Antibody drug conjugates (ADCs) charged with HDAC inhibitor for targeted epigenetic modulation

Article abstract of DOI:10.1039/c7sc05266a

We describe here two novel antibody-drug conjugates loaded with the HDAC inhibitor ST7612AA1 (IC₅₀ equal to 0.07 μM on NCI-H460 cells), a thiol-based molecule with a moderate toxicity in vivo. Two payloads were prepared using cleavable and non-cleavable linkers. After anchoring to cetuximab through amide bond with lysines, the resulting HDAC inhibitor-antibody conjugates showed ability to recognize EGFR and efficient internalization in tumor cells. Both ADCs induced sensible increment of histones 3 and 4 and alpha-tubulin acetylation. Animal models of human solid tumors showed high anti-tumor efficacy of the conjugates without the toxicity generally observed with traditional ADCs delivering highly potent cytotoxic drugs. These compounds, the first ADCs charged with not highly cytotoxic warheads, are potentially suitable for epigenetic modulation, extending the ADC strategy to the targeted delivery of HDAC inhibitors with many possible therapeutic applications beyond cancer.

Full text of DOI:10.1039/c7sc05266a

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Antibody Drug Conjugates (ADCs) Charged with HDAC Inhibitor for
Targeted Epigenetic Modulation
Elena Cini,b Valentina Faltoni,a Elena Petricci,a Maurizio Taddei,*a Laura Salvini,c Giuseppe
Giannini,*d Loredana Vesci,d Ferdinando Maria Milazzo,d Anna Maria Anastasi,d Gianfranco
Battistuzzi,d and Rita De Santisd
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
We describe here two novel antibody-drug conjugates loaded with the HDAC inhibitor ST7612AA1 (IC50 equal to 0.07 ꢀM
on NCI-H460 cells), a thiol-based molecule with a moderate toxicity in vivo. Two payloads were prepared using cleavable
and non-cleavable linkers. After anchoring to cetuximab through amide bond with lysines, the resulting HDAC inhibitor-
antibody conjugates showed ability to recognize EGFR and efficient internalization in tumor cells. Both ADCs induced
sensible increment of Histones 3 and 4 and alpha-tubulin acetylation. Animal models of human solid tumors showed high
anti-tumor efficacy of the conjugates without the toxicity generally observed with traditional ADCs delivering highly potent
cytotoxic drugs. These compounds, the first ADCs charged with not highly cytotoxic warheads, are potentially suitable for
epigenetic modulation, extending the ADC strategy to the targeted delivery of HDAC inhibitors with many possible
therapeutic applications beyond cancer.
deriving from off-target effects due to linker instability. ADCs
containing microtubule inhibitors induce peripheral
neuropathy, neutropenia, gastrointestinal thrombocytopenia,7
Introduction
With more than 50 antibody−drug conjugates (ADC) in clinical
trials for treatment of oncological diseases, the ADC approach
to chemotherapy is finding renewed interest especially after
Adcetris (FDA, 2011), Kadcyla (FDA, 2013) and more recently
Besponsa (FDA, 2017) approval for treatment of CD30+
Hodgkin lymphoma, Her-2-positive metastatic breast cancer
and acute lymphoblastic leukemia (ALL), respectively.1 While
the use of monoclonal antibodies (mAbs) and molecularly
diverse likers has been widely represented, only highly potent
cytotoxic drugs like microtubule inhibitors maytansines
(DM1/DM4) or auristatins (MMAE/MMAF) dominate the
current ADC landscape. Despite a relatively poor clinical
hepatic and ocular toxicities8 while calicheamicin based ADCs
cause thrombocytopenia and hepatic dysfunction.9 With very
few exceptions, until now the paradigm that an efficient ADC
must be charged with a highly potent payload has guided ADC
research and development. However, the side effects
associated with intrinsic cytotoxicity of the payloads are a
serious limit to the ADC applicability in therapies beyond
cancer.10 In addition, the potency of the cytotoxic payload
implies manufacturing problems in ADC production, because
of containment restrictions required to guarantee protection
of operators and environment.11 Compared to the progress in
development of ADCs in oncology, few ADC candidates have
been investigated for the treatment of other diseases.
Dexamethasone and budesonide have been linked to anti-
success rate,
∼70% of the ADCs currently in clinical trials
contains payloads belonging to these classes of molecules.2
Other cytotoxic drugs include molecules targeting the DNA
minor groove,3,4 and topoisomerase I inhibitors.5 Since only
<1% of the injected dose is expected to target the tumor, the
presence of payloads active in nano- or picomolar ranges is
thought to be a strict requirement for ADC-based therapies.6
Consequently, most of the ADC toxicity observed in patients is
CD70
antibodies
for
anti-inflammatory
and
immunosuppressive therapies.12 The highly cytotoxic Src
kinase inhibitor dasatinib (IC50 < 1nM) has been also linked to
an antibody to produce an immunosuppressive ADC.13 Finally,
the recent discovery that a modified rifampicin bonded to an
anti-Streptococcus Aureus antibody eradicates intracellular
infection has raised great expectations for fighting bacterial
antibiotic resistance with ADCs.14
Epigenetic aberrations contribute to the onset and progression
of several diseases via the gain or loss of function in epigenetic
regulatory proteins.15 Deacetylating enzymes are valuable
targets to treat aberrant deacetylations occurring in
neurological disorders, inflammation, viral and protozoal
infections, cardiovascular disorders and cancer.16 We recently
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discovered ST7612AA117
(1, Scheme 1), a new thiol-based almost complete transformation into the monoamide of
histone deacetylase (HDAC) inhibitor that slows down in vivo succinic acid in 12 h, as revealed by NMR and MS/ESI (see SI).
O
O
and in vitro growth of several tumors such as Ras-mutant
colon carcinoma, non-small cell lung tumors, ovarian cancer,
triple-negative breast cancer (TNBC), acute myeloid leukaemia,
NH
NH
CH3SNa, MeOH,
90%
HN
O
HN
O
H
H
N
S
N
SH
and diffuse large B cell lymphoma. Moreover,
1 showed to
O
O
O
1: ST7612AA1
2
modulate the NF-κB pathway and epithelial-mesenchymal
transition (EMT), as well as transcripts involved in immune
response and in key pathogenic pathways, suggesting the
potential use in management of inflammatory diseases.18
O
O
O
N
O
HN
N
H
HN
NH
HO
3
O
O
O
MeOH rt
69%
Compound
potential applications in therapies aiming at the eradication of
the viral reservoirs.19 Compound
is the pro-drug of the
corresponding thiol (Scheme 1) that is rapidly formed in
plasma after the injection. A high affinity of with some
1 also proved to be active in HIV reactivation, with
S
O
O
N
N
H
1
O
4
O
2
HO
2
histones isoforms (IC50 13, 5, 3 and 11 nM on HDAC-1, -3, -6
and -10 respectively) was observed together with a promising
activity of 1 on tumour cell lines (IC50= 0.07 ꢀM on NCI-H460
cells) and a moderate toxicity in vivo.17
Results and discussion
Starting from this standpoint, we thought to exploit the
features of compound into a new concept of ADC directed
towards histone targets. The release of thiol upon
1
2
internalization of a HDAC charged ADC, may open the way to
selectively target different HDAC isoforms with many potential
applications in cancer therapy and beyond, and other
indications that might possibly have benefit from epigenetic
modulations.
The clue of an effective ADC lies in its linker. Generally, linkers
are designed in such a way to discharge the drug intracellularly
through a controlled process. More stable linkers can release
the free toxin by unspecific endosomal degradation,2 while
Cathepsin B cleavable linkers, discharge the payloads upon
Scheme 1 Preparation of payloads/linkers for conjugation.
This opened succinimide is stable in solution as no trace of
compound was observed in the further 72 h.
2
This increased stability of ring opened succinimides have been
already documented in ADCs where maleimide was employed
for anchoring payloads to cysteines residues in the mAb.25
lysosomal processing.20 As the active form of ST7612AA1 (
thiol ), two alternative conjugation strategies were
exploited: i) a linker based on the Michael addition of the thiol
1) is
a
(2
Lysine anchoring was carried out with compounds
4 and 7
to maleimide that might be cleaved by catabolism (sharing after activation with NHS and incubation in DMSO/H2O/PBS
features with the linker present in Kadcyla);21 ii) a cleavable
buffer (pH 7.4) with Ctx at room temperature using a 20-molar
fold excess of the linker respect to the mAb.
linker based on the Val-Cit dipeptide22 bonded to a p-amino-
benzyl (PAB) self-immolative group.23,24 Both linkers were
attached through
a stable amide bond with lysines to
cetuximab (Ctx), a monoclonal antibody (mAb) specific for the
Epidermal Growth Factor Receptor (EGFR). The linker-payloads
4
and
alkaline deacetylation of
gave product ready for lysine coupling after N-
hydroxysuccinimide (NHS) in situ activation. Compound was
prepared starting from dipeptide after deprotection and
coupling with monoethyl pimelate in the presence of EEDQ to
give . The hydroxy group was then transformed into the
corresponding bromide not stable enough for isolation.
However, direct nucleophilic substitution with thiol , followed
by ester hydrolysis, gave acid (Scheme 1). Compounds and
were stable in PBS or in mouse plasma. However, after 5 h,
7
were thus prepared as described in Scheme 1. After
1
, Michael addition of thiol to
2
3
4
7
5
6
Scheme 2: Preparation of ADCs: 8: DAR= 8 (±1), average on 6 batches; and ADC 9:
2
DAR=6 (±0.5), average on 3 batches].
7
4
7
hydrolysis of the five-membered ring of
4 started giving an
2 | J. Name., 2012, 00, 1-3
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a
b
Figure 1: a) Internalization of Ctx and ADCs 8 and 9 in Capan-1 (pancreas carcinoma), A549 and NCI-H1975 (lung carcinoma) human cells, and b) their effect on acetylation of
HDAC-target proteins in NCI-H1975 cells. Insets show specific fluorescence signals within the cells. Each image is representative of at least 5 fields of duplicate wells. Magnification
60x. Data are from one representative experiment out of two.
Purification of
conjugation and DAR were determined by MALDI analysis that
showed DAR= 8(±1) for compound and DAR= 6(±0.5) for
(Fig. S1 and S2). As expected, by HIC analysis the
and showed a large distribution of molecular
weights with less than 10 % of unreacted antibody in any
sample. Size exclusion chromatography showed a major peak
of the size expected for conjugates 8 and 9 with minor peaks
8 and 9 was carried out by dialysis while
alpha-tubulin and histones, a clear mark of specific inhibition
of HDAC6 and nuclear HDAC isoforms,26 respectively (Fig. 1b).
Fluorescence data showed that all tested ADCs induced a
relevant increase in the acetylation level of both alpha-tubulin
and histones H3 and H4, as result of direct enzymatic inhibition
of HDAC6 and class I HDACs, respectively while no effect was
observed in cells treated with Ctx alone (Fig. 1b), in EGFR-
negative cells and in other cell lines assessed (Fig. S7 and S8).
Increased acetylation of tubulin and histone H4 was also
confirmed in A549 cells by Western blot analysis of total
protein lysate (Fig.2). Comparison of band intensity clearly
shows an increment of acetylation when cells are treated with
8
compound
conjugates
9
8
9
referable to aggregated and degraded forms, respectively (Fig.
S3).
The binding specificity of conjugates
8 and 9 was than
confirmed by flow-cytometry (FACS analysis) on Capan-1
(human pancreas carcinoma), NCI-H1975, A549 (human lung
carcinoma), and SK-MEL-28 (human melanoma) cell lines, a
panel of EGFR positive and negative tumour cells (Fig. S4).
Immunoreactivity was tested by antigen-specific ELISA on a 96-
well plate coated with 50 ng/well of recombinant human EGF-
R/ErbB1 Fc chimera. Both ADCs showed reactivity with their
ADCs
8 and 9 if with the vehicle-treated cells and with cells
treated with Ctx.
specific target with potency comparable (ADC
8) or slightly but
significantly lower (ADC to that of Ctx (Fig. S5a).
9)
Consistently, affinity measurements by surface plasmon
resonance (Biacore system) showed an apparent higher
Figure 2: Effect of native Ctx (lane 2), ADCs 8 or 9 (lanes 3 and 4, respectively) on
acetylation of alpha-tubulin and histone H4 in A549 (human lung carcinoma) cells. Cells
were cultivated 3 hours at 37°C with medium (lane 1) or antibodies (20 µg/mL) and
then Western Blot analysis was carried out on total protein lysates. Beta-actin was used
for normalization. One representative blot is shown.
affinity of
8 compared to 9 although both derivatives exhibit
antigen interaction kinetics in the same sub-nanomolar range
of Ctx (Fig. S5b).
After securing that conjugation did not modify the Ctx
properties, ADC internalization, an essential step for biological
function, was investigated on the EGFR-expressing tumour cell
lines Capan-1, NCI-H1975, A549 (Fig. 1a), and on EGFR-
negative cells (Fig. S6b). After cell treatment with Alexa
Fluor488-labeled ADCs, fluorescence was observed by High
Content Screening (HCS) imaging analysis. Results show that,
This result demonstrates the ability of ADCs to be internalized
and to acts as a HDAC inhibitor, due to the presence of
compound
Release of
2
2
indeed.27
from ADC
8
occurred by treatment with human
hepatic microsomes.28 After 72 h of incubation at 37 °C,
quantitative HRMS analysis showed the presence of the peaks
at M/z =362.1548 and 328.1671 referable exclusively to
upon binding to EGFR, ADCs
8 and 9 are internalized in a
comparable manner respect to Ctx, resulting in accumulation
within the internal vesicles of the multivescicular bodies and
subsequent translocation to the lysosomal compartment (Fig.
1a).
compound
to the amount of payload present in the starting ADC.) When
ADC was submitted to the same procedure, no evidence of
2 in concentration 1.9 ꢀM (29% of release referred
9
2
was found. The linker Val-Cit-PAB conjugated wit
be cleaved by lysosomal Cathepsin B.
2 is known to
To demonstrate that the HDAC inhibitor charged ADCs
8
and
9
are suitable for epigenetic modulation, the release of
2
was
then proven in NCI-H1975 cells by detecting acetylation of
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a
b
c
d
Figure 3: a) Effects of ADC 8 and 9 in tumours developed in Nu/Nu mice after s.c. injection of 5x106 NCI-H1975 cells. Lesion development and response to antibody treatment was
monitored using a Vernier calliper. Mice injected i.p. with either 8, 9, Ctx (4 doses of 50 mg/kg once every 4 days) or 1 (4 doses of 120 mg/kg once every 4 days) and PBS (n=8
mice/group; mean and SEM, °°°p < 0.001 and °°p<0.01 vs Ctx alone, Mann-Whitney’s test). b) Effects of ADC 8 in tumours developed in Nu/Nu mice for 13 days after s.c. injection
of 5x106 A549 cells. Lesion development and response to antibody treatment was monitored using a Vernier calliper. Mice injected i.p. with either 8 and Ctx (4 doses of 50 mg/kg
once every 4 days) or PBS (n=10 mice/group; mean and SEM, °p < 0.05 vs Ctx; **p<0.01 and *p<0.05 vs vehicle, Mann-Whitney’s test. c) Artificial metastatic lung cancer
experiment carried out with 5x106 A549-luc-C8 (A549luc) cells into the tail vein of immunodeficient SCID/beige mice. Tumour bioluminescence imaging (BLI) was recorded by
Xenogen IVIS Imaging System 200, at different time points (+35, +49 and +56 days from cell injection), after i.p. injection of luciferin (150 µg/mouse). Mice were treated by aerosol
with PBS or ADC 8 or Ctx (3.5 mL of 100 µg/mL solution) q7dx4 (n=12 mice/group; mean and SEM, °°p < 0.01 vs cetuximab; *p < 0.05 and **p<0.01 vs vehicle). d) Orthotopic
tumour pancreas experiment performed with 1x106 tumor Capan-1 cells injected into pancreas. Tumor weight was evaluated 90 days after tumor injection. Mice treated
intraperitoneally with 8 or Ctx (4 doses of 40 mg/kg once every 4 days), PBS and 1 (200 mg/kg, q4dx4) (n=10 mice/group); mean and SEM, °p<0.05 vs Ctx,*p<0.05 and **p<0.01 vs
vehicle)
However, in our hands, different incubation experiments a zinc binding group (ZBG) that coordinates to the catalytic
carried out on compound
or hepatic microsomes, never gave convincing proofs of the the hydrophobic channel and helps Cap and ZBG to find the
presence of or, at least, of the PAB-thioether derived from
correct position.33 The accommodation of the ZBG into the
7 or ADC 9 with the isolate enzyme metal atom within the active site, and (c) a linker that binds to
2
2
by amide cleavage. Cellular metabolism might generate the HDAC catalytic site is a crucial step of the inhibition process
drug attached to amino acid fragments derived from the and is finely controlled by its zinc coordination ability and by
antibody.29–31 and, successively,
to MetaSite,
transformations related to cytochrome and monooxygenase between 6.6 and 7.2 A in order to have molecules with activity
mediated reactions,32 is revealed as a possible metabolite as HDAC inhibitors.34,35 These molecular features do not fit
(SI). This putative metabolic mediated release of from ADC with amino acid conjugate coming from a partial metabolism
may explain why this ADC is only slightly more therapeutically of ADC
potent than Ctx alone suggesting also that the linkage through The putative anti-proliferative activity of conjugates was
Val-Cit PAB was not a good choice for a correct release. evaluated on two lung adenocarcinoma cell lines (NCI-H1975
However, the activity of ADC in epigenetic modulation can be and Calu-3), treated up to 6 days with ADC or ADC , as well
explained exclusively assuming that is released. The largely as with equivalent doses of the parental antibody (free Ctx). As
accepted pharmacophore model for most of the known determined by cell proliferation curves, although at different
hydroxamic acid inhibitors consists of (a) a capping group that extent ADC inhibited tumour cell proliferation of both cell
interacts with the residues at the active site entrance (cap), (b) lines, showing IC50 value of 250 nM on NCI-H1975 cells and
2. Submitting the structure
7
key interactions with the surrounding protein residues.
a
software that predicts metabolic Moreover, the distance between the ZBG and the cap must be
2
2
9
9.
8
9
2
8
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40% of inhibition at the highest measured dose (500 nM) on was not affected throughout all the study duration, indicating
Calu-3 cells. Although not extremely potent, conjugates and that the treatment is well tolerated (Fig. 4).
resulted much more active than Ctx alone that, at the same In vivo combination study with Ctx and ST7612AA1 alone,
8
9
concentration, was not effective (Fig S9).
demonstrated also that an equimolar mixture of the
However, ADC
animal models (Fig. 3). Their efficacy in comparison with Ctx effective in the animal model. Overall, in vitro and in vivo
and was evaluated in a mouse tumour xenograft model (non- results show that ADC is a promising lead for further
8 and 9 showed a high antitumor activity in uncoupled drug and the antibody was well tolerated but not
1
8
small cell lung). The NCI-H1975 tumour cells were injected therapeutic applications and that its high activity is clearly due
subcutaneously into nude Nu/Nu mice (day zero). By day 11, to conjugation of the HDAC inhibitor to the antibody.
treatment was initiated when tumour lesions reached ~100
mm3. Mice were randomized and injected with vehicle (PBS),
Conclusions
8
,
9
,
1
and Ctx (Fig. 3a). Mice treated with
of tumour in 50% of mice up to 90 days after the tumour
injection. Although at a lower extent, also ADC exhibited an
anti-tumour activity, significantly higher than Ctx. No activity
was observed with PBS or , although the latter was used at
high dose.18 Based on this result, ADC
was investigated in
8 showed absence
In conclusion, we have developed a new class of ADCs charged
with HDAC inhibitors. Conjugates (ST8154AA1) and
9
8
9
(ST8155AA1), the first example of an ADC for epigenetic
modulation, delivered the HDAC inhibition to cells expressing
the antibody antigens, inducing significant increment of
1
8
additional tumour models. In A549 NSCLC injected in nude
Nu/Nu mice, the compound showed significant antitumor
Histones 3 and 4 and α-tubulin acetylation. Animal models of
human solid tumours indicate anti-tumour efficacy of such
conjugates without the toxicity generally observed with
traditional ADCs charged highly potent cytotoxic drugs.
activity compared with Ctx
, (Fig. 3b). We were also delighted
to observe that ADC 8 proved to be effective in a severe
metastatic lung cancer model done by injecting type A549-luc-
C8 (A549luc) cells into the tail vein of immunodeficient
SCID/beige mice. After 1 week from tumour injection, animals
Overall, comparison of data suggests that ADC
, probably due to the influence of the linker design and the
release processes. With the preparation of ADCs and we
8 is superior to
9
8
9
were treated with ADC
aerosol (Fig. 3c). The evaluation of bioluminescence, analysed
at three different times (35-49-56 days), showed that ADC
8 or unconjugated Ctx by whole body
have disclosed that it is possible to obtain active ADCs even
with not highly cytotoxic warheads, with exceptional potential
of decreasing the side effects of this class of drugs. This work
clearly demonstrates that the paradigm ADC-cytotoxic payload
can be overcome with many advantages for applications of
ADCs beyond cancer therapy. Further work is in progress to
understand why HDAC inhibitor payloads are different from
numerous other tested in ADC field and to ascertain if the
possibility to successfully conjugate medium/low cytotoxic
drugs is limited to HDAC inhibitors or may be extended to
other biologically active compounds.
8
significantly inhibits tumour metastases with a higher potency
in comparison with Ctx alone (Fig. 3c). Comparison among ADC
8,
1
and Ctx was finally evaluated in a mouse model of the
highly aggressive CAPAN-1 orthotopic pancreatic tumour.
Treatments with ADC and Ctx started 6 days after tumour
inoculation and, 90 days after tumour injection, mice were
sacrificed to analyse the pancreas tumour weight. ADC
8, 1
8
showed to inhibit the tumour growth of 84% with 6 complete
responses (6 pancreas free from tumour), while Ctx gave 50%
of tumour growth inhibition with 2 complete responses.
Live subject statement
In animal models, all the procedures adopted for housing and
handling of animals were in strict compliance
with Italian and European guidelines for Laboratory Animal
Welfare. Studies were performed in accordance
with the “Directive 2010/63/UE” on the protection of animals
used for scientific purposes, made effective in Italy by the
Legislative Decree 4 March 2014, n. 26, and ARRIVE
guidelines.2
Figure 4: Body weight of NCI-H1975 tumor bearing mice, throughout the experiment
described in Fig 2a
Conflicts of interest
G.G, L.V., F.M.M., A.M.A., G.B., R.D.S. are employees of
Alfasigma srl. No conflict of interest is declared by all the other
authors.
Compound
(38%) although with five complete responses (Fig. 3d). Finally,
it is worth noting that compound showed the same toxicity
1 alone showed a lower activity on tumour growth
8
than Ctx alone (Fig 4). The body weight of mice treated with
8
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Lyssikatos, M. Strandh, K. Koefoed, P. S. Andersen, J. A.
Acknowledgements
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We thank Caterina Chiapparino and Antonio Rosi for excellent
technical support.
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ADC
A new weapon is added to the ADC arsenal, a thiol based HDAC inhibitor. Low
toxic, hits the target and stops tumor growth in many mouse models.
HDAC