A dual-enzyme cleavable linker for antibody-drug conjugates

Article abstract of DOI:10.1039/d1cc00957e

A novel enzyme cleavable linker for antibody-drug conjugates is reported. The 3-O-sulfo-β-galactose linker is cleaved sequentially by two lysosomal enzymes - arylsulfatase A and β-galactosidase - to release the payload in targeted cells. An α-HER2 antibod

Full text of DOI:10.1039/d1cc00957e

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A dual-enzyme cleavable linker for antibody–drug
conjugates†
Cite this: Chem. Commun., 2021,
7, 3457
5
a
ab
a
Jonathan D. Bargh,
Albert Isidro-Llobet,
Stephen J. Walsh,
Jason S. Carroll
Nicola Ashman,
and David R. Spring
c
b
a
Received 19th February 2021,
Accepted 2nd March 2021
*
DOI: 10.1039/d1cc00957e
rsc.li/chemcomm
15
A novel enzyme cleavable linker for antibody–drug conjugates is cytotoxicity and cell-selectivity. The arylsulfate construct offered
reported. The 3-O-sulfo-b-galactose linker is cleaved sequentially improved mouse plasma stability and hydrophilicity versus dipep-
by two lysosomal enzymes – arylsulfatase A and b-galactosidase – tide linkers. However, the arylsulfate motif only represents a
to release the payload in targeted cells. An a-HER2 antibody–drug pseudo-substrate for lysosomal sulfatases, whose natural sub-
20
conjugate synthesised using this highly hydrophilic dual-cleavable strates are alkylsulfates.
linker exhibited excellent cytotoxicity and selectivity.
We looked to expand the toolbox of cleavable ADC linkers
by mimicking sulfatide, the natural substrate of arylsulfatase
2
1
Antibody–drug conjugates (ADCs) now represent a major class A (ARSA). In its metabolic pathway, sulfatide is first hydro-
of biotherapeutics, with nine approved by the Food and Drug lysed by ARSA to reveal a b-galactosyl ceramide, which is then
1
–3
Administration (FDA) and 460 more in clinical trials.
By susceptible to b-galactosidase (b-gal)-mediated cleavage, producing
22
combining the tumour selectivity of monoclonal antibodies the ceramide metabolite (Fig. 1A). We therefore designed a
and the antitumour activity of small molecule payloads, a wide 3-O-sulfo-b-galactose linker, envisaged to hijack the natural dual
4
,5
therapeutic window can be achieved. Key to the efficacy and enzymatic ARSA/b-gal metabolic cascade, thus releasing a payload
safety of an ADC is a covalent linker between the two therapeutic in the target cell (Fig. 1B). This cleavable group was anticipated to
components, which should help solubilise the typically hydro- exhibit particularly high hydrophilicity, due to the presence of both
6
phobic payload and provide a stable linkage in circulation.
an anionic sulfate and a pyranose functionality. Furthermore, the
Importantly, the linker usually also contains a cleavable trigger, dual-enzymatic process would afford excellent lysosome-selectivity
7
which selectively releases the payload at target tumour cells.
to the conjugate. Given that efficient lysosomal cleavage of
Enzyme-cleavable linkers represent a particularly attractive cleavable b-galactosyl ADC linkers has been previously reported, drug release
option, given their potential for high plasma stability and selective from the linker was expected to be fast, provided that the first,
1
1
payload release in the lysosomes of target cells.
ARSA-mediated cleavage was also efficient.
We first investigated linker-payload 1, bearing 7-amino-4-methyl
Thus far, the toolbox of enzyme-cleavable ADC linkers has
8
–10
11,12
13
included protease-,
glycosidase-,
pyrophosphatase-, acid coumarin (AMC) as a model payload, to measure real-time
14
15,16
phosphatase- and sulfatase-cleavable
motifs. Of particular
prevalence are cathepsin-cleavable dipeptides, now present in the
majority of ADCs in clinical development. However, the mouse
plasma instability and hydrophobicity of the dipeptide linkers can
impede accurate preclinical evaluations and cause ADC
17–19
aggregation.
To address these shortcomings, we recently
reported the arylsulfate motif as an effective sulfatase-cleavable
group, capable of releasing drugs from ADCs with excellent
a
Department of Chemistry, University of Cambridge, Lensfield Road,
Cambridge, CB2 1EW, UK. E-mail: spring@ch.cam.ac.uk
b
Cancer Research UK Cambridge Institute, University of Cambridge,
Robinson Way, Cambridge, CB2 0RE, UK
c
GSK, Gunnels Wood Road, Stevenage, SG1 2NY, UK
†
Electronic supplementary information (ESI) available. See DOI: 10.1039/
Fig. 1 (A) The ARSA/b-gal-mediated metabolism of sulfatide. (B) An
a-HER2 ADC employing the 3-O-sulfo-b-galactose linker motif.
d1cc00957e
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Scheme 1 Synthesis of 3-O-sulfo-galactose-AMC 1 and linker-payloads 2 and 8.
enzymolysis by fluorimetry. An analogous linker-payload 2 featuring
the potent tubulin binder MMAE was also envisioned for in vitro
ADC biological evaluation. This linker-MMAE construct therefore
required antibody attachment, so a divinylpyrimidine (DVP)
23
conjugation motif was also incorporated.
Synthesis of both linker-payloads proceeded from acetyl-
1
1
galactose 3 (Scheme 1). Carbamate 4a was synthesised by
reaction with AMC-isocyanate 5 in the presence of a dibutyltin
dilaurate catalyst. For the MMAE carbamate 4b, 3 was instead
reacted with 4-chlorophenyl chloroformate to afford the
4-nitrophenyl carbonate 6, before displacement with MMAE.
The acetyl-galactose-carbamates 4a and 4b were then hydro-
1
1
lysed to reveal tetrahydroxypyranoses 6a and 6b. In the key
step of the synthesis, 6a and 6b were first reacted with dibu-
tyltin oxide to form dibutylstannylene acetals, before reaction
2
4,25
with SO
equatorial 3-O-position, affording sulfates 1 and 7 in 67% and
5% yields, respectively. The sulfogalactose-MMAE 7 was
3
ÁNMe
3
.
This procedure selectively sulfated at the
5
further functionalised with a DVP conjugation motif, using
copper-catalyzed azide–alkyne cycloaddition (CuAAC) chemistry,
affording linker-payload 2. This route also facilitated the func-
tionalisation of intermediate galactosyl 6b with DVP, affording
galactosyl linker-payload 8, thus allowing a direct comparison
between 3-O-sulfo-b-galactose and b-galactose ADC linkers.
Fig. 2 Fluorometric measurement of AMC release from probe 1 by
incubation with ARSA and b-gal enzymes. lex = 350 nm, lem = 460 nm.
To validate its enzyme sensitivity, sulfogalactose-AMC 1 was release observed over 20 hours in human or mouse plasma
incubated with isolated ARSA and b-gal enzymes and the (Fig. S3, ESI†). Together, these results suggest the 3-O-sulfo-b-
fluorescence monitored over time (Fig. 2). As expected, no galactose motif will facilitate tumour cell targeting by discrimi-
significant increase in fluorescence was observed upon incuba- nating between lysosomal and circulatory conditions.
tion with either ARSA or b-gal alone. In contrast, incubation
To evaluate the biological properties of the 3-O-sulfo-b-
with both enzymes resulted in a dramatic increase in fluores- galactose linker, linker-payloads 2 and 8 were conjugated to
cence intensity, suggesting that dual-enzyme catalysis is a-HER2 antibody trastuzumab (Fig. 3). As with previous IgG
1
5,23,26,27
required for the hydrolysis of 1. Moreover, the extracellular conjugations employing the DVP motif,
the four inter-
stability of 1 was demonstrated, with no observable AMC chain disulfides of trastuzumab were first reduced by treatment
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Fig. 3 Bioconjugation of linker-payloads 2 and 8 to trastuzumab to afford
ADC 1 and ADC 2 respectively.
with tris(2-carboxyethyl)phosphine hydrochloride (TCEP).
Upon subsequent addition of 2 or 8, the reduced disulfides
were rebridged via the bis-reactive DVP, affording conjugates
ADC 1 and ADC 2 with four drugs/antibody. The excellent Fig. 4 In vitro biological evaluation of ADC
1 and ADC 2 in (A)
aqueous solubility of sulfo-galactose 2 was exemplified by its HER2-positive SKBR3 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.
complete solubility in water at 20 mM, thereby facilitating
protein bioconjugation at 0% organic co-solvent. Conversely,
preparation of a 20 mM stock of galactose 8 required DMSO for
dissolution, thus the ensuing bioconjugation was not 100%
aqueous. Although the small amount of co-solvent (3% DMSO) was
linkers’ selectivity for intracellular cleavage.
not deleterious to protein stability in this case, such differences in
both exhibiting relatively low potency and thus indicating their
In conclusion, the 3-O-sulfo-b-galactose motif is a highly
hydrophilic and cleavable ADC linker motif. The ARSA/b-gal
linker-payload solubilities suggest that a 3-O-sulfo-b-galactose lin-
ker may facilitate bioconjugation with a more lipophilic payload.
dual-enzymatic cleavage was confirmed by virtue of fluores-
Next, the two ADCs were tested against HER2-positive SKBR3
cence probe 1, signifying the linker’s strict requirement for
cleavage by both lysosomal enzymes for drug release. The motif
cells to determine their dose-dependent cytotoxicity (Fig. 4A).
We have previously demonstrated that under the same assay
was successfully attached to trastuzumab in a completely aqu-
conditions and with analogous non-cleavable or poorly-
eous bioconjugation reaction and the resulting ADC 1 was
highly potent and selective. It is anticipated that the motif will
be widely applicable to a range of hydrophobic payloads and
antibodies, given its favourable properties.
cleavable ADCs, ADC potency is dramatically reduced versus
rapidly cleavable ADCs, thus confirming the relation-
1
5
ship between linker cleavage efficiency and cytotoxicity.
Gratifyingly, sulfogalactose ADC 1 was extremely potent, with
IC50 = 49 pM. This cytotoxicity is comparable to our most potent
analogous arylsulfate ADC, (IC50 [SKBR3] = 40 pM), as well as
the cathepsin-cleavable Val-Ala-PABC-MMAE ADC (IC50
J. D. B. acknowledges an iCASE studentship from GlaxoSmithK-
line/EPSRC. N. A. acknowledges an iCASE studentship from
AstraZeneca. D. R. S. acknowledges support from the Engineering
and Physical Sciences Research Council (EP/P020291/1) and
Royal Society (Wolfson Research Merit Award). The Spring lab
acknowledges general lab support from the EPSRC, BBSRC, MRC
and Royal Society.
1
5
[SKBR3] = 41 pM). It is therefore probable that the ARSA/
b-gal dual-enzymatic cascade occurs in lysosomes at a compar-
able rate to the enzymolysis of arylsulfate linkers and the
Val-Ala dipeptide linkers. Although the non-sulfo ADC 2 was
slightly more cytotoxic (IC50 = 23 pM) (Fig. 4A), we postulate
that the hydrophilicity gained upon sulfation of the galactosyl
motif may deem the 3-O-sulfo-b-galactose motif more appro-
priate for certain hydrophobic linker-payloads. The ADCs were
Conflicts of interest
also tested against HER2-negative MCF7 cells (Fig. 4B), with There are no conflicts to declare.
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