Sulfatase-cleavable linkers for antibody-drug conjugates

Article abstract of DOI:10.1039/c9sc06410a

Antibody-drug conjugates (ADCs) are a class of targeted drug delivery agents combining the cell-selectivity of monoclonal antibodies (mAbs) and the cytotoxicity of small molecules. These two components are joined by a covalent linker, whose nature is critical to the efficacy and safety of the ADC. Enzyme-cleavable dipeptidic linkers have emerged as a particularly effective ADC linker type due to their ability to selectively release the payload in the lysosomes of target cells. However, these linkers have a number of drawbacks, including instability in rodent plasma and their inherently high hydrophobicity. Here we show that arylsulfate-containing ADC linkers are cleaved by lysosomal sulfatase enzymes to tracelessly release their payload, while circumventing the instability problems associated with dipeptide-linkers. When incorporated with trastuzumab and the highly potent monomethyl auristatin E (MMAE) payload, the arylsulfate-containing ADC 2 and ADC 3 were more cytotoxic than the non-cleavable ADC 4 against HER2-positive cells, while maintaining selectivity over HER2-negative cells. We propose that the stability, solubility and synthetic tractability of our arylsulfate linkers make them an attractive new motif for cleavable ADC linkers, with clear benefits over the widely used dipeptidic linkers.

Full text of DOI:10.1039/c9sc06410a

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Sulfatase-cleavable linkers for antibody-drug
conjugates†
Cite this: DOI: 10.1039/c9sc06410a
a
ab
c
All publication charges for this article
have been paid for by the Royal Society
of Chemistry
Jonathan D. Bargh,
Soleilmane Omarjee,
Stephen J. Walsh,
Jason S. Carroll
Albert Isidro-Llobet,
b
b
a
*
and David R. Spring
Antibody-drug conjugates (ADCs) are a class of targeted drug delivery agents combining the cell-selectivity
of monoclonal antibodies (mAbs) and the cytotoxicity of small molecules. These two components are
joined by a covalent linker, whose nature is critical to the efficacy and safety of the ADC. Enzyme-
cleavable dipeptidic linkers have emerged as a particularly effective ADC linker type due to their ability to
selectively release the payload in the lysosomes of target cells. However, these linkers have a number of
drawbacks, including instability in rodent plasma and their inherently high hydrophobicity. Here we show
that arylsulfate-containing ADC linkers are cleaved by lysosomal sulfatase enzymes to tracelessly release
their payload, while circumventing the instability problems associated with dipeptide-linkers. When
incorporated with trastuzumab and the highly potent monomethyl auristatin E (MMAE) payload, the
arylsulfate-containing ADC 2 and ADC 3 were more cytotoxic than the non-cleavable ADC 4 against
HER2-positive cells, while maintaining selectivity over HER2-negative cells. We propose that the stability,
solubility and synthetic tractability of our arylsulfate linkers make them an attractive new motif for
cleavable ADC linkers, with clear benefits over the widely used dipeptidic linkers.
Received 18th December 2019
Accepted 27th January 2020
DOI: 10.1039/c9sc06410a
rsc.li/chemical-science
constituent amino acids.3 In the case of a non-cleavable linker,
the active intracellular cytotoxin is released as the linker-payload
with the terminal amino acid from the mAb still attached.4
Introduction
Antibody-drug conjugates (ADCs) are now established as an
important class of therapeutics for the treatment of cancer. There
are currently seven FDA-approved ADCs, with at least 60 more in
clinical development.1 The success of ADCs arises from the
combination of the exquisite cell-selectivity of monoclonal anti-
bodies and the cytotoxicity of small molecule chemotherapies.
However, for the mAb and drug to exert their maximum thera-
peutic potential, the covalent linker between the two groups must
exhibit the following properties: (1) high plasma stability; the
long circulatory lifetimes (t1/2 > 1 week) of ADCs places stringent
stability requirements on the linker to avoid off-target payload
release. (2) High aqueous solubility to aid bioconjugation of
lipophilic payloads and avoid antibody aggregation. (3) Release of
a potent cytotoxin from the antibody in the target cell.2
Conversely, a cleavable linker is designed to release the unmod-
ied drug at this point, or upon subsequent entry to the cytosol.
Cleavable linkers are generally preferred to non-cleavable
linkers in ADC research for a number of reasons.5 First, trace-
less drug release allows the unmodied payload to perform its
intracellular function without an unwanted linker appendage,
thereby maximising cytotoxicity. Second, the unmodied
payload can be uncharged, allowing it to diffuse into ‘bystander’
tumour cells that may not be expressing the target antigen. This
‘bystander effect’ provides an important mechanism for eradi-
cating tumours with heterogeneous antigen expression.6
Enzyme-cleavable linkers are the most widely used group of
ADC linkers. Other cleavable linkers, such as reducible disul-
des or acid-sensitive motifs have been developed but their
stability in human plasma is generally inferior.7,8 The benets of
enzyme-cleavable linkers include their ability to selectively
induce drug release at target cells rather than in circulation.
Thus far, the only clinically explored enzyme-cleavable linkers
are peptides, sensitive to cleavage by lysosomal cathepsin
proteases.9–13 Linkers containing Val–Cit or Val–Ala sequences
are most widely employed, due to their high stability in human
plasma and efficient drug release within the lysosomes of target
cells (Fig. 1a). A self-immolative para-aminobenzoyl carbamate
(PABC) spacer is also required, to ensure the cathepsin-
mediated cleavage is unimpeded by the payload.
The majority of ADCs employ antibodies that target internal-
ising antigens, overexpressed on the surface of certain cancer
cells. Upon antibody-antigen binding at the target cell, the ADC is
endocytosed and trafficked to the highly hydrolytic lysosomal
compartments, where the antibody is broken down into its
aDepartment of Chemistry, University of Cambridge, Lenseld Road, Cambridge, CB2
1EW, UK. E-mail: spring@ch.cam.ac.uk
bCancer Research UK Cambridge Institute, University of Cambridge, Robinson Way,
Cambridge, CB2 0RE, UK
cGSK, Gunnels Wood Road, Stevenage, SG1 2NY, UK
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9sc06410a
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target cells, efficiently releasing their payloads. Sulfatases
therefore offer an opportunity for selective payload release due
to their high activity within the lysosomes and low activity in
human and rodent plasma.21 A number of different sulfatases
reside in the lysosome, catalysing the hydrolysis of alkylsulfate
esters to alcohols.22 Although selective towards their natural
substrates, they also each display arylsulfatase activity.
Furthermore, sulfatases are overexpressed in a number of
different cancer types, thereby offering the possibility of addi-
tional selectivity for arylsulfate-containing ADCs towards
tumours.23 It was anticipated that the charged sulfates would be
amenable to bioconjugation in aqueous media and their
synthesis simplied by previously reported protecting group
strategies.24,25 Accordingly, arylsulfate ADC linkers would
potentially display signicant advantages over dipeptide-based
linkers.
Fig. 1 (a) Previously developed dipeptide linkers, cleaved by lysosomal
cathepsins and mouse plasma enzyme Ces1C; (b) arylsulfate linkers for
use in ADCs are cleaved by lysosomal sulfatases.
Arylsulfate linker motifs were designed such that, upon
hydrolysis, a 4-alkoxybenzyl carbamate would be revealed,
primed for spontaneous 1,6-elimination of an amine-linked
payload (Fig. 1b). To link to the antibody, we were rst
inspired by b-glucuronidase-cleavable linkers, which are linked
to the mAb from an amide ortho- to the enzyme cleavable group
(Fig. 2, blue route).18 It was also postulated that arylsulfates
linked to the antibody by branching from the benzyl position
would be less hindered at the cleavable sulfate, potentially
improving sensitivity towards sulfatases (Fig. 2, red route). For
our preliminary stability and release studies, we employed the
uorescent 7-amino-4-methyl coumarin (AMC) group as
a model payload. AMC is widely used within enzymatic probes,
as uorescence is only observed upon release of the amino-
coumarin from an amide or carbamate precursor.26 Therefore,
through the use of uorimetry, payload release at physiologi-
cally relevant concentrations could be measured in enzyme
solutions and plasma. Model linkers 7 and 12 incorporate the
two antibody-attachment designs, with AMC as their payload
(Fig. 3).
Unfortunately, dipeptidic linkers feature a number of major
drawbacks. First, they are unstable in rodent blood, due to their
susceptibility to the Ces1C hydrolase enzyme in rodent
plasma.7,14,15 This hydrolysis causes premature payload release
in vivo and limits the accuracy of preclinical efficacy and safety
studies. Second, these linkers are hydrophobic, thereby
hindering bioconjugation of lipophilic payloads and causing
high levels of antibody aggregation upon conjugation.16,17
Despite these shortcomings, Val–Cit and Val–Ala dipeptides
continue to be widely used in ADC research, routinely alongside
monomethyl auristatin E (MMAE) or pyrrolobenzodiazepine-
(PBD) dimer payloads respectively.
Other non-peptidic linkers have also been developed for
ADCs. Linkers sensitive to the lysosomal b-glucuronidase and b-
galactosidase enzymes have been described, which appear to
address the problems associated with linker-payload hydro-
phobicity and rodent-plasma instability.11,18 However, their
stereochemical complexity and possible overreliance on one
specic lysosomal enzyme may explain their lack of develop-
ment in clinically tested ADCs. Additionally, pyrophosphatase-
cleavable motifs have also been described, for use with
alcohol-linked glucocorticoid payloads.19,20
Novel linkers employing different modes of action are
required to expand the ADC toolbox, given the therapeutic
importance of the linker and the shortcomings of current
cleavable linker technology.5 Herein, we describe the develop-
ment of novel sulfatase-cleavable linkers for ADCs (Fig. 1b).
Initial linker studies, facilitated by their simple synthesis, reveal
the highly soluble arylsulfate linkers are stable in both human
and mouse plasma. In addition, a series of arylsulfate-ADCs
employing a trastuzumab antibody and MMAE as a payload
were prepared and their cytotoxicity assessed against HER2-
positive and HER2-negative cells.
Synthesis of linker-coumarins
The synthesis of the arylsulfate linkers required a sulfate pro-
tecting group strategy to limit the number of steps requiring
purication of charged molecules. The neopentyl moiety was
chosen as an appropriate protecting group due to its stability,
facile installation and functional group compatibility.25
Although harsh deprotection conditions are typically required,
a procedure described by Simpson et al. employing aqueous
NH4OAc appeared to offer a mild alternative.27 Synthesis of
Results and discussion
Design of linkers
Fig. 2 Design of linker-AMC model compounds. It was envisaged that
the linker-payloads could be joined to the antibody through an ortho-
amide (blue route) or a benzyl-alkyl (red route).
Effective enzyme-cleavable ADC linkers must be highly stable in
circulatory conditions but labile upon entry to the lysosomes of
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Fig. 3 (a) Synthesis of linker-AMC 7 and (b) synthesis of linker-AMC 12. Np ¼ neopentyl.
linker-AMC 7 was achieved in six steps from nitroarene 1 were incubated with the sulfatase and the uorescence of the
(Fig. 3a). First, 1 was hydrogenated to yield aniline 2, which was released AMC payload was measured over 8 h (Fig. 4b). The
then modied via HATU-mediated amide coupling to yield resulting increase in uorescence conrmed that both linkers
alkyne 3. Next, installation of the neopentyl-protected sulfate are substrates for sulfatases and the subsequent 1,6-elimination
was achieved by reaction with neopentyl sulfurochloridate. The occurs as expected. Although both linkers were hydrolysed by
resulting methyl ester 4 was then selectively reduced with the sulfatase, the enzymolysis rate of benzyl-linked 12 (t1/2 ¼ 24
LiAlH4 before reaction with AMC-isocyanate in the presence of min) was dramatically superior to that of ortho-amide linked 7
a dibutyltin dilaurate catalyst afforded carbamate 6. Gratify- (t1/2 > 12 h) under the same conditions. These results suggest an
ingly, deprotection of the neopentyl sulfate with aqueous ADC linker based on 12 would release its payload much more
NH4OAc occurred smoothly to afford linker-AMC 7 in good efficiently in the lysosome than 7, increasing the cytotoxicity of
overall yield. Linker-AMC 12 was synthesised via an analogous the ADC.
synthetic route (Fig. 3b). First, the neopentyl sulfate was intro-
To investigate the enzymatic nature of the hydrolysis,
duced as before, with subsequent Barbier reaction with prop- sulfates 7 and 12 were incubated with the sulfatase in the
argyl bromide and zinc powder affording alcohol 10. Finally, presence of phenyl sulfamate, a known sulfatase inhibitor
reaction with AMC-isocyanate was followed by neopentyl (Fig. S3†).29 Pleasingly, no uorescence was observed under
deprotection to generate linker-AMC 12 with moderate to good these conditions, conrming that enzymolysis is required for
yields throughout.
release. Furthermore, incubation of 12 at pH 7.4 and pH 9
demonstrated the acidic optimum pH of the sulfatase hydro-
lysis, with the cleavage rate decreasing with increasing pH
(Fig. S4†). Finally, to affirm its susceptibility towards human
lysosomal sulfatases, 12 was incubated with recombinant
human lysosomal arylsulfatase A (ARSA) and arylsulfatase B
(ARSB), upon which, uorophore release was observed in both
cases (Fig. S5†).
Stability and release studies
With constructs 7 and 12 in hand, the lability of the linkers
under conditions representative of the lysosomal compart-
ments and blood plasma was evaluated (Fig. 4a). First, to
approximate the lysosomal environment, the susceptibility of
the arylsulfate linkers 7 and 12 to sulfatase cleavage was
determined using sulfatase from Helix pomatia (EC 3.1.6.1).
This enzyme has been used previously to approximate sulfate
hydrolysis, given its general sulfatase activity.28 The arylsulfates
The plasma stability of arylsulfate-AMC constructs 7 and 12
was then investigated, rst by incubation in mouse plasma
(Fig. 4c). Under these conditions, the sulfate linkers 7 and 12
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Fig. 4 (a) Structures of model linker-AMC constructs including arylsulfates 7 and 12 and Val–Ala 13 and Val–Cit 14. Upon hydrolysis and
subsequent 1,6-elimination, the AMC payload is released and fluoresces. (b) Comparison of enzymolysis rates of 7 versus 12 when incubated with
sulfatase from Helix pomatia. (c) Stability comparison of arylsulfates 7 and 12 versus dipeptides 13 and 14 in mouse plasma over 8 hours. (d)
Stability comparison in mouse plasma over 7 days. The t ¼ 0 reading was taken after adding plasma to all the samples, by which time 13 and 14
were already partially hydrolysed. (e) Stability comparison in human plasma over 7 days.
were highly stable, with no signicant increase in uorescence.
Contrastingly, dipeptides 13 and 14 were rapidly hydrolysed in
mouse plasma, with half-lives of less than one hour. The
exceptional stability of 7 and 12 was further conrmed by
incubation in both human and mouse plasma over seven days,
with minimal hydrolysis observed (Fig. 4d and e). Moreover,
sulfates 7 and 12 were shown to be stable in the presence of an
intracellular nucleophile (glutathione) (Fig. S6†).
Design of linker-MMAE constructs
Having conrmed the favourable properties of arylsulfates 7
and 12, the linkers were elaborated to include an antibody
attachment group and a cytotoxic payload, for biological eval-
uation of the proceeding ADCs. The divinylpyrimidine (DVP)
group has been reported by our group as an effective method for
creating stable, homogeneous ADCs with an average drug-
antibody ratio (DAR) of four.30 We therefore included this
functional handle, as well as the cytotoxic MMAE payload for
the full linker-payloads. Linker-payloads 15 and 16a are derived
from the model compounds 7 and 12 respectively, and were
expected to exhibit similar plasma stability and sensitivity
towards sulfatases. Linker-payload 16b is an analogue of 16a
with an added electron-withdrawing NO2 group on the arylsul-
fate, included because of the increased activity of sulfatases
towards electron-poor arylsulfates.31 Non-cleavable analogue 17
and Val–Ala-containing linker payload 18 were also synthesised
so that their biological properties could be compared to the
arylsulfates in vitro (structures in Fig. 5, full synthetic details
described in the ESI†).
Fig. 5 Bioconjugation of linker-payloads 15, 16a, 16b, 17 and 18 to
trastuzumab to afford ADCs 1–5.
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binding activity is expected to be lower. Gratifyingly, arylsulfate-
containing ADC 2 and ADC 3 compared similarly with the
dipeptidic ADC 5; both were also 5–10 times more cytotoxic than
non-cleavable ADC 4 (Fig. 6a). The similar performance of these
arylsulfate- and dipeptide-ADCs suggests that the arylsulfate
linkers are also being cleaved in the cells following ADC inter-
nalisation. Contrastingly, ADC 1 was non-toxic across both HER2-
positive cell lines, demonstrating that ortho-amide containing
arylsulfate linkers are unsuitable for ADCs. The complete lack of
cytotoxicity indicates that the in vitro sulfatase cleavage of ADC 1
is occurring at an insufficient rate and the maintained presence
of the anionic sulfate group adjacent to the auristatin payload
inhibits its tubulin-binding ability.
All ve ADCs were non-toxic towards the HER2-negative
MCF7 cells up to 3 nM, validating the stability of the linkers
in media and the retained functionality of the mAb to bind and
internalise with the HER2 antigen (Fig. 6b). Furthermore, tras-
tuzumab's lack of cytotoxicity against HER2-positive BT474 cells
conrms that the MMAE payload is critical to the cell-killing
ability of the ADCs. The results of our in vitro evaluations
were conrmed by testing the ADCs in additional HER2-positive
(SKBR3) and HER2-negative (T47D) cell lines, where similar
trends were observed (Fig. S7 and Table S1†).
Bioconjugation
The linker-payloads were conjugated to trastuzumab, an FDA-
approved IgG mAb which targets the HER2 receptor, an inter-
nalising antigen that is overexpressed in around 20% of breast
cancers.32 Our earlier studies conrmed the excellent retention
of antibody binding affinity and internalisation of DVP-derived
trastuzumab ADCs, as well as the high plasma stability of the
cysteine-DVP linkage.30 First, the four interchain disuldes were
reduced by TCEP for 1 h to reveal eight reactive cysteine resi-
dues (Fig. 5). Then linker-payloads 15, 16a, 16b, 17 and 18 were
incubated for 3 h to react with the cysteine residues, rebridging
them to form ve ADCs (ADCs 1–5). Efficient conversion (>95%)
to the desired conjugates was conrmed by LC-MS and SDS-
PAGE analysis (Fig. S1 and S2†).
In vitro cytotoxicity
Having successfully synthesised the cleavable and non-cleavable
ADCs (ADCs 1–5), their cytotoxicity against HER2-positive
(BT474) and HER2-negative (MCF7) cells was evaluated (Fig. 6).
As expected, the cathepsin-cleavable ADC 5 demonstrated
increased cytotoxicity (IC50 ¼ 92 pM) towards the HER2-positive
BT474 cells compared with the non-cleavable ADC 4 (IC50
¼
609 pM). This six-fold increase in potency can be explained by the
traceless release of MMAE, compared with the modied MMAE-
metabolite released from non-cleavable ADC 4, whose tubulin-
Conclusions
In conclusion, we have demonstrated the suitability of arylsul-
fates as cleavable linkers for ADCs. Model studies of the linkers
with a uorometric AMC payload validate their susceptibility
towards sulfatase enzymes to tracelessly release amine-linked
payloads, as well as their vastly superior mouse plasma
stability compared to dipeptidic linkers. Upon conjugation to
trastuzumab with an MMAE payload, all ve ADCs retained
selectivity towards HER2-positive cells. The arylsulfate-
containing ADC 2 and ADC 3 were signicantly more potent
against HER2-positive cells than the non-cleavable ADC 4,
suggesting the highly potent MMAE payload is being released
inside the cells. The dramatic difference in cytotoxicity between
ADC 1 and ADCs 2 and 3 reects the difference in enzymolysis
rates of 7 and 12, suggesting that rapid payload release rates are
crucial to the cell-killing capacity of the arylsulfate-ADCs. An
appropriate choice of aryl-substitution is therefore vital to the
cytotoxicity of sulfatase-cleavable linkers.
We envision that the arylsulfate linkers should be applicable
to other lipophilic payloads, given their solubility and synthetic
viability. Furthermore, the ubiquity of lysosomal sulfatases
should enable pairing of the arylsulfate linkers with alternative
antibodies for the treatment of a range of cancer types.
Conflicts of interest
There are no conicts to declare.
Fig. 6 In vitro biological evaluation of ADCs 1–5 in (a) HER2-positive
BT474 cells and (b) HER2-negative MCF7 cells. Viability data shows the
mean of three independent experiments and error bars represent
standard error of the mean. For BT474 cells, IC50 values are as follows:
ADC 1 ¼ N/A; ADC 2 ¼ 111 pM; ADC 3 ¼ 61 pM; ADC 4 ¼ 609 pM; ADC
5 ¼ 92 pM.
Acknowledgements
¨
We are grateful to Dr Marko Hyvonen for allowing us access to
the Pherastar FS plate reader and Teodors Pantelejevs for his
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guidance with its operation. J. D. B. acknowledges an iCASE 17 P. J. Burke, P. D. Senter, D. W. Meyer, J. B. Miyamoto,
studentship from GlaxoSmithKline/EPSRC. D. R. S. acknowl-
edges support from the Engineering and Physical Sciences
Research Council (EP/P020291/1) and Royal Society (Wolfson
M. Anderson, B. E. Toki, G. Manikumar, M. C. Wani,
D. J. Kroll and S. C. Jeffrey, Bioconjugate Chem., 2009, 20,
1242–1250.
Research Merit Award). The Spring lab acknowledges general 18 S. C. Jeffrey, J. B. Andreyka, S. X. Bernhardt, K. M. Kissler,
lab support from the EPSRC, BBSRC, MRC and Royal Society.
T. Kline, J. S. Lenox, R. F. Moser, M. T. Nguyen,
N. M. Okeley, I. J. Stone, X. Zhang and P. D. Senter,
Bioconjugate Chem., 2006, 17, 831–840.
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