The Methylene Alkoxy Carbamate Self-Immolative Unit: Utilization for the Targeted Delivery of Alcohol-Containing Payloads with Antibody–Drug Conjugates

Article abstract of DOI:10.1002/anie.201601506

A strategy for the conjugation of alcohol-containing payloads to antibodies has been developed and involves the methylene alkoxy carbamate (MAC) self-immolative unit. A series of MAC β-glucuronide model constructs were prepared to evaluate stability and enzymatic release, and the results demonstrated high stability at physiological pH in a substitution-dependent manner. All the MAC model compounds efficiently released alcohol drug surrogates under the action of β-glucuronidase. To assess the MAC technology for ADCs, the potent microtubule-disrupting agent auristatin E (AE) was incorporated through the norephedrine alcohol. Conjugation of the MAC β-glucuronide AE drug linker to the anti-CD30 antibody cAC10, and an IgG control antibody, gave potent and immunologically specific activities in vitro and in vivo. These studies validate the MAC self-immolative unit for alcohol-containing payloads within ADCs, a class that has not been widely exploited.

Full text of DOI:10.1002/anie.201601506

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201601506
German Edition: DOI: 10.1002/ange.201601506
Drug Delivery
The Methylene Alkoxy Carbamate Self-Immolative Unit: Utilization
for the Targeted Delivery of Alcohol-Containing Payloads with
Antibody–Drug Conjugates
Robert V. Kolakowski,* Karl T. Haelsig, Kim K. Emmerton, Chris I. Leiske, Jamie B. Miyamoto,
Julia H. Cochran, Robert P. Lyon, Peter D. Senter, and Scott C. Jeffrey*
Abstract: A strategy for the conjugation of alcohol-containing
payloads to antibodies has been developed and involves the
methylene alkoxy carbamate (MAC) self-immolative unit. A
series of MAC b-glucuronide model constructs were prepared
to evaluate stability and enzymatic release, and the results
demonstrated high stability at physiological pH in a substitu-
tion-dependent manner. All the MAC model compounds
efficiently released alcohol drug surrogates under the action
of b-glucuronidase. To assess the MAC technology for ADCs,
the potent microtubule-disrupting agent auristatin E (AE) was
incorporated through the norephedrine alcohol. Conjugation
of the MAC b-glucuronide AE drug linker to the anti-CD30
antibody cAC10, and an IgG control antibody, gave potent and
immunologically specific activities in vitro and in vivo. These
studies validate the MAC self-immolative unit for alcohol-
containing payloads within ADCs, a class that has not been
widely exploited.
I
nterest in antibody–drug conjugate (ADC) technology has
greatly increased in recent years because of the approval of
ꢀ
[
1]
ꢀ
ADCETRIS (brentuximab vedotin) and KADCYLA
[
2]
(
ado-trastuzimab emtansine) for the treatment of various
Figure 1. Methods for conjugation of amine- and alcohol-containing
payloads for ADCs.
malignancies, and numerous other ADCs having promising
[
3]
preclinical and early-stage clinical profiles. Consequently,
considerable attention has been directed towards evaluating
[
4]
[8e]
new payloads to expand the scope and utility of ADCs. The
installed such residues synthetically. However, introducing
an amine functional group may not always be synthetically
feasible, and it may have a detrimental impact on the
pharmacology of the resulting drug analogue.
Several strategies have been explored for the utilization of
alcohol-containing agents as ADC payloads. A carbonate of
[
5]
most commonly used drugs for ADCs are the auristatins,
[6]
[7]
[8]
maytansines, g-calicheamicin, camptothecins, duocarmy-
cin analogues, pyrrolobenzodiazepine dimers, and anthra-
cyclines, which have demonstrated pronounced activities.
Typically, these agents are linked through a reactive residue,
and emphasis has been placed on maximizing linker stability
[9]
[10]
[4b]
[
8b]
[8b]
a camptothecin analogue, SN-38,
has been linked to
[6]
in circulation. To deliver amine-containing payloads, we and
monoclonal antibodies (mAbs), but was found to be labile
under physiological conditions. Self-immolative dicarbama-
[11]
others have employed peptide-
or b-glucuronide-based
[
12]
[8b,9,13]
[14]
linker systems, both of which are stable in circulation and
give facile drug release once the ADC is internalized into
cancer cells (Figure 1). When an amine for conjugating is not
present in a molecule of interest, we have, when possible,
tes
(Figure 1) and ether linkages
have also been
employed, but these approaches are limited in scope, thus
efficiently releasing only phenolic payloads.
We surmised that a general solution for utilizing alcohol-
containing drugs within ADCs could involve a methylene
alkoxy carbamate (MAC) construct, a method which has been
used in several drug delivery strategies. One of the first
applications involved a MAC-type unit to release 5-fluoro-
[
*] Dr. R. V. Kolakowski, K. T. Haelsig, K. K. Emmerton, C. I. Leiske,
J. B. Miyamoto, J. H. Cochran, Dr. R. P. Lyon, Dr. P. D. Senter,
Dr. S. C. Jeffrey
[
15]
uracil from a glucuronide prodrug. Shabat and co-workers
further developed the unit for the release of mercaptoacetic
Seattle Genetics, 21823 30th Dr SE, Bothell, WA 98021 (USA)
E-mail: rkolakowski@seagen.com
[16]
[17]
sjeffrey@seagen.com
acid
and glucose.
Subsequently, Santi and co-workers
utilized a MAC construct for b-elimination-promoted release
of the potent cytotoxic agent SN-38.
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201601506.
[18]
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The goal of our work was to utilize a MAC construct for
antitumor ADCs. To achieve this goal, the MAC unit was
configured for stability during prolonged serum exposure
because of the long circulation time of ADCs. In addition, we
protecting groups gave the target MAC b-glucuronides, the
model compounds 10a–g.
A final model compound was prepared by Curtius
[
17a]
rearrangement
to provide the secondary carbamate com-
[12]
utilized a b-glucuronidase-promoted release mechanism to
trigger MAC self-immolation, to enable rapid and efficient
payload release upon target cell delivery. Herein, we describe
a novel MAC construct of the potent microtubule-disrupting
pound 11 (Scheme 2). Rhodium-acetate-promoted alkylation
of 7a with tert-butyl azidoacetate (12), followed by tert-butyl
ester removal, gave the acid 13, which was subjected to
Curtius rearrangement conditions using diphenylphosphoryl
azide. The intermediate isocyanate was trapped with the
alcohol 14 in the presence of dibutyltin dilaurate to afford the
carbamate 15. Nitro group reduction gave the aniline 16,
which was acetylated and saponified, thus providing the
model MAC construct 11.
[
5]
agent auristatin E and demonstrate that the derived con-
jugates are stable under physiological conditions, highly
potent, and immunologically specific in vitro and in vivo.
The results support the use of this linker technology for
alcohol-containing drugs within ADCs.
Our design of a MAC unit for ADCs centered on
incorporating the payload alcohol oxygen atom in a stable
manner to allow efficient release upon enzymatic
processing. To explore the viability of the MAC
approach for conjugating alcohols to ADCs, a series
of model compounds was prepared starting with the
To assess stability, each of the MAC b-glucuronide model
constructs 10a–g and 11 was dissolved in phosphate-buffered
[
12]
known para-nitrophenylcarbonate
3 (Scheme 1).
Addition of ethyl amine (4a), N,N-dimethylethylenedi-
amine (4b), and 2-(methylsulfonyl)ethylamine (4c)
gave the compounds 5a–c, respectively, which were
converted into the corresponding chloromethyl adducts
6
a–c through reaction with chlorotrimethylsilane and
[
19]
paraformaldehyde. The N-ethyl chloromethyl adduct
a was reacted with alcohols 7a–d, to provide the
6
corresponding intermediates 8a–d. The carbamate 6b
was reacted with 7a and 7c to give compounds 8e and
8
f, respectively. Reaction of the methanesulfonyl
adduct 6c with 7a afforded 8g. Nitro group reduction
of 8a–g gave the corresponding anilines 9a–g, which
were capped as acetamides by treatment with acetic
Scheme 2. Synthesis of a MAC-based substrate by a Curtius rearrangement.
anhydride, and saponification of the b-glucuronide DCM=dichloromethane, TFA=trifluoroacetic acid.
Scheme 1. Synthesis of model MAC-linked test substrates and an auristatin E based drug linker. DIPEA=diisopropylethylamine, DMF=N,N-
dimethylformamide, EEDQ=ethyl-2-ethoxy-1,2-dihydro-1-quinolinecarboxylate, Fmoc=fluorenylmethoxycarbonyl, TMS=trimethylsilyl.
2
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Table 1: MAC analogue structures and hydrolysis under physiological
conditions.
MS, presumably because of rapid decomposition and release
of the free alcohol.
We next turned our attention toward the utility of the
MAC unit for ADCs using the potent microtubule-disrupting
[
5]
agent AE (2). The dimethylaminoethyl-containing construct
was selected, based on the stability characteristics from the
model compounds 10e and 10 f. Reaction of the chloromethyl
adduct 6b with 2 gave the AE-alkoxy carbamate 8h
1
2
3
[a]
Compound
R
R
R
Hydrolysis (%)
/14 days
7
(Scheme 1). Nitro group reduction afforded 9h, which was
coupled to Fmoc b-alanine (17) using EEDQ, to give 18.
Saponification with concomitant removal of the Fmoc
protecting group using aqueous lithium hydroxide, followed
by maleimide installation using the N-hydroxysuccinimide
ester 19, gave the fully elaborated MAC b-glucuronide AE
drug linker 1. The compound 1 was incubated in phosphate-
buffered saline at 378C, and like the model MAC constructs
possessing the dimethylaminoethyl residue (10e and 10 f), no
hydrolysis of the MAC-AE unit was observed over the 14 day
incubation period (Table 1; see Figure S1 in the Supporting
Information). As expected, b-glucuronidase treatment
resulted in clean and facile formation of AE (2; see Fig-
ure S2).
1
1
1
0a
0b
0c
Et
Et
Et
Ac
Ac
Ac
44/n.d.
0/0
61/n.d.
10d
Et
Ac
0/0
1
1
1
1
0e
0f
0g
1
Ac
Ac
Ac
Ac
MP
0/0
0/0
0/0
The method for conjugating 1 was as previously de-
H
100/n.d.
0/0
[
12]
[1]
scribed. Briefly, the anti-CD30 mAb cAC10 was partially
reduced with tris(2-carboxyethyl)phosphine and then purified
by gel filtration, thus affording approximately four free thiol
residues/mAb. Addition of a slight excess of 1 to the reduced
mAb resulted in conjugation with an average of 4 drugs/mAb.
The cAC10-1 conjugate was evaluated against CD30-
positive and -negative cell lines (Table 2). As a positive
[
b]
1
AE
[
a] In Gibco pH 7.4 (10X) PBS at 378C, as determined by LC-MS. [b] The
compound 1 undergoes maleimide hydrolysis to the ring-open form
during the course of the stability study. n.d.=not determined because of
instability at the 7 day time point.
saline and incubated at 378C for up to 14 days. Changes were
monitored by liquid chromatography/mass spectroscopy (LC/
MS) and integration of the parent peak of each model
compound was used to determine the degree of hydrolysis.
The results for the substituted carbamate model compounds
are shown in Table 1. The compounds 10a–d, with N-ethyl
carbamate residues derived from primary, secondary, tertiary,
and 1-napthyl alcohols (7a–d), were differentially stable. The
compounds 10b and 10d were completely stable up to 14
days, while the primary and tertiary alcohol constructs 10a
and 10c showed significant hydrolysis at 7 days.
[
a]
Table 2: In vitro activity and immunologic specificity.
ADC
Karpas 299
CD30 290K
ALCL
L540cy
CD30 433K
HL
Ramos
CD30 (À)
Burkitt’s
cAC10-1
hIgG-1
brentuximab vedotin
0.5
>1000
0.5
2
>1000
4
>1000
>1000
>1000
À1
[
a] Activity reported as IC50 value in ngmL of ADC. Cells were treated
with ADC for 96 h and viability was assessed using Cell TiterGlo.
Anaplastic large cell lymphoma (ALCL), Hodgkin lymphoma (HL).
To stabilize the MAC linkage, we introduced both basic
and electron-withdrawing groups proximal to the aminal
linkage. Replacement of the R ethyl group of 10a and 10c,
control for the assay, the anti-CD30 ADC brentuximab
vedotin, employing monomethyl auristatin E (MMAE) at-
tached by a valine-citrulline para-aminobenzyl carbamate
1
with a dimethylaminoethyl group, resulted in the compounds
[1]
1
0e and 10 f, respectively, which were stable for the 14 day
linker, was included (Figure 1). The ADC cAC10-1 was
comparable to brentuximab vedotin in activity on both the
antigen positive cells, and on the antigen negative cell line, in
which both constructs were relatively inactive. Further
evidence for antigen-dependent activity was obtained with
the nonbinding hIgG-1 conjugate, which was inactive (IC >
incubation period. Likewise, incorporation of the methane-
sulfonylethyl group in 10g also improved stability relative to
the N-ethyl compound 10a (and 10c), presumably by
reducing electron density at the carbamate nitrogen atom.
High electron density at the carbamate nitrogen atom may
explain why the secondary carbamate, 11, was not stable and
showed complete hydrolysis at 7 days.
Each model construct released the corresponding alcohol
compound when treated with bovine liver b-glucuronidase.
As anticipated, the intermediate aminal released from the
b-glucuronide MAC construct could not be detected by LC/
5
0
À1
1000 ngmL ) on all three cell lines tested. These results are
consistent with the high degree of stability observed with 1 in
aqueous buffer (Table 1). A stability assessment under
physiological-type conditions was also obtained by incubating
cAC10-1 in mouse plasma at 378C. An LC/MS assay detected
less than 1% AE (2) liberated after a 7 day incubation (see
Figure S3).
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curative at 1 mgkg (6/6 animals), and partially curative at
À1
0
.5 mgkg (3/6 animals). The effects were immunologically
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À1
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[
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[
[
[
Keywords: alcohols · antibodies · cancer · drug delivery ·
glycoconjugate
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Received: February 11, 2016
Revised: April 4, 2016
Published online: && &&, &&&&
4
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Communications
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Communications
Drug Delivery
R. V. Kolakowski,* K. T. Haelsig,
K. K. Emmerton, C. I. Leiske,
J. B. Miyamoto, J. H. Cochran, R. P. Lyon,
P. D. Senter, S. C. Jeffrey*
&&&& — &&&&
MAC out: A strategy for the conjugation
of alcohol-containing payloads to anti-
bodies has been developed using the
methylene alkoxy carbamate (MAC) self-
immolative unit. A series of MAC b-
glucuronide model constructs were pre-
The Methylene Alkoxy Carbamate Self-
Immolative Unit: Utilization for the
Targeted Delivery of Alcohol-Containing
Payloads with Antibody–Drug Conjugates
pared and demonstrated high stability at
physiological pH, and efficiently released
alcohol drug surrogates under the action
of b-glucuronidase.
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!