Reduction-rebridging strategy for the preparation of ADPN-based antibody-drug conjugates

Article abstract of DOI:10.1039/c8md00141c

The reduction-rebridging strategy is a powerful method for the preparation of stable and homogeneous antibody-drug conjugates (ADCs). In this communication, we describe the development of the arylene-dipropiolonitrile (ADPN) functional group for the rebri

Full text of DOI:10.1039/c8md00141c

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RESEARCH ARTICLE
Reduction–rebridging strategy for the preparation
of ADPN-based antibody–drug conjugates†‡
Cite this: DOI: 10.1039/c8md00141c
Oleksandr Koniev,a Igor Dovgan,b Brigitte Renoux,c Anthony Ehkirch,de
a
Jitka Eberova,b Sarah Cianférani,de Sergii Kolodych,
*
b
Sébastien Papot
c and Alain Wagner
*
Received 14th March 2018,
Accepted 7th April 2018
The reduction–rebridging strategy is a powerful method for the preparation of stable and homogeneous
antibody–drug conjugates (ADCs). In this communication, we describe the development of the arylene-
dipropiolonitrile (ADPN) functional group for the rebridging of reduced disulphide bonds and its application
in the preparation of potent and selective ADCs.
DOI: 10.1039/c8md00141c
rsc.li/medchemcomm
Antibody–drug conjugates (ADCs) are efficient therapies that
combine the targeting specificity of monoclonal antibodies
with the potency of small molecular drugs. Four ADCs have
been approved by the FDA and more than 65 are currently in
clinical trials, illustrating the potential of these powerful
sulphide bonds located in the hinge region of monoclonal
antibodies. These disulphide bonds can be easily reduced un-
der mild conditions without disrupting the intrachain di-
sulphide bonds or the secondary structure of the antibody.
Further rebridging can be achieved through a reaction with
targeted therapies.1 Recent research has shown that
a
3,4-disubstituted
maleimides,9–11
bissulfones,12
number of parameters, such as the drug-to-antibody ratio
(DAR),2 site of conjugation3 and linker chemistry,4,5 can influ-
ence the efficacy of ADCs. Currently marketed ADCs are
heterogeneous mixtures of species with different DARs and
conjugation sites. The heterogeneity complicates the develop-
ment of ADCs, since species with different DARs and conjuga-
tion sites have different pharmacokinetic and toxicity pro-
files.6 Therefore, reducing the heterogeneity of ADCs can
yield more efficient and predictable therapies.7 One of the
possible ways to reduce the ADC heterogeneity is the site-
specific introduction of amino acids or peptide sequences
that can be further modified using enzymatic or chemical
conjugation approaches.8 However, this approach requires
the use of costly and laborious techniques for protein engi-
neering in order to produce the modified antibodies.
dibromopyridazinediones13–16 or alkynes under UV irradia-
tion.17 In addition to offering a higher homogeneity, ADCs
prepared using the reduction–rebridging strategy retain the
covalent links between the chains of the antibody, which im-
proves the stability of their secondary structures.
Recently, we have described the use of the
3-arylpropiolonitrile (APN) moiety for the preparation of ADCs
with improved plasma stability.18–20 The S–Csp2 bond formed
upon conjugation of the APN group with cysteine residues
has shown excellent stability in the presence of other amino
acid residues, such as lysine. In this paper, we discuss the de-
velopment and application of the 3,3′-arylene-dipropiolonitrile
(ADPN) functional group for the preparation of ADCs using
the reduction–rebridging approach (Fig. 1).
We hypothesized that the introduction of
a second
An elegant alternative strategy is reduction–rebridging,
which exploits the higher accessibility of interchain di-
propiolonitrile functional group into the aromatic ring would
provide bifunctional reagents suitable for rebridging reduced
disulphide bonds. In this pilot study, the potential of ADPN
as a novel bioconjugation functional group was explored with
ADCs consisting of trastuzumab, monomethyl auristatin E
(MMAE) and our β-galactosidase-responsive self-immolative
linker (Fig. 1).17–19 Following selective internalization inside
HER2-positive cancer cells, these ADCs are designed to be ac-
tivated by lysosomal β-galactosidase leading to the release of
MMAE, thereby restoring its cytotoxic activity in a stringently
controlled fashion.
a Syndivia SAS, 650 Bd Gonthier d'Andernach, 67400 Illkirch, France.
E-mail: sk@syndivia.com
b Laboratory of Functional ChemoSystems (UMR 7199), Labex Medalis, University
of Strasbourg, 74 route du Rhin, 67401 Illkirch-Graffenstaden, France
c Institut de Chimie des Milieux et des Matériaux de Poitiers, IC2MP, Université
de Poitiers, UMR-CNRS 7285, 4 Rue Michel Brunet, 86022 Poitiers, France
d BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, Université de
Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France
e IPHC, CNRS, UMR7178, 67087 Strasbourg, France
For our initial study, we have prepared meta, ortho and
para isomers of ADPN (Fig. 2). The products were synthesized
† The authors contributed equally to this work.
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8md00141c
following
a
previously described procedure.20 Briefly,
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exclusion chromatography and analysed using SDS-PAGE un-
der reducing conditions (Fig. 3). This allowed the identifica-
tion of antibody fragments that were covalently bound by
ADPN isomers. Surprisingly, para-ADPN yielded only trace
amounts of the rebridged antibody fragments. Ortho-ADPN
demonstrated higher efficiency in the rebridging, but sub-
stantial amounts of free heavy and light chains were still pres-
ent. This lack of efficiency of para- and ortho-ADPN may be
explained by the fact that the electron-withdrawing substitu-
ents in these positions reduce the hydrolytic stability of the
propiolonitrile functional group, making it less compatible
with the aqueous buffers used for conjugation. Meta-ADPN
appeared to be the most efficient rebridging reagent, since
SDS-PAGE revealed no free heavy or light chain fragments.
Encouraged by these results, we proceeded to the synthe-
sis of cytotoxic payloads containing meta-ADPN for antibody
rebridging. First, we prepared the trifunctional precursor
containing meta-ADPN and an azide group, suitable for cou-
pling various drugs (Scheme 1).
Thus, commercially available 2,6-diiodo-4-nitroaniline (1)
was diazotized by NaNO2 in H2SO4 and reduced by heating in
EtOH in the presence of CuSO4 to give 2. The nitro group in
2 was reduced by SnCl2 and NaBH4 in EtOH to provide prod-
uct 3. Double Sonogashira coupling of 3 with propargyl alco-
hol yielded intermediate 4, which was oxidized by MnO2 in
the presence of NH3 and MgSO4 to give product 5. Finally, an
azido group was introduced by reaction of 5 with NaNO2/HCl
followed by NaN3, giving the trifunctional precursor 6. An-
other trifunctional compound, 7, containing a short PEG
linker, was prepared from 5 by reaction with triphosgene
followed by N3-PEG-OH.
Fig.
1 Reduction–alkylation strategy (left) and rebridging strategy
(right) for the preparation of ADCs.
In order to prepare payloads for antibody rebridging, we
used the galactoside (8) that was previously described by our
corresponding phenyl diiodides underwent Sonogashira cou-
pling with propargyl alcohol to provide intermediates that
were oxidized in the presence of ammonia to yield the iso-
mers of ADPN.
In order to choose the most suitable isomer for disulphide
rebridging, we carried out
a comparative study using
trastuzumab (obtained from a specialty pharmacy and puri-
fied by ultrafiltration) as a model antibody. This monoclonal
IgG1 antibody consists of two heavy chains (H) and two light
chains (L), linked together by four interchain disulphide brid-
ges. Rebridging of the reduced antibody can yield the total
antibody (HHLL) as well as HHL, HL and HH fragments.
We incubated the reduced trastuzumab with each ADPN
isomer for 12 h. The conjugates were purified by size-
Fig. 3 Reducing SDS-PAGE analysis of the rebridged trastuzumab.
Lane
c – reduced trastuzumab. Lanes m, o and p – reduced
Fig. 2 Structures of the model ADPN isomers used in the initial
trastuzumab rebridged with meta-, ortho- and para-ADPN,
studies.
respectively.
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Table 1 IC50 values of the ADPN-based ADCs compared to those of T-
DM1
IC50 (nM)
IC50 (nM)
SK-BR-3 (HER2+)
cell line
MDA-MB-231 (HER2−)
ADC
cell line
T-ADPN-Gal-MMAE
T-ADPN-PEG4-Gal-MMAE 0.019
T-DM1 0.032
0.022
4.39
3.43
19.03
the enzyme substrate could hamper the recognition of the
carbohydrate moiety by β-galactosidase. Therefore, to prevent
this possible issue, we synthesized payload 10, which con-
tains a short PEG linke
r, by coupling compounds 7 and 8 under the same conditions
as those used for the preparation of 9.
Trastuzumab was used as a model antibody for the prepa-
ration of new ADCs. The ADCs were prepared following the
optimized reduction–rebridging protocol (ESI‡). Briefly, the
interchain disulphide bonds of the antibody were reduced by
incubation with 5 eq. of TCEP in PBS at 37 °C. The reduced
antibody was subsequently incubated with 9 or 10 for 12 h at
25 °C. The resulting rebridged ADCs (T-ADPN-Gal-MMAE pre-
pared from 9 and T-ADPN-PEG4-Gal-MMAE prepared from 10)
were purified by size-exclusion chromatography. According to
native mass spectrometry analysis, the average DAR values
were 4.5 for T-ADPN-Gal-MMAE and 4.0 for T-ADPN-PEG4-Gal-
MMAE. Although the major species in the DAR distribution
of each ADC corresponded to DAR 4, small amounts of DAR
5 species were also observed (Fig. S1‡). The formation of DAR
5 species may occur if the reduced disulphide reacts with two
different molecules of ADPN-based payload. Further optimi-
zation of the DAR distribution may be achieved through fine-
tuning of the antibody and payload concentrations.
Scheme 1 Synthesis of the trifunctional compound 6. a) NaNO2,
H2SO4, 0 °C, 2 h; b) CuSO4·5H2O, EtOH, reflux, 2 h, 36%; c) SnCl2
·2H2O, NaBH4, EtOH, reflux, 0.5 h, 93%; d) propargyl alcohol (3 eq.),
NEt3 (4 eq.) PdIJPPh3)2Cl2 (2 mol%), CuI (4 mol%), DMF, r.t., 14 h, 96%;
e) MnO2 (30 eq.), MgSO4 (30 eq.), NH3 (8 eq.,
2 M isopropanol
solution), THF, r.t., 2 h, 11%; f) NaNO2 (1.1 eq.), NaN3 (2 eq.), HCl (15%),
H2O, 0 °C, 5 min, 96%.
group (Scheme 2).19,20 This has already been employed with
success for the preparation of β-galactosidase-responsive
ADCs that showed significant anticancer activity in vivo.
Thus, the cytotoxic payload 9 was synthesized by coupling
compounds 6 and 8 using the CuAAC reaction.
Since the galactoside trigger must be readily accessible to
lysosomal β-galactosidase for inducing efficient drug release,
we also investigated the preparation of payload 10. Indeed, in
payload 9, the short distance between the bulky antibody and
The in vitro cytotoxicity of the new ADCs was evaluated on
SK-BR-3 HER2-positive and MDA-MB-231 HER2-negative can-
cer cell lines, obtained from the American Tissue Culture Col-
lection (ATCC, Manassas, VA, USA). The FDA-approved
trastuzumab emtansine (T-DM1, obtained from a specialty
pharmacy and purified by ultrafiltration) was used as the
benchmark in our studies. Both rebridged ADCs showed
strong activity on HER2-positive cancer cells (Fig. S1‡), with
IC50 values comparable with those of T-DM1 (Table 1). Fur-
thermore, they were approximately 200 times less toxic for
MDA-MB-231 cells, demonstrating their selectivity for killing
HER2-positive cancer cells. Interestingly, there was no signifi-
cant difference between conjugates with and without the PEG
linker, suggesting that the hinge region of the antibody is
sufficiently accessible for β-galactosidase to perform efficient
drug release.
Conclusions
Scheme 2 Synthesis of cytotoxic payloads for antibody rebridging. a)
CuIJMeCN)4PF6 (1.5 eq.), CH2Cl2/MeOH (1 : 0.2), r.t., 18 h, 75%; b)
CuIJMeCN)4PF6 (1.5 eq.), CH2Cl2/MeOH (1 : 0.2), r.t., 6 h, 17% after
purification by preparative reverse-phase HPLC.
In conclusion, we have developed a new functional group for
the rebridging of reduced disulphide bonds. The ADPN func-
tional group was successfully combined with
a
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β-galactosidase-cleavable linker and MMAE to yield a series
of novel ADC payloads. The resulting payloads were used for
the preparation of potent and selective ADCs via the
rebridging of reduced trastuzumab.
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Conflicts of interest
The authors declare no competing interest.
Acknowledgements
This work was supported by CNRS, University of Strasbourg,
Région Alsace, La Ligue contre le Cancer (comités Deux-
Sèvres et Vienne), “Agence Nationale de la Recherche” (ANR)
and the French Proteomic Infrastructure (ProFI; ANR-10-INBS-
08-03). The International Center for Frontier Research in
Chemistry (icFRC) is also acknowledged for financial support.
A. E. acknowledges the «Association Nationale de la
Recherche et de la Technologie» (ANRT) and Syndivia for
funding his PhD fellowship. We thank Dr. Wojciech Krezel
for the assistance with the in vitro experiments.
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