Antibody–Drug Conjugates with Pyrrole-Based KSP Inhibitors as the Payload Class

Article abstract of DOI:10.1002/anie.201807619

The number of cytotoxic payload classes successfully employed in antibody–drug conjugates (ADCs) is still rather limited. The identification of ADC payloads with a novel mode of action will increase therapeutic options and potentially increase the therapeutic window. Herein, we describe the utilization of kinesin spindle protein inhibitors (KSPi) as a novel payload class providing highly potent ADCs against different targets, for instance HER-2 or TWEAKR/Fn14. Aspects of technical optimization include the development of different linker attachment sites, the stabilization of ADC linkage to avoid payload deconjugation and finally, the tailor-made design of active metabolites with a long lasting intracellular exposure in the tumor matching the mode of action of KSP inhibition. These KSPi-ADCs are highly potent and selective in vitro and demonstrate in vivo efficacy in a broad panel of tumor models including complete regressions in a patient-derived urothelial cancer model.

Full text of DOI:10.1002/anie.201807619

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Antibody-drug conjugates with pyrrole-based KSP inhibitors as
novel payload class
Authors: Hans-Georg Lerchen, Sven Wittrock, Beatrix Stelte-Ludwig,
Anette Sommer, Sandra Berndt, Nils Griebenow, Anne-
Sophie Rebstock, Sarah Johannes, Yolanda Cancho-Grande,
Christoph Mahlert, Simone Greven, and Carsten Terjung
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201807619
Angew. Chem. 10.1002/ange.201807619
Link to VoR: http://dx.doi.org/10.1002/anie.201807619
http://dx.doi.org/10.1002/ange.201807619

10.1002/anie.201807619
Angewandte Chemie International Edition
COMMUNICATION
Antibody-drug conjugates with pyrrole-based KSP
inhibitors as the payload class
Hans-Georg Lerchen*[a], Sven Wittrock[b], Beatrix Stelte-Ludwig[a], Anette Sommer[b], Sandra Berndt[b],
Nils Griebenow[c], Anne-Sophie Rebstock[d], Sarah Johannes[a], Yolanda Cancho-Grande[a], Christoph
Mahlert[a], Simone Greven[a] and Carsten Terjung[a]
Abstract: The number of cytotoxic payload classes successfully
employed in antibody-drug conjugates (ADCs) is still rather limited.
The identification of ADC payloads with a novel mode of action will
increase therapeutic options and potentially increase the therapeutic
window. Herein, we describe the utilization of kinesin spindle protein
inhibitors (KSPi) as a novel payload class providing highly potent
ADCs against different targets, for instance cerbB2/HER-2 or
TWEAKR/Fn14. Aspects of technical optimization comprise the
development of different linker attachment sites, the stabilization of
ADC linkage to avoid payload deconjugation and finally, the tailor-
made design of active metabolites with a long lasting intracellular
exposure in the tumor matching the mode of action of KSP inhibition.
These KSP-ADCs are highly potent and selective in vitro and
demonstrate in vivo efficacy in a broad panel of tumor models
including complete regressions in a patient-derived urothelial cancer
model.
inhibitors (KSPis) such as ispinesib results in high antitumor
potency which raised great excitement to this compound class.[7]
However, transferring the preclinical potency of SMOL KSP
inhibitors into highly efficient clinical regimens with an
acceptable therapeutic window has remained challenging.[8]
Inspired by previous work of Abrams and Wang et al.[9], we
discovered a new pyrrole subclass of KSPis, i.e. compound A,
showing sub-nanomolar potency against a large panel of tumor
cell lines (Scheme 1 and Table 1, column 4) which we
investigated for their applicability as a novel payload class for
ADCs. For initial investigations of technical aspects such as
finding appropriate attachment sites for the linker and
optimization of the effector chemistry with a focus on ADC
stability we selected the monoclonal antibody (mAb) employed
in trastuzumab emtansine (Kadcyla).[2a] For our investigations
we used an in house produced version of trastuzumab called
BAY-865 having the same protein sequence as trastuzumab.
Optimized effector chemistries were subsequently employed in
ADCs which are targeting the receptor of TWEAK (tumor
necrosis factor [TNF]-like weak inducer of apoptosis). The
TWEAK receptor (TWEAKR, Fn14), the smallest member of the
The recent clinical success and approval of brentuximab vedotin
(Adcetris, 2011) in relapsed Hodgkin lymphoma and anaplastic
large cell lymphoma[1], trastuzumab emtansine (Kadcyla, 2013)
in HER-2 positive metastatic breast cancer[2], as well as re-
approval of gemtuzumab ozogamicin (Mylotarg) and approval of
TNF receptor superfamily is
a tumor antigen which is
inotuzumab ozogamicin
(Besponsa) in 2017[3] stimulated
overexpressed in several solid tumor indications including non-
small cell lung, bladder, colorectal and pancreatic cancer.[10] This
raised our interest in exploring TWEAKR-targeted ADCs with the
KSPi payload as anticancer agents and results are also
described in this manuscript.
extensive research efforts in the ADC field. Goals of technical
innovation include the identification of novel targets and the
improvement of the ADC components, antibody, linker and
payload, which resulted in a pipeline with currently more than 60
ADCs in clinical trials.[4] The ADC payload classes presently
tested in clinical trials are dominated by microtubule binders and
DNA binding agents. Key challenges of those are hydrophobicity
leading to aggregation as well as the management of off-target
toxicities.[5] Despite tremendous efforts, the success rate in the
discovery of cytotoxic payload classes with a novel mode of
action resulting in ADCs with an improved therapeutic window
has been rather low.
The KSPi with a pyrrole core (compound A) was synthesized
starting with the benzylation of the methyl 4-bromo-1H-pyrrole-2-
carboxylate educt followed by Suzuki coupling with (2,5-
difluorophenyl)boronic acid, a reduction-oxidation sequence and
an enantioselective installation of the amino and tert.-butyl
moiety via Ellmann’s tert.-butanesulfinamide methodology[11]
(Scheme 1 and Supporting Information). For applicability of
compound A as payload in ADCs, we investigated different
positions in the molecule which are compatible with the
attachment of cleavable and/or non-cleavable linkers. Initial SAR
studies with regard to linker attachment retaining the strong
binding affinity to KSP, revealed two appropriate positions
marked by the arrows in scheme 1, whereas the basic amino
group is important for binding (data not shown). Reductive
alkylation of intermediate A with suitably protected amino acid
derived aldehydes, acylation and partial deprotection allowed for
the synthesis of either intermediate B or intermediate C. These
intermediates were further modified with a variety of linkers
carrying thiol reactive groups. For ADC synthesis, the anti-HER2
antibody BAY-865 has been incubated with Tris(2-
carboxyethyl)phosphine (TCEP) in PBS phosphate buffer at pH
7 to reduce the 4 interchain disulfide bridges and subsequently
reacted with 6-8 equivalents of the electrophilic precursor
molecules to provide the ADCs. The drug-to-antibody ratios
(DARs) are given in scheme 1 and details of the synthesis are
Kinesin spindle protein (KSP, synonyms: Eg5, KIF11) is an ATP-
dependent motor protein involved in the separation of
centrosomes in the G2/M phase of the cell cycle.[6] The blockade
of this essential event in mitosis with small molecule KSP
[a]
Dr. H.-G. Lerchen*, Dr. Beatrix Stelte-Ludwig, Dr. S. Johannes, Dr.
Y. Cancho-Grande, Dr. C. Mahlert, S. Greven, Dr. C. Terjung
Bayer AG R&D Pharmaceuticals, Aprather Weg 18a, 42113
Wuppertal (Germany) E-Mail: hans-georg.lerchen@bayer.com
[b]
[c]
[d]
Dr. S. Wittrock, Dr. A. Sommer, Dr. S. Berndt
Bayer AG R&D Pharmaceuticals, Müllerstr., 13353 Berlin (Germany)
Dr. N. Griebenow
Bayer AG Animal Health, Lead Discovery, Monheim (Germany)
Dr. A.-S. Rebstock, Bayer AG Crop Science, 14, impasse Pierre
Baizet, Lyon (France)
Supporting Information for this article is given via a link at the end of
the document.
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10.1002/anie.201807619
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COMMUNICATION
provided in the supporting information. All ADCs were highly
potent against the HER2 positive breast cancer cell line KPL-4
with IC50 values in the picomolar range.
succinimide thioether very much depends on the length of the
alkyl chain between succinimide and amide bond. When ADC
precursor P1.4 with a maleidoacetyl amide linker is conjugated
to the HER2 antibody, nearly quantitative ring-opening of the
obtained ADC is achieved under mild conditions just by stirring
overnight in PBS buffer at pH 8 and room temperature providing
the ADC 1.4 (Scheme 1b). In contrast, no ring opening was
found under the same conditions with the chain-elongated ADC
1.2
Consequently, the ADC 1.4 with the ring-opened succinic acid
amide linker does not show significant de-conjugation upon
stirring for 12 days in TRIS-buffer in the presence of N-
acetylcysteine. In contrast, under the same conditions, a loss of
~60% of its initial drug load was observed with ADC 1.2 (Fig. S5).
Cysteine-linked ADCs conjugated via maleidocaproyl linker as
employed in many ADCs in the clinic and also in ADC 1.2. are
susceptible to de-conjugation by retro-Michael reaction which
may be associated with off-target side effects.[12] Since ring-
opened succinimides are no longer susceptible to retro-Michael
type reactions, ring opening was strived for by e.g. Tumey et al.
via stirring ADCs for 48 h at a pH of 9.2 at elevated
temperatures.[13] Lyon et al. described the utilization of adjacent
amino groups to provide self-hydrolizing maleimides under much
milder conditions which, however, adds additional complexity.[14]
With KSP ADCs we observed that the ease of hydrolysis of the
a) Synthesis of KSPi SMOL (compound A) and ADCs (exemplified for HER-2 ADCs)
KSPi SMOL
R = H, R1 =
ADC
1.1
DAR KPL-4 cells
IC50 [nM]
3.0
0.7
1.2
4.2
0.8
compound A
(pyrrole based KSPi)
1.3
1.4
3.5
3.6
0.07
0.2
intermediate B
R1 = COOH
R = Teoc
intermediate A
R = H, R2 =
2.1
2.2
2.1
2.9
0.02
0.07
intermediate C
educt
R2=Cl
R = Teoc
4
b) Synthesis of ring-opened ADCs conjugated via succinimido acetamides
stirring overnight
in PBS buffer at pH 8
partially reduced mAb
mAb
mAb
P 1.4
ADC 1.4
Scheme 1a. Pyrrole-based inhibitors of kinesin spindle protein (KSP) and exploration of appropriate attachment sites and linker chemistries for highly potent
HER-2-targeting ADCs. Drug-to-antibody ratios (DARs) and potency of the ADCs in the HER2 positive breast cancer cell line KPL-4. Scheme 1b. Ring-opening of
ADCs conjugated via succinimido acetamides under mild conditions (exemplified for HER2 KSP ADC 1.4) providing stable ADCs (Fig. S4 and S5).
Inspired by the favorable technical profile of HER-2 targeted
KSP-ADCs with BAY-865 and particularly by the strong potency
observed in HER-2 positive tumor cells, we investigated the
extension to tumor targets so far unexplored in the ADC setting
such as the TWEAK receptor (TWEAKR / Fn14). By IHC, we
confirmed the TWEAKR antigen expression described for
different solid tumor indications also in a broad panel of cancer
cell lines representative for these tumors (Table 1 and Table S1,
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10.1002/anie.201807619
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COMMUNICATION
columns 1-3). Furthermore, the small molecule KSPi compound
A was also found to be highly potent with IC50 values in the
picomolar range against the cell panel (Table 1 and Table S1,
column 4).
For ADC synthesis, the fully humanized anti-TWEAKR hIgG1
antibody was prepared in its aglycosylated form (BAY-356)[15]
and showed good binding affinity (Kd ~ 13 nM) to TWEAKR and
TWEAKR-dependent internalization (Fig. S6). The thiol reactive
ADC precursor molecules were coupled to the anti-TWEAKR
antibody BAY-356 in analogy to the BAY-865-conjugated ADCs
described above. Ring-opening of the anti-TWEAKR ADCs 1.4,
2.1, and 2.2 after stirring overnight at pH 8 and room
temperature was higher than 90% for all ADCs. The DARs were
in the range of 3.4 - 4.6 without significant formation of
aggregates (Supporting Information). Overnight stirring at pH 8
had no significant influence on IEF profiles of the antibodies
showing that these conditions were well tolerated by the
antibodies (Figure S3).
Scheme 2a and 2b. Intracellular metabolism of ADCs 1.1 or 1.3 and detection of metabolites M1.1 and M1.3, respectively. After incubation of NCI-H292 cells in
vitro for 3, 24, or 48 h with the TWEAKR-ADCs 1.1 (DAR=1.8) or 1.3 (DAR=3.7) concentration of metabolites M1.1 and M1.3, respectively, increases over time in
the intracellular compartment and remains below the lower limit of quantitation (LLOQ=1µg/L) in the extracellular compartment. Scheme 2c. In vitro potency and
permeability profile of pyrrole-containing KSPi compound A and intracellularly formed metabolites M1.1 and M1.3.
Most of the ADCs exemplified in scheme 1 have non-cleavable
linkers, while ADC 1.3 contains a cathepsin B cleavable linker.
Representative routes of ADC metabolism after internalization
and trafficking into the lysosomal compartment are shown in
scheme 2a and 2b. Formation of the cysteine metabolite M1.1 is
expected after complete antibody degradation of ADC 1.1 in the
lysosomes, while metabolite M1.3 is formed upon cathepsin B
mediated cleavage of ADC 1.3.
The profile of these metabolites has been investigated in
comparison to the KSPi compound A (Scheme 2c). Similar to
compound A, metabolites M1.1 and M1.3 are highly potent in an
enzymatic KSP inhibition assay indicating that linker or linker
fragments attached to the KSPi molecule at that particular
position do not negatively impact KSP inhibition. However, when
cytotoxicity against tumor cells such as NCI-H292 is measured,
compound A is highly potent, whereas M1.1 is 100-fold and
M1.3 even 1000-fold less active. The reason for these
pronounced differences are different membrane permeabilities
which have been investigated in a Caco-2 assay as a surrogate.
In this assay, compound A shows high passive permeability and
pronounced transporter-mediated efflux, whereas both M1.1 and
M1.3 display very low permeability and low active efflux. These
different membrane permeability profiles of KSPi ADC
metabolites offer a great flexibility in adapting ADC performance
to the requirements of individual targets. Metabolites with low
permeability are trapped intracellularly thus enabling a long
lasting exposure inside the tumor cell and at the same time
reduce off-target toxicities due to low permeability into normal
cells. In contrast, metabolites with high permeability like
compound A may induce by-stander killing in tumors with
heterogeneous target expression.
In scheme 2a and 2b it is shown that upon incubation of NCI-
H292 cells with ADCs 1.1 and 1.3 the expected metabolites
M1.1 and M1.3 can be detected in a time-dependent manner
only in the intracellular fraction and not in the supernatant.
These findings are in alignment with the permeability data from
the Caco-2 assay and indicate an intracellular trapping of the
metabolites after binding and internalization of the ADCs. Taken
together, this profile allows for a long lasting intracellular
exposure of cytotoxic metabolites and may help to extend
activity also against cancer cells entering the G2/M phase of the
cell cycle relevant for KSP inhibition at a later point in time.
All TWEAKR ADCs were active against the TWEAKR-positive
NSCLC cell line NCI-H292, the pancreatic cancer cell line
BxPC3, and the colorectal cancer cell line LoVo. In this panel,
the TWEAKR ADCs 1.4 and 2.2 showed a very consistent
potency in the picomolar range with ~1000-fold selectivity versus
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10.1002/anie.201807619
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COMMUNICATION
respective isotype control ADCs containing identical linkers and
payloads (Table 1). Antigen-dependency of ADC potency was
furthermore shown by comparing the TWEAKR-negative cell line
NCI-H520 and the same cell line stably transfected with
TWEAKR. While the SMOL KSP inhibitor compound A is highly
potent in both cell lines, the TWEAKR ADC is selectively potent
in the transfected target positive variant only, clearly indicating
that target antigen expression is required for anti-tumor activity
(table S2). Extended in vitro testing confirmed the high potency
of the TWEAKR ADCs 1.4 and 2.2 in further tumor cell lines
from various solid cancer indications (Table S1).
Table 1. In vitro potency of SMOL KSPi compound A and of TWEAKR-KSP ADCs 1.1., 1.2, 1.3, 1.4, 2.1, and 2.2 in TWEAKR-expressing cell lines versus
selected respective isotype control ADCs 1.4 and 2.2 with same linker payload chemistry
In conclusion, we have shown that KSPis are a versatile new
payload class for the generation of highly potent and selective
ADCs against different targets as shown for HER-2 and the
TWEAK receptor. The small molecule KSPi (compound A) offers
high flexibility in terms of linker attachment site and linker
composition. The stability of cysteine-linked KSPi ADCs is
improved by ring-opening of thiosuccinimide which can be
achieved under mild conditions by stirring overnight at pH 8
when maleimido acetyl amide linkers are employed.
Variation of the linker chemistry of KSPi ADCs allows for a
flexible design of intracellularly formed active metabolites with
high affinity to the KSP enzyme and different membrane
permeability profiles. KSPi metabolites with low efflux
accumulate in tumor cells intracellularly after ADC administration,
which may extend activity and reduce off-target toxicities. KSP-
ADCs targeting TWEAKR demonstrate potent and selective anti-
proliferative activity in a panel of TWEAKR-positive cancer cell
lines in vitro and high antitumor efficacy in vivo. Thus, ADCs with
KSPi as payload offer a promising new approach for tumor
treatment and further evaluation in additional cancer types is
warranted and will be reported elsewhere.
In vivo efficacy of the TWEAKR targeting ADCs was investigated
in the non-small cell lung cancer (NSCLC) model NCI-H292.
Tumor regressions were observed after 3 treatments with ADC
1.4 (5 and 10 mg/kg) or ADC 2.2 (10 mg/kg; Figure 1a). The
isotype control ADC of 2.2 was ineffective at the same doses
and ispinesib, a small molecule KSPi which had been tested in
clinical trials, had only moderate efficacy at doses of 10 mg/kg.
The repeated doses of ADC did not induce a body weight loss of
the mice. In the TWEAKR-expressing patient-derived urothelial
cell cancer (UCC) xenograft model BXF 1218, the KSP ADC 1.4
administered at 5 or 7.5 mg/kg showed high efficacy with
complete tumor eradication with both dose levels. Ispinesib as
SMOL KSP inhibitor at 10 mg/kg and gemcitabine (standard of
care in UCC) showed moderate activity whereas the isotype
control ADC was inactive which again demonstrates the
specificity of the ADC induced tumor cell killing (Figure 1b).
N C I-H 2 92
8 0 0
V e h ic le
C o n tro l-K S P -A D C 2 .2 1 0 m g /k g
Is p in e s ib 1 0 m g /k g
6 0 0
4 0 0
2 0 0
0
T W E A K R -K S P -A D C 1 .4
5 m g /k g
***
T W E A K R -K S P -A D C 1 .4 1 0 m g /k g
T W E A K R -K S P -A D C 2 .2 1 0 m g /k g
Acknowledgements
***
***
***
We thank Bertolt Kreft, Rolf Jautelat, Hilmar Weinmann and
Heiner Apeler for fruitful discussions and Hannah Jörißen for
metabolite analytics. For excellent technical assistance we thank
S. Bendix, H. Bubik, A. DiBetta, T. Boldt, S. Deitz, N. Dittmer, B.
Dübel, D. Grab, K. Gräwinger, D. Hauert, M. Jaspers, B. König,
S. Nesan, M. Ritter, C. Siefert, R. Tamm, F. Tesche and D.
Wolter.
0
1 0
2 0
3 0
4 0
D a y s a fte r s ta rt o f tre a tm e n t
Figure 1a. Efficacy of TWEAKR-ADCs 1.4 and 2.2 with aglycosylated
TWEAKR mAb BAY-356 in the NCI-H292 NSCLC xenograft model in NMRI
nude mice.
B F X 1 218
4 0 0 0
V e h ic le
3 0 0 0
C o n tro l-K S P I-A D C 1 .4 , 1 5 /7 .5 m g /k g
Keywords: ADC, kinesin spindle protein, TWEAK receptor, drug
Is p in e s ib , 1 0 m g /k g
2 0 0 0
discovery, cancer, cytotoxicity, bioconjugate
G e m c ita b in e , 2 0 0 m g /k g
***
***
T W E A K R -K S P I-A D C 1 .4 ,
5 m g /k g
1 0 0 0
0
T W E A K R -K S P I-A D C 1 .4 , 7 .5 m g /k g
[1]
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***
***
0
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Figure 1b. Efficacy of TWEAKR-KSP ADC 1.4 in the BXF1218 UCC PDX
model in NMRI nude mice.
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10.1002/anie.201807619
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COMMUNICATION
Entry for the Table of Contents
Layout 2:
COMMUNICATION
Hans-Georg Lerchen*, Sven Wittrock,
Beatrix Stelte-Ludwig, Anette Sommer,
Sandra Berndt, Nils Griebenow, Anne-
Sophie Rebstock, Sarah Johannes,
Yolanda Cancho-Grande, Christoph
Mahlert, Simone Greven and Carsten
Terjung
Flexible - Stable - Potent! Inhibitors of kinesin spindle protein (KSP) have been
developed as a novel payload class in antibody drug conjugates (ADC) to increase
the therapeutic window. Flexibility in linker attachment and tuning of the profile of
active metabolites fitting to the KSPi mode of action as well as high linker stability
provide potent ADCs against different targets.
Page No. – Page No.
Antibody-drug conjugates with
pyrrole-based KSP inhibitors as the
payload class
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