short (n = 5) peptide linker, which drives the
noncovalent, yet stable, homodimerization of two VH−VL poly-
peptides. In the Cys-Db(F8) variants, the N-terminal Cys residue is
depicted in order to illustrate the site for selective thiazolidine
formation. (c) SDS−PAGE analysis of Cys-Db(F8); lanes 2−3, Cys-
Db(F8) in nonreducing and reducing conditions;17 lanes 4−5, Cys-
Db(F8) after pretreatment with DTT and MeONH2·HCl (non-
reducing and reducing conditions). (d and e) ESI−MS of Cys-
Db(F8) immediately after purification on Protein A resin and after
pretreatment with DTT and MeONH2·HCl. (f) SDS−PAGE and
(g and h) ESI−MS analysis of Cys-Gly-Db(F8), in similar conditions
as described for the Cys-Db(F8) mutant.
Figure 3. (a) Synthetic scheme for the site-selective 1,2-aminothiol
incorporation at a C-terminal cysteine residue in the diabody Db(F8)-
Cys. (b) SDS−PAGE monitoring of the Db(F8)-Cys modification
process; lanes 1−2, Db(F8)-Cys in nonreducing and reducing
conditions; lane 3, Db(F8)-Cys-M-thz, a reaction intermediate run
under nonreducing conditions; lanes 4−5, Db(F8)-Cys-M-Cys
(nonreducing and reducing conditions). (c and d) ESI−MS of
reduced Db(F8)-Cys-M-thz and Db(F8)-Cys-M-Cys, respectively.
1,2-Aminothiol Functional Group Introduced with a Cross-
Linker. The F8 antibody in diabody format, carrying a cysteine residue
at the C-terminus (Db(F8)-Cys),17 dissolved in PBS (29 μM, 0.058 μmol,
0.73 mg/mL, 2 mL), was reduced with tris(2-carbox-yethyl)phosphine
hydrochloride (TCEP·HCl) (20 equiv, 11.6 μL from 100 mM solution in
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water, 4 °C, 9 h, Figure 3). To that mixture, N-(2-(2,5-dioxo-2,5-dihydro-
1H-pyrrol-1-yl)ethyl)thiazolidine-4-carboxamide (40 equiv, 23 μL from
100 mM solution in water) was added and incubated at 4 °C. After 15 h,
the excess of the maleimido reagent was quenched with
β-mercaptoethanol (16 μL, 100 equiv with respect to the malei-
mide reagent) during 1 h at 4 °C. This intermediate product was analyzed
by ESI−MS (calcd 25259.0 Da, found 25258.0 Da, Figure 3c), and
directly treated with MeONH2·HCl at pH 3−4 (400 mM final
concentration, 16 h) for the thiazolidine opening reaction. The protein
was subsequently purified by FPLC (HiTrap desalting columns, AcOK
pH 4.5 eluting buffer). ESI−MS (calcd 25247.0 Da, found 25246.0 Da,
Figure 3d). The overall yield of modified antibody recovered was 71%.
General Procedure for Thiazolidine formation. The protein
solution in AcOK (pH 4.5, 100 mM) was concentrated with
centrifugal filter units from Millipore (Amicon, Ultra centrifugal filter,
0.5 mL; Ultracel, 3 kDa membrane; Millipore; UFC500324) until an
antibody concentration of 20 μM was reached. The concentrated
proteins were first reduced with 1 mM DTT or 1 mM TCEP·HCl (16 h),
and then incubated with cemadotin aldehyde (Cem-CHO, 40 mM
stock in EtOH, 1−4 mM final concentration, 10% max EtOH con-
centration, 40−200 equiv). The reaction mixture was incubated at
4 °C for 2.5−4 days, monitoring conversion by LC−MS, and then
purified by FPLC (Pharmacia Akta-FPLC system) with two HiTrap
desalting columns in series (GE Healthcare) of 5 mL volume equilibra-
ted with PBS as running buffer at a flow rate of 1 mL/min. The
fractions containing the antibody were collected, concentrated
(absorbance at 280 nm), and used for release experiments or in vitro
cytotoxicity assay. ESI−MS: Cem-thz-Db(F8) (calcd 25541.0 Da, found
25542.0 Da, Figure 5b). Cem-thz-Gly-Db(F8) (calcd 25598.0 Da, found
25598 Da, Figure 5c); Db(F8)-Cys-M-thz-Cem (calcd 25898.0 Da, found
25900.0 Da, Figure 6b).
Drug Release Experiments. ADCs were tested for stability and
drug release. Aliquots (110 μL, 5−10 μM concentration in PBS) were in-
cubated at three different temperatures (i.e., 37, 4, −80 °C) and treated at
given time points as follows. The aliquots (thawed if necessary) were shortly
centrifuged (4 °C, 1 min, 14 000 rpm), the supernatant was transferred to a
1.5 mL Eppendorf tube, then 5.5 μL of DMSO was added (to reduce
unspecific interactions with resin particles). The solution was incubated with
protein A resin (50 μL from 50% slurry, prewashed twice with 2×
500 μL of 5% DMSO in PBS). The mixture was incubated at 4 °C for 20
min in a thermomixer; then, the resin was removed by gentle centrifugation
(1 min, 600g). For each time point in the study, 100 μL of supernatant was
withdrawn, shock frozen, and stored at −20 °C until further analysis. The
concentration of released Cem-CHO was determined by tandem LC−MS/
MS analysis, using a Multiple Reaction Monitoring (MRM) technique.
Standard solutions of the synthetic Cem-CHO (25−0.05 μM) were used to
obtain a calibration curve and samples were treated exactly as for the drug
aliquots released from the corresponding ADC. Prior to analysis, samples
were acidified (final [HCl] = 100 mM) and analyzed on a Micromass
Quattro micro API using an XTerra MS C18 column, 3.5 μM, 1.0 × 50 mm
(Waters; 186000386) and run on a linear gradient from 0 to 90%
acetonitrile (B) in 0.1% formic acid (A) in 11 min. For MRM analysis,
daughter ions with m/z 85.98, 100.3, 227.43, and 340.5 of the parent ion
669.43 from Cem-CHO were monitored, comparing peak areas to those of
the Cem-CHO calibration series.
Twenty microliters of this solution was added to each well, and after 2 h
of incubation at 37 °C in 5% CO2, absorption was measured at 490 nm.
The percentage of cell growth inhibition was calculated as a ratio of the
counts between treated cells over untreated cells.
RESULTS
■
Preparation of 1,2-Aminothiol Containing Antibodies.
To facilitate analytical studies in the absence of protein glycosyla-
tion, we used recombinant antibodies in diabody format,18 pro-
duced in CHO cells using transient gene expression (TGE).16 For
chemical transformations, we used the clinical-stage human
monoclonal antibody F8, which is specific to the alternatively
spliced EDA domain of fibronectin, a marker of tumor angio-
genesis.19,20
One strategy for the introduction of a 1,2-aminothiol moiety
in the antibody molecule consisted in the introduction of a
cysteine residue immediately after the cleavage site of the leader
peptide required for antibody secretion (Figure 2). This strategy
led to the production of F8 variants (termed Cys-Db(F8)), which
contained an N-terminal cysteine residue separated from the main
antibody by a variable number of glycine residues (n = 0, 1, 4).
The best results in terms of protein homogeneity and expression
yields were obtained with the shortest N-terminal sequences (Cys-
or Cys-Gly-, Figure 2). Closely related protein species were
observed by MS analysis after antibody purification (Figure 2d,g),
which were converted into a single molecular species of the
predicted molecular weight upon treatment with DTT (10 mM)
as reducing agent, followed by treatment with MeONH2·HCl
(400 mM, Figure 2e,h).21
A second strategy for the introduction of the 1,2-aminothiol
moiety into the F8 antibody in diabody format featured the
chemical modification of an engineered C-terminal cysteine residue
(Db(F8)-Cys)17 by means of a newly developed maleimide/
thiazolidine (M-thz) heterobifunctional linker (Figure 3). After a
mild reduction of the covalent interchain disulfide bond17,22,23 of
Db(F8)-Cys with TCEP·HCl (1 mM), the resulting thiol was
reacted with the M-thz linker, yielding a modified antibody carrying
a thiazolidine moiety (Db(F8)-Cys-M-thz; Figure 3a). The reaction
was selective and quantitative, as shown by SDS−PAGE and ESI−
MS analysis (Figure 3b,c). The thiazolidine group was then
deprotected with a molar excess of MeONH2·HCl (400 mM final
concentration, pH 3−4) to afford the desired 1,2-aminothiol
(Db(F8)-Cys-M-Cys, Figure 3d), suitable for subsequent mod-
ification with aldehydes, in good yields.
Cemadotin Aldehyde (Cem-CHO). To generate a potent
cytotoxic drug containing an aldehyde moiety for thiazolidine
formation, we synthesized a dolastatin-15 analogue (Figure 4a,
RCHO; see SI for synthetic details), based on a conservative
modification of the corresponding cytotoxic drug cemadotin
(Figure 4a, RH).24 Dolastatins are a class of small peptides,
isolated from the shell-less mollusk Dolabella auricularia, that
bind to tubulin subunits, inhibit microtubule assembly, and de-
polymerize existing microtubules, thus, blocking cell cycle pro-
gression during mitosis at the G2−M phase. The depsipeptide
dolastatin-15 is one of the most studied members of this family.
However, this drug showed no significant benefits for patients
with advanced solid malignancies, possibly due to poor cellular
uptake and inadequate pharmacokinetics.25 Cemadotin is a highly
toxic, water-soluble analogue of dolastatin-15 with simplified
peptidic structure and a C-terminal benzylamine moiety in place of
the original pyrrolidone. This drug has been studied as anticancer
agent in numerous clinical trials, but its narrow therapeutic
window has hampered advanced clinical developments.25 The
In Vitro Cytotoxicity Assays. HEK 293, HL60 (CCL-240, ATCC),
and WI38VA-13 cells (CCL-75.1, ATCC) were grown in Dulbecco’s
Modified Eagle Medium (DMEM; Gibco-Invitrogen) medium supple-
mented with 10% fetal calf serum (FCS), 100 U/mL penicillin, and
100 μg/mL streptomycin (Gibco-Invitrogen). F9 murine teratocarcinoma
cells (CRL-1720, ATCC) were grown in RPMI (Gibco-Invitrogen) media
supplemented with 10% FCS, 100 U/mL penicillin, and 100 μg/mL
streptomycin (Gibco-Invitrogen) in culture dishes coated with 0.1%
gelatin. All cell lines were maintained at 37 °C in 5% CO2 atmosphere.
For the in vitro cytotoxicity assay, 10 000−15 000 cells of each cell line
were seeded in 100 μL into a multiwell plate and a defined volume
of the appropriate dilution of cemadotin or cemadotin products (e.g.,
Cem-CHO, and Cemadotin ADCs) was added. After incubation at 37 °C
in 5% CO2 for 72 h, cell viability was determined with the Cell
Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega).
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Figure 4. (a) Chemical structure of cemadotin (RH, Cem) and the
corresponding aldehyde derivative (RCHO, Cem-CHO). (b) In
vitro cytotoxicity test. Cytotoxicity of Cem and Cem-CHO was
measured by adding the compounds at various concentrations to F9,
HL60, and HEK 293 cell lines. Cells were incubated for 72 h at 37 °C
in 5% CO2 atmosphere. Cell viability was then monitored with the
Cell Titer 96 Aqueous One Solution Cell Proliferation Assay
(Promega); values were read after 2 h. The figure shows that the
cell killing profiles for Cem and Cem-CHO are comparable.
Figure 6. (a) Schematic representation of Db(F8)-Cys-M-Cys diabody
reacting with Cem-CHO to form chemically defined thiazolidine
conjugates. (b) Db(F8)-Cys-M-thz-Cem coupling product, analyzed by
SDS−PAGE (lane 2, nonreducing conditions), ESI−MS, size-exclusion
chromatography (Superdex 75 HR 10/300 GL column), and Biacore on
microsensor chip coated with EDA-containing 11A12 fragment.17
MS (Figures 5b,c and 6b). The resulting chemically defined
conjugates, Cem-thz-Db(F8), Cem-thz-Gly-Db(F8), and Db-
(F8)-Cys-M-thz-Cem, displayed excellent purity, stability, and
antigen binding properties, as confirmed by SDS−PAGE,
ESI−MS, size-exclusion chromatography, and Biacore analysis
(Figures 5b,c and 6b).
cemadotin aldehyde analogue (Figure 4a; hereafter named Cem-
CHO) retained the cell killing activity of the parental cemadotin
compound toward a different panel of cell lines, with significant
potency in the low nanomolar range (Figure 4b).
Thiazolidine Linkage Formation. Our general conjuga-
tion strategy (Figures 5a and 6a) relied on thiazolidine linkage
Cem-CHO Release Experiments. Having established
reliable methods for the site-specific modification of antibodies
with a potent analogue of cemadotin, we evaluated the kinetics
of drug release and the resulting cytotoxicity of the cleavable
ADCs synthesized from N-terminal cysteine modified antibod-
ies (Cem-thz-Db(F8) and Cem-thz-Gly-Db(F8)). Cem-CHO
release from both chemically modified antibodies was monitored
at 37 °C in PBS by tandem LC−MS/MS spectrometry (Figure 7a).
The half-life for the Cem-thz-Gly-Db(F8) conjugate (45 h)
yielded ∼65% of free drug after 75 h (pure Cem-CHO was
used as positive control for MS quantification of the release
process). Both ADCs were stable when stored at low tem-
perature, as confirmed by release kinetics measured at
4 °C and −80 °C (SI).
We evaluated the Cem-CHO release process in cytotoxicity
assays with WI38VA-13 and HL60 cell lines. These cells do not
enable an antibody-mediated internalization of the drug and were
chosen to test in vitro the cell killing ability of Cem-CHO released
from the corresponding thiazolidine-based ADC. Unmodified
Cem-CHO was used as positive control in the experiment, while
antibody conjugates with cemadotin linked via a noncleavable
amide bond, synthesized by native chemical ligation (SI), were
used as negative controls. As expected, Cem-CHO released from
the thiazolidine-based conjugates mediated a cell killing activity
almost comparable to the one of the same drug administered at
once to the cell culture as pure chemical reagent. By contrast, as
expected, ADCs with stable amide bonds exhibited a substantially
lower cytotoxicity (Figure 7b).
Figure 5. (a) Schematic representation of Cys-Db(F8) and Cys-Gly-
Db(F8) diabodies reacting with Cem-CHO to form chemically
defined thiazolidine conjugates. (b and c) Cys-Db(F8) and Cys-Gly-
Db(F8) coupling products with Cem-CHO, respectively, analyzed by
SDS−PAGE (lanes 1 and 2, nonreducing and reducing conditions),
ESI−MS, size-exclusion chromatography (Superdex 75 HR 10/300 GL
column and Superdex 200 HR 10/300 column), and Biacore technology
on microsensor chip coated with EDA-containing 11A12 fragment.17
DISCUSSION
■
Chemoselective reactions are of fundamental importance for
the generation of post-translationally modified proteins and for
the production of antibody−drugs conjugates. The thiazolidine
formation reaction has been known to chemists for decades but
had previously never been used for the site-selective preparation of
antibody−drug conjugates, capable of a slow release of a potent
formation between the 1,2-aminothiol containing diabodies
and Cem-CHO in AcOK buffer at pH 4.5 under mild reducing
conditions (1 mM DTT or TCEP·HCl). After 2.5−4 days,
we observed conversions to the desired antibody-drug
conjugates with yields in excess of 90% as confirmed by ESI−
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ideally suited for the site-specific coupling of cytotoxic aldehydes to
tumor-targeting antibodies (such as F8) in the diabody format.
Diabodies display superior biodistribution properties compared to
other antibody formats, which have already been validated in
quantitative biodistribution experiments with radiolabeled antibody
preparations.28−30 The introduction of an N-terminal cysteine
residue, following leader peptide cleavage, is a general strategy
which is applicable to other antibody formats (Figure 1) and is
compatible with standard antibody manufacturing procedures.
Alternatively, 1,2-aminothiols can be introduced by chemical trans-
formation of cysteine-modified recombinant antibodies using the
newly developed M-thz bifunctional linker (Figure 3), but the
biosynthetic approach described in Figure 2 is more attractive, as it
requires fewer chemical modification steps.
To our knowledge, this is the first example in which a
thiazolidine linkage was used as cleavable linker for the targeted
delivery and slow release of cytotoxic drugs.
The antibody−drug conjugates, prepared using antibodies
carrying an engineered Cys residue at the N-terminus, were
assembled in a single site-specific, chemoselective step. Upon
thiazolidine cleavage, the native antibody and the free drug are
restored, leaving no “scar” from the original linkage. This
chemical feature facilitates the pharmacokinetic analysis of the
drug release process and limits immunogenic reactions, which
may be associated with the presence of residual linker moieties
on the antibody.31−33
We have recently generated and characterized antibody
conjugates based on thiol-containing drugs and mixed disulfides
as a second example of cleavable traceless linkers.34 Pharmaceu-
tical agents containing an aldehyde moiety have been approved
in the categories of antibiotics,35 antibacterials,35 and antialler-
gics.36 Additional aldehyde drugs are under active development.
One of them, Gossypol (AT101), is a potent Bcl-2 antagonist
which is currently being investigated in phase II studies against
various tumor types. AT101 possesses two aldehyde groups
that are responsible for interactions with different biomolecular
targets.37 Masking aldehydes by conjugation to antibodies is an
attractive pharmaceutical strategy, as it protects the aldehyde
function from reaction with bioavailable nucleophiles and from
oxidation.
The use of aldehyde drugs and of thiazolidine linkers may be
particularly suited for the selective killing of cancer stem cells
by antibody−drug conjugates, considering the fact that alde-
hyde dehydrogenase (ALDH) activity represents one of the
most prominent characteristics of “cell stemness”.38,39 ALDH-
mediated chemical transformation of membrane-permeable
aldehyde derivatives of fluorophores into their corresponding
carboxylic acids (which remain trapped in the cell) has long
been used to selectively label stem cells and for fluorescence-
activated cell sorting procedures.40,41 It would therefore be
feasible to use thiazolidine linkers for the coupling of aldehyde
drugs with internalizing human antibodies capable of selective
recognition of cancer stem cells. Those tumor cells are char-
acterized by an unlimited regeneration potential and are
suspected to be responsible for resistance to chemotherapeutic
treatment and relapse.42−44 The intracellular conversion of
aldehyde drugs into the corresponding carboxylic acids is likely
to be associated with a potent cytotoxic effect, if the acids
remain bioactive and if their exit from the cell is hindered by
their negative charge at neutral pH.
Figure 7. Drug release experiments from thiazolidine-based ADCs.
(a) LC−MS/MS quantification of Cem-CHO released from Cem-thz-
■
Db(F8) ( ; left y axis; starting ADC concentration of 5.0 μM) or
●
Cem-thz-Gly-Db(F8) ( ; right y axis; starting ADC concentration of
10.0 μM). Samples were incubated at 37 °C in PBS, the residual ADC
and the free antibody were removed by affinity capture on protein-A
resin, and the released Cem-CHO was analyzed and quantitated at
different time points using Multiple Reaction Monitoring (MRM)
technique. Values measured were compared with a calibration curve
generated from a standard dilution series of the free Cem-CHO drug.
(b) In vitro cytotoxicity assay of Cys-Db(F8) (blue) and Cys-Gly-
Db(F8) (green) conjugates with Cem-CHO, performed on WI38VA-
13 and HL60 cells. The cell killing activity mediated by Cem-CHO
released from thiazolidine-based conjugates (solid lines) was compared
to the one observed with amide bond-based conjugates (dashed lines)
and the free Cem-CHO drug (black). Cytotoxicity of Cem-CHO and
of the conjugates was measured by adding the compounds at various
concentrations to WI38VA-13 and HL60 cell lines and determining
their vitality, as described in Methods. Amino acids are indicated using
single letter codes: C = Cys; G = Gly; CONH indicates the amide
bond.
cytotoxic agent. Oximes, hydrazones, and thiazolidines are stable
aldehyde derivatives, with the latter being the most stable form in a
subclass of glyoxal derivatives.8 The stability of hydrazones formed
from aldehydes with electron-withdrawing substituents is max-
imal.26 In this context, the benzaldehyde moiety present in Cem-
CHO displays intermediate reactivity values, thus, yielding deriva-
tives sufficiently stable for synthesis and storage, while permitting a
hydrolytic drug release under physiological conditions at 37 °C.
The half-life of Cem-thz-Gly-Db(F8) (45 h) is long compared to
the circulatory half-life of Db(F8) in vivo following intravenous
administration in tumor-bearing mice (T1/2 ∼ 30 min), but is short
compared to the residence time of the antibody at the tumor site
(several days).20,27 Thus, the thiazolidine linker appears to be
5891
dx.doi.org/10.1021/ja211589m | J. Am. Chem. Soc. 2012, 134, 5887−5892

Journal of the American Chemical Society
Article
(24) de Arruda, M.; Cocchiaro, C. A.; Nelson, C. M.; Grinnell, C. M.;
Janssen, B.; Haupt, A.; Barlozzari, T. Cancer Res. 1995, 55, 3085.
(25) Simmons, T. L.; Andrianasolo, E.; McPhail, K.; Flatt, P.;
Gerwick, W. H. Mol. Cancer. Ther. 2005, 4, 333.
(26) King, T. P.; Zhao, S. W.; Lam, T. Biochemistry 1986, 25, 5774.
(27) Schwager, K.; Kaspar, M.; Bootz, F.; Marcolongo, R.; Paresce,
E.; Neri, D.; Trachsel, E. Arthritis Res. Ther. 2009, 11, R142.
(28) Wu, A. M.; Senter, P. D. Nat. Biotechnol. 2005, 23, 1137.
(29) Borsi, L.; Balza, E.; Bestagno, M.; Castellani, P.; Carnemolla, B.;
Biro, A.; Leprini, A.; Sepulveda, J.; Burrone, O.; Neri, D.; Zardi, L. Int.
J. Cancer 2002, 102, 75.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, characterization data, 1,2-aminothiol
incorporation with cross-linkers, native chemical ligation
conditions. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
dario.neri@pharma.ethz.ch
(30) Berndorff, D.; Borkowski, S.; Moosmayer, D.; Viti, F.; Muller-
̈
Tiemann, B.; Sieger, S.; Friebe, M.; Hilger, C. S.; Zardi, L.; Neri, D.;
Dinkelborg, L. M. J. Nucl. Med. 2006, 47, 1707.
(31) Kosmas, C.; Snook, D.; Gooden, C. S.; Courtenayluck, N. S.;
McCall, M. J.; Meares, C. F.; Epenetos, A. A. Cancer Res. 1992, 52,
904.
Notes
The authors declare the following competing financial
interest(s): Giulio Casi is employed at Philochem AG, Dario
Neri is a co-founder and shareholder of Philogen SpA.
(32) Buskas, T.; Li, Y. H.; Boons, G. J. Chem.Eur. J. 2004, 10, 3517.
(33) Boeckler, C.; Frisch, B.; Muller, S.; Schuber, F. J. Immunol.
Methods 1996, 191, 1.
ACKNOWLEDGMENTS
■
We thank Dr. Sarah Wulhfard, Dr. Alessandra Villa, Dr. Kathrin
Schwager and Nadine Pasche for their help during protein
engineering. Financial contribution from ETH Zurich, Swiss
(34) Bernardes, G. J. L.; Casi, G.; Hartmann, I.; Trussel, S.; Schwager,
̈
K.; Scheuermann, J.; Neri, D. Angew. Chem., Int. Ed. 2012, 51, 941.
(35) Morikawa, K.; Oseko, F.; Morikawa, S.; Iwamoto, K. Antimicrob.
Agents Chemother. 1994, 38, 2643.
̈
National Science Foundation, Swiss-Bridge/Stammbach
Stiftung, Kommission fur Technologie und Innovation (KTI)
̈
(36) Bohets, H.; McGowan, C.; Mannens, G.; Schroeder, N.;
Edwards-Swanson, K.; Shapiro, A. J. Ocul. Pharmacol. Ther. 2011, 27,
187.
and Philochem AG is gratefully acknowledged.
(37) Kitada, S.; Leone, M.; Sareth, S.; Zhai, D. Y.; Reed, J. C.;
Pellecchia, M. J. Med. Chem. 2003, 46, 4259.
(38) Marcato, P.; Dean, C. A.; Pan, D.; Araslanova, R.; Gillis, M.;
Joshi, M.; Helyer, L.; Pan, L.; Leidal, A.; Gujar, S.; Giacomantonio,
C. A.; Lee, P. W. K. Stem Cells 2011, 29, 32.
(39) van den Hoogen, C.; van der Horst, G.; Cheung, H.; Buijs, J. T.;
Pelger, R. C. M.; van der Pluijm, G. Clin. Exp. Metastasis 2011, 28, 615.
(40) Jones, R. J.; Barber, J. P.; Vala, M. S.; Collector, M. I.;
Kaufmann, S. H.; Ludeman, S. M.; Colvin, Q. M.; Hilton, J. Blood
1995, 85, 2742.
(41) Storms, R. W.; Trujillo, A. P.; Springer, J. B.; Shah, L.; Colvin,
O. M.; Ludeman, S. M.; Smith, C. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 9118.
REFERENCES
■
(1) Hackenberger, C. P. R; Schwarzer, D. Angew. Chem., Int. Ed.
2008, 47, 10030.
(2) Stephanopoulos, N.; Francis, M. B. Nat. Chem. Biol. 2011, 7, 876.
(3) Chari, R. V. J. Acc. Chem. Res. 2008, 41, 98.
(4) Alley, S. C.; Okeley, N. M.; Senter, P. D. Curr. Opin. Chem. Biol.
2010, 14, 529.
(5) Webb, S. Nat. Biotechnol. 2011, 29, 297.
(6) Senter, P. D. Curr. Opin. Chem. Biol. 2009, 13, 235.
(7) Ducry, L.; Stump, B. Bioconjugate Chem. 2010, 21, 5.
(8) Tam, J. P.; Lu, Y. A.; Liu, C. F.; Shao, J. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 12485.
(9) Carrico, I. S.; Carlson, B. L.; Bertozzi, C. R. Nat. Chem. Biol.
2007, 3, 321.
(10) Zeng, Y.; Ramya, T. N. C.; Dirksen, A.; Dawson, P. E.; Paulson,
J. C. Nat. Methods 2009, 6, 207.
(11) Villain, M.; Vizzavona, J.; Rose, K. Chem. Biol. 2001, 8, 673.
(12) Muir, T. W. Annu. Rev. Biochem. 2003, 72, 249.
(13) Casi, G.; Hilvert, D. Curr. Opin. Struct. Biol. 2003, 13, 589.
(14) Nguyen, D. P.; Elliott, T.; Holt, M.; Muir, T. W.; Chin, J. W.
J. Am. Chem. Soc. 2011, 133, 11418.
(42) Dalerba, P.; Cho, R. W.; Clarke, M. F. Annu. Rev. Med. 2007, 58,
267.
(43) Dean, M.; Fojo, T.; Bates, S. Nat. Rev. Cancer 2005, 5, 275.
(44) Zhou, B.-B. S.; Zhang, H.; Damelin, M.; Geles, K. G.; Grindley,
J. C.; Dirks, P. B. Nat. Rev. Drug Discovery 2009, 8, 806.
(15) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H.
Science 1994, 266, 776.
(16) Muller, N.; Derouazi, M.; Van Tilborgh, F.; Wulhfard, S.;
Hacker, D. L.; Jordan, M.; Wurm, F. M. Biotechnol. Lett. 2007, 29, 703.
(17) Trussel, S.; Dumelin, C.; Frey, K.; Villa, A.; Buller, F.; Neri, D.
̈
Bioconjugate Chem. 2009, 20, 2286.
(18) Holliger, P.; Prospero, T.; Winter, G. Proc. Natl. Acad. Sci. U.S.A.
1993, 90, 6444.
(19) Rybak, J.-N.; Roesli, C.; Kaspar, M.; Villa, A.; Neri, D. Cancer
Res. 2007, 67, 10948.
(20) Villa, A.; Trachsel, E.; Kaspar, M.; Schliemann, C.; Sommavilla,
R.; Rybak, J.-N.; Rosli, C.; Borsi, L.; Neri, D. Int. J. Cancer 2008, 122,
̈
2405.
(21) Gentle, I. E.; De Souza, D. P.; Baca, M. Bioconjugate Chem.
2004, 15, 658.
(22) Niesner, U.; Halin, C.; Lozzi, L.; Gunthert, M.; Neri, P.;
̈
Wunderli-Allenspach, H.; Zardi, L.; Neri, D. Bioconjugate Chem. 2002,
13, 729.
(23) Liberatore, M.; Neri, D.; Neri, G.; Pini, A.; Iurilli, A. P.; Ponzo,
F.; Spampinato, G.; Padula, F.; Pala, A.; Colella, A. C. Eur. J. Nucl. Med.
1995, 22, 1326.
5892
dx.doi.org/10.1021/ja211589m | J. Am. Chem. Soc. 2012, 134, 5887−5892