Ethynylphosphonamidates for the Rapid and Cysteine-Selective Generation of Efficacious Antibody–Drug Conjugates

Article abstract of DOI:10.1002/anie.201904193

Requirements for novel bioconjugation reactions for the synthesis of antibody–drug conjugates (ADCs) are exceptionally high, since conjugation selectivity as well as the stability and hydrophobicity of linkers and payloads drastically influence the performance and safety profile of the final product. We report Cys-selective ethynylphosphonamidates as new reagents for the rapid generation of efficacious ADCs from native non-engineered monoclonal antibodies through a simple one-pot reduction and alkylation. Ethynylphosphonamidates can be easily substituted with hydrophilic residues, giving rise to electrophilic labeling reagents with tunable solubility properties. We demonstrate that ethynylphosphonamidate-linked ADCs have excellent properties for next-generation antibody therapeutics in terms of serum stability and in vivo antitumor activity.

Full text of DOI:10.1002/anie.201904193

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Accepted Article
Title: Ethynylphosphonamidates for the rapid and cysteine selective
generation of efficacious Antibody-Drug-Conjugates
Authors: Marc-André Kasper, Andreas Stengl, Philipp Ochtrop, Marcus
Gerlach, Tina Stoschek, Dominik Schumacher, Jonas Helma,
Martin Penkert, Eberhard Krause, Heinrich Leonhardt, and
Christian Hackenberger
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201904193
Angew. Chem. 10.1002/ange.201904193
Link to VoR: http://dx.doi.org/10.1002/anie.201904193
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Ethynylphosphonamidates for the rapid and cysteine selective
generation of efficacious Antibody-Drug-Conjugates
Marc-André Kasper,[a][b] Andreas Stengl, Philipp Ochtrop, Marcus Gerlach, Tina Stoschek, Dominik
Schumacher,[a][b][c] Jonas Helma,[c] Martin Penkert,[a][b] Eberhard Krause,[b] Heinrich Leonhardt*[c] and
Christian P. R. Hackenberger*[a][b]
[c]
[a]
[c]
[c]
Abstract: Requirements for novel bioconjugation reactions for the
Maleimides have become the prime linker reagents for the
®
synthesis of Antibody-Drug-Conjugates (ADCs) are exceptionally high, generation of ADCs including two approved ADCs – Kadcyla
(trastuzumab emtansine) and Adcetris® (brentuximab vedotin).
[8]
since conjugation selectivity as well as stability and hydrophobicity of
linkers and payloads drastically influence the performance and safety
profile of the final product. Herein we describe Cys-selective
ethynylphosphonamidates as new reagents for the rapid generation
of efficacious ADCs from native non-engineered monoclonal
antibodies, applying a simple one-pot reduction and alkylation
protocol. Ethynylphosphonamidates can be easily substituted with
hydrophilic residues, giving rise to electrophilic labeling reagents with
Maleimides can be applied to either modify native IgG antibodies
via interchain-disulfide reduction and alkylation[9] or to engineered
antibodies via addition to an additionally incorporated cysteine
(Thiomab™).[ Nevertheless, one of the biggest drawbacks of
maleimide-linkages is their susceptibility towards retro-Michael-
additions leading to premature drug cleavage during circulation
7a]
[
7a,
and reattachment to cysteine containing proteins like albumin.
1
0]
tunable
solubility
properties.
We
demonstrate
that
Even though consequences arising from such payload transfer
ethynylphosphonamidate-linked ADCs have excellent properties for
next generation antibody therapeutics in terms of serum stability and
in vivo antitumor activity.
are not yet fully understood, it is anticipated that the anti-tumor
efficacy might be lowered due to a decreased drug delivery to
targeted cells. Furthermore toxic side effects might occur.[
Several compound classes have been developed to overcome
this issue, including self-hydrolyzing-maleimides[11] and
structurally refined Michael-type acceptors such as carbonyl
acrylic derivatives[12] or exocyclic maleimides[13]. All of these
methods yield stable sulfhydryl-adducts; however, synthetic
incorporation of these electrophiles into functional molecules
remains challenging.[14]
11]
Antibody-conjugates consisting of a drug linked to a tumor
selective antibody, so called Antibody Drug Conjugates (ADCs),
represent an emerging class of targeted therapeutics.[1] While
most of the ADCs in clinical development contain cytotoxic
molecules, recent studies also include the treatment of infectious
diseases by Antibody Antibiotic Conjugates (AACs).[2] ADCs are
particular interesting for the treatment of cancer, since they
combine the high potency of cytotoxic molecules with the tumor
specificity of monoclonal antibodies. Thus, ADCs have the
potential to significantly broaden the therapeutic window
compared to standard chemotherapy.[1, 3] Recent progress in
clinical development include the approval of inotuzumab
Undesired aggregation of ADCs is another challenge, since many
drugs used in the context of ADCs are hydrophobic.[ The
addition of organic co-solvents to the conjugation mixture is
commonly employed to enable conjugation of hydrophobic drugs,
15]
which however may affect the structural integrity of the
[4]
[16]
ozogamicin (Besponsa™) and the re-approval of gemtuzumab
ozogamicin (Mylotarg™).[ Still, challenges remain especially in
improving the linkage between drug and antibody.[6] Commonly
used linker systems face problems such as insufficient serum
stability and undesired aggregation behavior, limiting the number
of drug molecules linked to an antibody and leading to undesired
off-target toxicity.[7]
antibody.
Additionally, the hydrophobic nature of drugs
5]
increases the formation of High Molecular Weight Species
(HMWS) in the final product.[17] Those aggregates impair the
pharmacokinetic profile and efficacy[ of ADCs and often limit the
Drug to Antibody Ratio (DAR) to a maximum of 4.[ To overcome
this issue, hydrophilic PEG linkers have been developed that
compensate the lipophilic nature of the drug.[20] However, it has
recently been shown that PEG can negatively affect
pharmacokinetics when incorporated as a linear spacer between
antibody and drug.[21] Increasing the solvent exposure of the drug
most likely facilitates unspecific hydrophobic interactions. This
18]
19]
[a]
M.-A. Kasper, P. Ochtrop, Dr. D. Schumacher, M. Penkert, Dr. E.
Krause, Prof. Dr. C.P.R. Hackenberger*
Chemical Biology Department, Leibniz-Forschungsinstitut für
Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125
Berlin (Germany)
unwanted effect was successfully mitigated by side-chain
attachment of the solubilizing polymer.[21]
*
E-mail: hackenbe@fmp-berlin.de
[
b]
c]
M.-A. Kasper, Dr. D. Schumacher, M. Penkert, Prof. Dr. C.P.R.
Hackenberger
Department of Chemistry, Humboldt Universität zu Berlin, Brook-
Taylor-Str. 2, 12489 Berlin (Germany)
A. Stengl, Dr. M. Gerlach, T. Stoschek, Dr. D. Schumacher, Dr. J.
Helma, Prof. Dr. H. Leonhardt*
Department of Biology II, and Center for Integrated Protein Science
Munich, Ludwig-Maximilians-Universität München, Großhadenerstr.
In this study we apply ethynylphosphonamidates as a novel
compound class for the generation of stable Cys-linked ADCs.
We demonstrate that our previously reported phosphonamidate
based labelling protocol is suitable for conjugation of cytotoxic
payloads to two model antibodies, brentuximab and trastuzumab
which form the basis of two marketed ADCs, facilitating the
[
[22]
2, 82152 Martinsried (Germany)
*
E-mail: h.leonhardt@lmu.de
Supporting information for this article is given via a link at the end of
the document.
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Figure 1. a) Synthetic scheme for the attachment of 4 to trastuzumab b) Antiproliferative potency of trastuzumab-4 on two Her2 overexpressing cell lines (SKBR3,
BT474) and a control (MDAMB468). Plots depict the number of proliferating cells after 4 days of antibody treatment in dependency of the antibody concentration.
Trastuzumab alone (magenta), trastuzumab-4 (green) and trastuzumab without disulfide reduction prior incubation with 4 (cyan). c) Effect of trastuzumab-4
treatment on mitotic tubulin organization in BT474 (Her2+) cells. Shown are representative images of mitotic BT474 cells after 4 days of treatment with 0.3 nM
trastuzumab-4 and untreated.
construction of ADCs that efficiently and selectively kill targeted
cells in vitro. In addition, we introduce ethynylphosphonamidates,
trastuzumab-4 and by 42-fold from 800 to 19 pM for BT474 cells
(Her2+). An effect on the proliferation of the control cell line
MDAMB468 was only observed at very high ADC concentrations
(IC50 > 100 nM). Notably, trastuzumab-4 inhibits the proliferation
close to 100% of cell population for SKBR3 cells, while
trastuzumab alone only inhibits up to 50%. As an additional
control, trastuzumab was treated with 4 without prior disulfide
reduction. Those constructs behaved similar to the non-modified
antibodies, highlighting the high Cys-selectivity, efficient removal
of excessive toxin and gentle reaction conditions of our method
(Figure 1b). We additionally validated our measured IC50-values
in a standard cell viability assay and obtained similar IC50- growth
inhibition constants for trastuzumab-4 (Figure S3 in the
supporting information). The mode of action of MMAF is
modified with
a side-chain ethylene glycol unit promoting
improved payload solubility under aqueous conditions without
increasing linker length between antibody and drug in the final
conjugate. With this, we synthesized a vedotin analog and
conjugated it to brentuximab, furnishing a phosphonamidate-
®
linked ADC that is structurally analogous to Adcetris . Our
protocol enables straightforward ADC synthesis, starting from
non-engineered native antibodies that can be labelled in a one pot
protocol with only minimal reagent excess. In extensive profiling
of our ethynylphosphonamidate linked ADC we were able to
demonstrate an excellent linkage stability in rat serum and in vivo
antitumor activity, as investigated in two xenograft mouse model
experiments.
destabilizing
microtubules
by
inhibition
of
tubulin
polymerization.[ Along this line we observed by fluorescence
microscopy a disturbance of tubulin organization in BT474 cells
upon treatment with 0.3 nM trastuzumab-4 for 4 days in contrast
to proper spindle formation in untreated mitotic control cells
26]
We initiated our studies by conjugating the antimitotic agent
Monomethyl auristatin
F
(MMAF),[23]
,
to Her2 targeting
trastuzumab using a phosphonamidate functionalized cathepsin
B cleavable linker 4, which was synthesized based on previously
published procedures for Fmoc-protected Val-Cit dipeptides[24]
(
Figure 1c and Figure S4 in the supporting information).
(
Figure S1 in the supporting information). In a first proof-of-
Furthermore, we anticipated that ethynylphosphonamidates,
bearing O-substituents with increased hydrophilicity might serve
as powerful building blocks to increase the polarity of the whole
linker system without increasing the overall linker length. Since
the Staudinger-phosphonite reaction (SPhR) comprises a very
concept study, we conjugated 4 to trastuzumab following our
previously established protocol applying 10 eq. labeling reagent
per Cys[22], giving an average DAR of 4.6 (Figure 1a and S2 in the
supporting information). To validate the functionality of
trastuzumab-4, it was evaluated in a Her2-based proliferation
assay with two Her2-overexpressing cell lines BT474 and SKBR3
as well as a Her2-negative cell line as a control (MDAMB468).[
Since trastuzumab alone exhibits antiproliferative potency, cell
viability was measured via a sensitive, high content assay to
asses retained antibody functionality after exposure to the
conjugation procedure. Antibody concentrations leading to fifty
percent of maximal growth inhibition (IC50) was decreased by 81-
fold from 900 to 11 pM for SKBR3 cells (Her2++) with
convenient
synthetic
route
to
incorporate
the
ethynylphosphonamidate moiety into a given molecule, we
focused on the synthesis of a hydrophilic phosphonite. Following
our previous protocols, we were able to synthesize the diethylene
glycol substituted phosphonite 5 in a one-pot procedure.[
Subsequent SPhR was performed with the NHS-modified azide 6
and yielded the desired diethyleneglycol-phosphonamidate 7 in
31% yield (Figure 2a).
25]
27]
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Figure 2. a) Synthesis of an ethylene glycol substituted ethynylphosphonamidate building block 7 and structural comparison to the corresponding ethyl substituted
[
22]
8.
b) Structure of vedotin and synthesis of ethyl- and diethylene glycol phosphonamidate-based vedotin analogs. c) RP-HPLC analysis of vedotin (cyan) and the
analogs 9 (red) and 10 (green).. d) Solubility in PBS with 5% DMSO of vedotin (cyan) and the analogs 9 (red) and 10 (green) in µM. Error bars originate from three
independent measurements.
To demonstrate the versatility of our method we proceeded with
the construction of a second antibody-drug pair. Since we wanted
to directly compare our linkage technology with the maleimide
linkage used in Adcetris® we continued our studies with
phosphonamidate-linked ADCs, structurally as close to Adcetris®
as possible. In addition to the MMAF constructs used in the
previous study, two ethynylphosphonamidate MMAE-derivatives
were synthesized, one with an ethyl substituent at phosphorous
in 9 and one with the diethylene glycol substituent in 10 (Figure
desired ADC brentuximab-10 with a DAR of 3.8-4.0. To
furthermore simplify the conjugation process, we applied partial
reduction of the interchain disulfide bonds with TCEP.[ By
screening TCEP equivalents needed to modify brentuximab, we
found out that a partial reduction protocol with 3. eq. TCEP, 5 eq.
10 and 5 mg/ml brentuximab, applied in a one pot process,
produces an ADC with a DAR of 3.9 without the need for removal
of reducing agent prior payload conjugation (Figure S8 in the
supporting information). This one-pot process can be problematic
with other cysteine labelling reagents, since it has been shown
that maleimides and vinyl sulfones for instance, irreversibly react
with TCEP.[30]
29]
2b). RP-HPLC analysis showed a reduced retention time for
compound 10, when compared to 9 or vedotin (Figure 2c).
Solubility measurements revealed that the aqueous solubility of 9
is drastically increased by the PEG substituent in 10 from 95 to
2
98 µM. Aqueous solubility of 10 is also twice as high compared
Taking advantage of our ADC synthesis protocols we then
evaluated commercially available Adcetris in comparison to our
to vedotin (Figure 2d and Figure S5 in the supporting information)
Based on these observations we decided to proceed with the
hydrophilic compound 10 for subsequent conjugation studies to
antibodies.
®
analog brentuximab-10 (Figure 3a). We started with a cell-based
viability assay with a CD30-overexpressing cell line Karpas299
and a CD30-negative cell line HL60 as a control. Both ADCs
showed similar toxicities of below 1 ng/mL in the antigen positive
®
Next, we tried to optimize our conjugation protocol to reduce the
drug equivalents needed for sufficient conjugation. Since
Adcetris® is modified with an average of four vedotin molecules
per antibody[28], we started by screening different equivalents of
cell line. Whereas, Adcetris slightly affected the antigen negative
cell line at high ADC concentrations we observed no such effect
for brentuximab-10. (Figure 3b).
1
0 to achieve similar modification and analyzed the DAR by intact
As mentioned earlier, stability of the linkage between drug and
antibody in serum might improve the properties of ADCs in terms
of off-target toxicity and anti-tumor efficacy.[11] In our experiment,
we observed that 90% of the phosphonamidate-linked MMAE was
still connected to brentuximab after 7 days of incubation in rat
serum at 37°C as measured by intact protein MS after pulldown
of the ADC from serum. Under the same incubation conditions,
Adcetris® lost more than 70% of its payload, already after three
days. (Figure 3c and S9 in the supporting information). This data
is in accordance, with previous observations, that describe a
drastic DAR decrease of similar maleimide linked ADCs following
protein MS (Figure S6 in the supporting information). We
estimated that 16 eq. of 10 per antibody (2 eq. per Cys-residue)
are needed to reach a DAR of 4 at 1 mg/ml antibody concentration.
We attribute the required excess of phosphonamidate to slower
reaction kinetics of ethynylphosphonamidates when compared to
maleimides.[22] To compensate for the slower kinetics, we
increased the antibody concentration in the conjugation reaction
to 5 mg/ml, a concentration that was previously also used for
maleimide conjugations.[9] With this, we were able to use as little
as 4.5 eq. of 10 per antibody to achieve a DAR of 4.0 (Figure S7
in the supporting information). After this, upscaling of the
conjugation reaction to 2.4 mg brentuximab was performed, at 1
6
days of incubation in serum at 37°C.[31] Although hydrolysis of
maleimides was shown to improve conjugate stability and
or
5
mg/ml followed by
a
preparative size-exclusion
generates ADCs with increased in vivo potency,[
11,
32]
the
chromatography step to ensure complete removal of the toxin
prior to subsequent functional evaluations, yielding 1.6 mg of the
occurrence of incomplete hydrolysis of many maleimides may
limit this approach.[33] Taken together, the stability experiments in
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Figure 3. a) Structural comparison of Adcetris® with brentuximab-10. b) Cell viability assays with a CD30 overexpressing cell line (Karpas299, top) and a control
®
(
HL60, bottom) for brentuximab-10 (green) and Adcetris (cyan) and brentuximab alone (magenta) c) Linkage-stability studies in rat serum. ADCs were incubated
in rat serum for 0, 3 and 7 days at 37 °C and analyzed by MS after pulldown, deglycosylation and reduction. Shown is the DAR relative to the average DAR of day
®
®
0
for brentuximab-10 (green) and Adcetris (cyan). d) Antitumor activity of brentuximab-10 (green) and Adcetris (cyan) and a PBS control (magenta) in a Karpas
299 tumor xenograft model in SCID mice. Treatment of 1 mg/kg was administered twice at day 7 and day 10 after tumor transplantation. Shown are tumor volumes
of four mice per group separately. e) Kaplan-Meier survival analysis of the study described in d. f) Treatment of 0.5 mg/kg at day 8 and day 12 after tumor
transplantation. Shown are tumor volumes of eight mice per group separately. g) Kaplan-Meier survival analysis of the study described in f.
serum clearly underlines our previously reported data on excellent
phosphonamidate linkage stability[22] in particular when compared
to maleimide linked ADCs. Additionally, we performed storage
and no relapse was observed over the whole observation period
of 58 days, while the untreated control showed an uncontrolled
tumor growth and had to be sacrificed within three weeks after
tumor transplantation (Figure 3d and S10 in the supporting
information). It was previously reported, that an ADC with an
average of four MMAE molecules connected to brentuximab via
maleimides is significantly less efficacious in a tumor xenograft
model when the dosing is lowered from 1 to 0.5 mg/kg.[35] From
tests with brentuximab-10 to analyze the formation of HMWS,
34]
similarly
as
reported
previously.[
Size-exclusion
chromatography revealed less than 9% HMWS formation with
respect to the monomeric species after storage at 40°C over two
weeks. No significant increase of HMWS was observed after two
weeks of storage at 4°C (Figure S10 in the supporting information). our serum stability studies (Figure 3c), we anticipated that
phosphonamidate linked ADCs might still be active at lower doses
due to a prolonged drug delivery in circulation. Therefore, we
Finally, brentuximab-10 was evaluated in vivo in a Karpas 299
initiated a second in vivo study with eight mice, treated with either
derived tumor xenograft model in immune-deficient female CB17-
SCID mice similar as previously reported.[35] In a first study, four
mice were treated twice, at day 7 and day 10, with 1 mg ADC per
kg bodyweight with brentuximab-10, Adcetris® or only PBS as a
controlt.. All 4 mice treated with brentuximab-10 or Adcetris®
showed an excellent response to the treatment. Treated mice
were in tumor remission already a few days after ADC injection
®
0
.5 mg/kg brentuximab-10 or Adcetris twice at day 8 and day 12.
As expected, we observed decreased antitumor activity for both
constructs when compared to the first study, leading to tumor
remission in two mice for brentuximab-10 and only one for
®
Adcetris . However, five out of eight mice did not show any
observable response to the treatment with Adcetris®. Here, the
tumor growth was as fast as in the PBS treated control group. In
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building block for the synthesis of hydrophilic, Cys-selective linker
systems for the conjugation of unpolar payloads. With this, we
synthesized an ADC from brentuximab and MMAE, and
demonstrated appropriate linkage stability combined with
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We thank K. K. Hassanin for excellent technical assistance. This
work was supported by grants from the Deutsche
Forschungsgemeinschaft (DFG) (SPP1623) to C.P.R.H. and (HA
4468/9-1), (LE 721/13-2) and (SFB1243/A01) to H.L., the Einstein
Foundation Berlin (Leibniz-Humboldt Professorship), the
Boehringer-Ingelheim Foundation (Plus 3 award) and the Fonds
der Chemischen Industrie to C.P.R.H, by the Leibniz Association
with the Leibniz Wettbewerb to C.P.R.H and H.L., by the German
Federal Ministry for Economic Affairs and Energy and the
European Social Fund with grants to D.S. and J.H. (EXIST FT I)
and by the Bavarian Ministry of Economic Affairs, Regional
Development and Energy with grants to D.S., J.H., H.L. and
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This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201904193
COMMUNICATION
COMMUNICATION
Marc-André Kasper, Andreas Stengl,
Philipp Ochtrop, Marcus Gerlach, Tina
Stoschek, Dominik Schumacher, Jonas
Helma, Martin Penkert, Eberhard
Krause, Heinrich Leonhardt*, Christian
P. R. Hackenberger*
ADphosphonamidateC:
Ethynylphosphonamidate-conjugation
enables
Page No. – Page No.
straightforward synthesis of ADCs with excellent linkage stability in serum and
promising antitumor activity in vivo. The phosphorous core structure of the labeling
reagent enables the attachment of an ethylene glycol motive to increase linkage
hydrophilicity.
Ethynylphosphonamidates for the rapid
and cysteine selective generation of
efficacious Antibody-Drug-Conjugates
This article is protected by copyright. All rights reserved.