Synthesis of Highly Potent N-10 Amino-Linked DNA-Alkylating Indolinobenzodiazepine Antibody-Drug Conjugates (ADCs)

Article abstract of DOI:10.1021/acsmedchemlett.9b00254

Indolinobenzodiazepine DNA alkylators (IGNs) are the cytotoxic payloads in antibody-drug conjugates (ADCs) currently undergoing Phase I clinical evaluation (IMGN779, IMGN632, and TAK164). These ADCs possess linkers that have been incorporated into a central substituted phenyl spacer. Here, we present an alternative strategy for the IGNs, linking through a carbamate at the readily available N-10 amine present in the monoimine containing dimer. As a result, we have designed a series of N-10 linked IGN ADCs with a wide range of in vitro potency and tolerability, which may allow us to better match an IGN with a particular target based on the potential dosing needs.

Full text of DOI:10.1021/acsmedchemlett.9b00254

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Letter
Synthesis of highly potent N-10 amino-linked DNA-alkylating
indolinobenzodiazepine antibody-drug conjugates (ADCs)
Katie E Archer, Emily E Reid, Manami Shizuka, James Woods, Luke Harris,
Erin K. Maloney, Laura M Bartle, Olga Ab, Alan Wilhelm, Yulius Setiady, Jose F.
Ponte, Rajeeva Singh, Thomas A Keating, Ravi V.J. Chari, and Michael L Miller
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00254 • Publication Date (Web): 22 Jul 2019
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Synthesis of highly potent N-10 amino-linked DNA-alkylating
indolinobenzodiazepine antibody-drug conjugates (ADCs)
†
‡
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Katie E. Archer, Emily E. Reid, Manami Shizuka, James Woods, Luke Harris, Erin K. Maloney, Laura
M. Bartle, Olga Ab, Alan Wilhelm, Yulius Setiady, Jose F. Ponte, Rajeeva Singh, Thomas A. Keating,
Ravi V.J. Chari, Michael L. Miller*
ImmunoGen, Inc., 830 Winter Street, Waltham, Massachusetts 02451, United States
KEYWORDS: Antibody-drug conjugates, ADCs, indolinobenzodiazepines.
ABSTRACT: Indolinobenzodiazepine DNA alkylators (IGNs) are the cytotoxic payloads in antibody-drug conjugates (ADCs)
currently undergoing Phase I clinical evaluation (IMGN779, IMGN632, and TAK164). These ADCs possess linkers which have been
incorporated into a central substituted phenyl spacer. Here we present an alternative strategy for the IGNs, linking through a carbamate
at the readily available N-10 amine present in the monoimine containing dimer. As a result, we have designed a series of N-10 linked
IGN ADCs with a wide range of in vitro potency and tolerability which may allow us to better match an IGN with a particular target
based on the potential dosing needs.
A majority of antibody-drug conjugates (ADCs)1-4 in clinical
-
SO3
H
O
O
S
N
O
N
S
evaluation use tubulin-interacting agents, the maytansinoids
and auristatins, as the cytotoxic payload.5-6 Recently, there has
been a surge of interest in payloads with alternative mechanisms
of action, such as topoisomerase 1 inhibitors, exemplified by
the camptothecins, and DNA crosslinkers, based on analogs of
the pyrrolobenzodiazepine (PBD) dimer, SJG-136 (Figure 1).7-
9 Emerging clinical data indicate that doses achieved with these
PBD ADCs are quite low, and treatment is often limited to two
cycles because of toxic side effects.10-11
O
N
N
N
O
O
H
10
11
N
N
O
O
11a N
N
OMe MeO
spacer
N
OMe
MeO
O
O
O
O
SJG-136
IMGN779 (anti-CD33 DGN462)
O
O
O
H
N
H
N
N
HN
N
H
S
O
O
O
H
N
N
O
O
N
N
OMe
MeO
O
O
Previously, we disclosed
a
new DNA alkylator class,
indolinobenzodiazepine pseudodimers (termed IGNs).12-13 One
major difference between the IGNs and PBDs is the mechanism of
action. While, both PBDs and IGNs bind to the minor groove of
DNA, only the PBDs, which contain a bis-imine, can crosslink with
inter-strand guanine residues at preferred GATC sequences.14
Through the controlled reduction of one of the two imine moieties,
the IGNs are only capable of DNA alkylation, providing ADCs
with improved tolerability in mice, and the potential to achieve
higher doses in the clinic. The IGN component of all three ADCs
of this payload class currently in clinical evaluation (IMGN779,
IMGN632 and TAK164) share two additional structural features
that are distinct from the PBDs: a) the pyrrolidine group was
replaced with an indolino moiety, which results in tighter binding
to DNA and a ~10-fold increase in in vitro cytotoxicity, b) the
introduction of a substituted phenyl spacer instead of the propyl
group between the two monomer sub-units which further increases
potency, and provides a site for linker attachment (Figure 1).
IMGN632 (anti-CD123 DGN549)
O
O
H
N
H
N
HN
N
H
O
O
H
N
N
O
O
N
N
OMe
MeO
O
O
TAK-164 (anti-GCC DGN549)
Figure 1. Structures of the DNA crosslinking PBD SJG-136 and
the clinical stage ADCs of mono-reduced IGN DNA alkylators.
While this initial strategy allowed for rapid evaluation of a variety
of linkers, we were limited in our ability to investigate the potential
of extensive modification to the spacer group, and perhaps access
to IGNs that would provide ADCs with a wider range of activity
and tolerability. In order to explore the role of linkage position and
spacer modification on preclinical activity as related to the IGN
DNA alkylators, we synthesized a small set of IGNs, wherein, we
fixed the linkage site at the N-10 amine of the reduced imine, while
altering the spacer connecting the two monomer sub-units. Here,
we report on the design, synthesis and preclinical evaluation of
these N-10 linked IGN ADCs.
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10 amino group; as upon ADC catabolism and linker cleavage,
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R
1
2
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a monoimine IGN that is capable of alkylating DNA would be
released. In addition, the presence of a free imine would allow
for reversible sulfonation which enhances aqueous solubility,
and facilitates conjugation to the antibody. In order to expand
the usefulness of this synthetic approach we developed an
unsymmetrical stepwise method, shown in Scheme 1, which
differed from our previous synthetic methodology. (see
Supplementary methods for the synthesis of catabolites 1-5).
H
N
N
O
O
1
2
3
(R = H)
(R = NH2)
(R = NH(CH2CH2O)3Me)
N
N
OMe
MeO
O
O
H
N
N
O
O
N
N
OMe
MeO
4
O
O
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H
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N
N
O
O
N
N
OMe MeO
O
O
5
Figure 2. Structure of expected IGN catabolites.
Our modular synthetic strategy focused on structural modifications
to the IGN psuedodimer to provide expected ADC catabolites with
a wide range of in vitro potency. We designed and synthesized a
small panel of these catabolites with spacers possessing either a
phenyl group (1), a substituted phenyl (2-3), or an alkyl group (4-
5) of different chain lengths (Figure 2). These catabolites were
tested in vitro against the KB and Namalwa cell lines, and as
expected, a range of potencies was observed. As shown in Table 1,
the phenyl containing IGNs 1 and 2 demonstrated identical in vitro
activity in the cell lines tested, whereas IGN 3, with the larger and
more polar PEG methyl ether substituent at the anilino nitrogen,
was about 3-fold less potent in the KB cell line. IGN 5, containing
the three carbon propyl spacer, was found to be the least potent
molecule in this series (10 to 30-fold less) in vitro. Interestingly,
we found that increasing the spacer length from three carbons (5)
to five (4) was accompanied by a significant increase in potency,
which agrees well with previously reported data for the PBDs.15 In
fact, IGN catabolite 4, with a five carbon pentyl spacer, was
identical in potency to the phenyl substituted IGN 3. In a separate
study, we investigated the DNA binding capability of the
catabolites in a competitive inhibition assay using a synthetic
hairpin oligonucleotide (Figure 3). This study revealed that the
Figure 3. DNA interaction of IGN catabolites. Competitive
inhibition by IGNs 2-5 at several concentrations to the binding
of biotinylated reference IGN with digoxigenin-labeled hairpin
oligonucleotide, measured as ELISA absorbance.
We prepared a set of IGNs wherein the dipeptide linker (e.g.,
Ala-Ala, Gln-Leu, Ala-Val) at the N-10 amino group was
varied. As a general example, the synthesis of the Ala-Val
methyl adipate IGN 12a containing an N-hydroxysuccinimide
(NHS) ester for conjugation to an antibody is described
(Scheme 1). The methyl adipate Ala-Val dipeptide 6, prepared
through a four-step process, was converted to the corresponding
p-aminobenzoyl derivative 7 in high yield by deprotection of
the t-butyl ester with TFA followed by coupling with (4-
aminophenyl)methanol using EEDQ. Treatment of the reduced
IGN monomer 8 with 4-nitrobenzyl carbochloridate gave the
activated carbamate 9 which was subsequently treated with
alcohol 7 in the presence of LiHMDS to give the benzyl
protected monomer 10 containing the desired linking group
attached via the reduced N-10 amine. In a separate effort, a
group of spacer modified imine containing IGN monomers (a-
c) were prepared which contained either an alkyl iodide or
bromide suitable for coupling reactions with a phenol.
catabolites possessing
demonstrated superior DNA binding, perhaps accounting for the
higher potency that was generally observed in vitro.
a substituted aromatic spacer (2-3)
Table 1. In Vitro Potency of IGN catabolites 1-5a
Catabolite, IC50 pM
Cell Line
KB
Indication
cervical
1
2
3
4
5
Hydrogenolysis of benzyl protected monomer 10 using Pd/C
generates the corresponding unprotected phenol which was then
coupled with the iodopropyl IGN monomer (a) using potassium
carbonate to give methyl ester 11a. Hydrolysis of 11a using
lithium hydroxide to generate the carboxylic acid and treatment
with N-hydroxysuccinimide gave the desired NHS ester 12a in
a 65% yield over two steps. With the successful preparation of
NHS ester 12a, conjugation was performed separately using
either an anti-FR or anti-EGFR antibody via lysine residues
giving the ADCs anti-FR 13a and anti-EGFR 14a,
respectively. Through a similar set of transformations the anti-
FR (13b-c) and anti-EGFR (14b-c) ADCs possessing a self-
immolative dipeptide linker at the N-10 amine and a variety of
spacer groups were prepared. All conjugates had an average of
8
8
20
20
200
Namalwa
lymphoma
1
2
3
3
30
aCancer cell lines were incubated with ADCs for 5 days at 37oC.
IC50 values were determined using a WST-based cell viability
assay.
With a series of catabolites possessing a range of potencies in
hand, we focused on developing a linking strategy. Using a
design similar to that described in the literature for PBD ADCs,
where the N-10 position is used as a linkage site,8 we postulated
that a self-immolative p-aminobenzyl (PAB) dipeptide could be
used. In contrast to the PBDs which are linked via the imine at
N-10, we chose to link IGNs 1-5 through a carbamate at the N-
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2.8 – 3.8 IGNs per antibody, were >97% monomeric by SEC
1
and contained less than 0.5% unconjugated IGN (free drug).
2
Scheme 1. Synthesis of N-10 linked mono-reduced IGN ADCs with varying spacers.
3
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5
6
7
8
9
O
O
O
O
H
N
H
N
H
N
I
O
O
N
a, b
N
O
N
O
H
H
N
N
MeO
O
O
O
O
O
a
7
6
OH
O
I
O
O
O
H
H
N
N
MeO
N
N
b
O
O2N
N
O
H
O
H
N
OBn
OMe
N
O
O
O
OBn
OMe
e-f
c
d
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O
O
O
O
N
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OBn
OMe
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Br
8
9
10
N
MeO
N
c
O
O
O
O
O
O
O
H
H
O
O
H
H
H
H
N
N
N
N
N
N
N
N
N
N
O
N
O
H
H
H
O
O
H
O
O
O
O
O
H2
N Lys
O
O
O
O
g, h
O
O
N
N
O
O
N
N
N
N
O
O
O
O
N
N
OMe
MeO
(Ab = anti-FR)
N
N
N
N
OMe MeO
OMe MeO
O
O
13a
14a
O
O
O
O
11a
12a
(Ab = anti-EGFR)
O
O
H
H
O
O
H
H
N
N
N
N
N
N
N
N
H
H
H
O
O
H
O
O
O
O
HN
O
O
O
O
O
N
N
N
N
O
O
O
O
N
N
N
N
OMe
MeO
OMe
MeO
O
O
O
O
13b
14b
(Ab = anti-FR)
(Ab = anti-EGFR)
13c
14c
(Ab = anti-FR)
(Ab = anti-EGFR)
Reagents and Conditions: (a) TFA, water, 84%, (b) EEDQ, DCM, (4-aminophenyl)methanol, 31%, (c) DIPEA, DCM, 4-nitrobenzyl
carbonochloridate, 71%, (d) 7, LiHMDS, THF, 0 oC, ~35%, (e) Pd/C, H2, CH3OH, 77%, (f) a, K2CO3, DMA, 30%, (g) LiOH, THF/water,
(h) N-hydroxysuccinimide, EDC, DCM, 65% over two steps.
Table 2. In Vitro Potency and tolerability of IGN ADCs
ADCs was found to correlate with the in vitro cytotoxicity of
the expected catabolites, and with the antigen level. Anti-FR
ADCs 13b-c had comparable in vitro potency in both cell lines
whereas ADC 13a, incorporating catabolite 5, was considerably
less potent. Additionally, anti-FR 13a was found to be
ineffective in the lower antigen expressing cell line T47D,
indicating the need for high antigen expression to be active.
Further evidence for this correlation was seen with the anti-
EGFR ADCs 14a-c when tested against three cells lines with
varying antigen expression. Again, the correlation of ADC
potency to the cytotoxicity of the catabolite and antigen
expression level was similar to that observed for the anti-
FR ADCs 13a-c. In all cases the ADCs were found to be
antigen-specific as the addition of unconjugated Ab (1 M)
abolished all activity (see supplementary Table S1).
ADC IC50 (pM)
KB
T47D
Pilot MTD
mg/kgb
>82
Anti-FR
ADC
DAR
2.9
(3,000k)a
100
(100k) a
>3000
13a
13b
13c
2.8
3.3
10
20
100
300
~40
~20
ADC IC50 (pM)
Anti-EGFR
ADC
HSC2
(1,000k) a
300
KB
(158k) a
3000
NCI-H2110
(26k) a
DAR
3.4
14a
>3000
In addition to the impact of catabolite potency on ADC activity,
we were also interested in determining its effect on bystander
activity. We selected two cell lines, MDA-MB-468 (EGFR-
positive) and Namalwa (EGFR-negative) to use in a bystander
killing assay. In vitro, only the EGFR-positive MDA-MB-468
cells were sensitive to the anti-EGFR ADCs 14a-c,
demonstrating antigen specific cell killing by the ADC. A co-
culture of MDA-MB-468 and Namalwa cells, representing the
capacity of an ADC to kill tumors with heterogeneous antigen
expression, was treated with ADCs 14a-c (see supplementary
Table S2). All three ADCs demonstrated potent bystander
activity in this study, indicating that the direct killing of antigen
14b
14c
3.2
3.8
6
6
30
300
100
40
aNumber of antibody molecules bound per cell. Dose based on
b
mg/kg ADC.
The anti-FR ADCs 13a-c were evaluated against two cell lines
which expressed either a high (KB) or low number (T47D) of
folate antigen receptors. As shown in Table 2, potency of the
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positive cells led to efficient generation of catabolites that were
capable of diffusing into, and killing proximal antigen negative
cells. Thus, the in vitro potency and bystander killing ability of
these IGN ADCs were found to correlate with catabolite
potency. We then conducted a pilot tolerability study in CD-1
mice. As shown in Table 2, the MTD (maximum tolerated dose)
of the anti-FR ADCs 13a-c correlated with the observed in
vitro activity, with the least potent ADC (13a) having the
highest MTD.
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O
NH2
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O
H
H
N
O
N
N
H
O
O
O
HN
O
O
N
O
HN
N
N
O
O
N
OMe
MeO
O
O
anti-FR 15

Figure 5. In vivo antitumor activity of the anti-FR 15 ADC in
Figure 4. Structure of IGN ADC anti-FR 15.
an ovarian cancer OV90 xenograft.
Encouraged by the generation of a series of N-10 amine linked
IGN ADCs which displayed a range of targeted in vitro potency,
strong bystander activity and in vivo tolerability we next
evaluated antitumor activity. For this study, we chose ADC 15,
which has a Gln-Leu linker, as this dipeptide was shown to be
cleaved very efficiently by proteases in an in vitro screening
assay. Anti-FR ADC 15 (Figure 4), showed identical in vitro
potency and tolerability to ADC 13c, that has an Ala-Val
dipeptide (see Supplementary Table S3). SCID mice bearing
OV90 subcutaneous ovarian xenografts which express folate
receptor alpha heterogeneously, at a very low level (H-Score
40)16 were treated with ADC 15 (DAR 3.2). As shown in Figure
5, in spite of the low antigen expression level, the ADC was
highly active, as treatment with a single i.v. dose of 0.7 mg/kg
(equivalent to 15 g/kg linked IGN 3 payload) resulted in 6/6
CRs with the higher doses (1.4 and 2.7 mg/kg) showing even
greater activity. In this low expressing OV90 tumor model the
anti-FR 15 ADC demonstrated a therapeutic index (TI),
defined as the ratio of MTD to MED (minimally effective dose),
of >30.
Figure 6. In vivo antitumor activity of the anti-EGFR 20 ADC
in a squamous cell carcinoma of the head and neck FaDu
Xenograft.
Since our goal was to develop ADCs with a wide range of
potency and tolerability, we then evaluated the anti-tumor
activity of the less potent, but better tolerated, anti-EGFR ADC
14a in a high antigen expressing model (Figure 6). Treatment
of SCID mice bearing FaDu subcutaneous SCCHN xenografts
(H-Score 225), with ADC 13a at a single i.v. dose of 3.7 mg/kg
(equivalent to 80 g/kg of the IGN 5 payload) resulted in high
activity, with 6/6 CRs, indicating that efficient antitumor
activity is achievable with a lower potency IGN payload when
the antigen expression level is properly matched.
In conclusion, we have developed a new linking strategy for
IGNs using the N-10 amine present in our monoimine
containing IGN pseudodimers. This has allowed us to explore
the impact of incorporating variable spacer units leading to the
synthesis of IGN ADCs which demonstrate a wide range of in
vitro potency that correlates with bystander activity and in vivo
tolerability. As a result, we can use this information to better
select an IGN for generating an ADC based on the needs of the
target, as demonstrated by the potent antitumor activity for the
anti-FR 15 ADC, where antigen expression is low and potent
bystander activity is needed, and the anti-EGFR 14a ADC
where the antigen expression in the xenograft model is high.
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A Novel HER2-Targeting ADC with a Novel DNA
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label, phase 1 study. Lancet Oncol. 2017, 18, 42-51.
(12) Miller, M. L.; Fishkin, N. E.; Li, W.; Whiteman, K. R.; Kovtun,
Y.; Reid, E. E.; Archer, K. E.; Maloney, E. K.; Audette, C. A.; Mayo,
M. F.; Wilhelm, A.; Modafferi, H. A.; Singh, R.; Pinkas, J.;
Goldmacher, V.; Lambert, J. M.; Chari, R. V. A New Class of
Antibody-Drug Conjugates with Potent DNA Alkylating Activity. Mol.
Cancer Ther. 2016, 15, 1870-8.
(13) Miller, M. L.; Shizuka, M.; Wilhelm, A.; Salomon, P.; Reid, E.
E.; Lanieri, L.; Sikka, S.; Maloney, E. K.; Harvey, L.; Qiu, Q.; Archer,
K. E.; Bai, C.; Vitharana, D.; Harris, L.; Singh, R.; Ponte, J. F.; Yoder,
N. C.; Kovtun, Y.; Lai, K. C.; Ab, O.; Pinkas, J.; Keating, T. A.; Chari,
R. V. J. A DNA-Interacting Payload Designed to Eliminate Cross-
Linking Improves the Therapeutic Index of Antibody-Drug Conjugates
(ADCs). Mol. Cancer Ther. 2018, 17, 650-660.
Corresponding Author
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* E-mail: michael.miller@immunogen.com
Present Address
†Pharmaron, Inc., Waltham, MA 02451. ‡Alkermes, Inc.,
Waltham, MA 02451.
Author Contributions
The manuscript was written through contributions of all authors.
All authors of submitting affiliation have given approval to the
final version of the manuscript.
Funding Sources
This study was supported by ImmunoGen, Inc.
ABBREVIATIONS
ADC, antibody-drug conjugate; PBD, pyrrolobenzodiazepine;
IGN, indolinobenzodiazepine; TMDD, target mediated drug
disposition; PAB, p-aminobenzoyl; TFA, triflouoroacetic acid;
EEDQ, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline; DCM,
dichloromethane; DIPEA, N,N-Diisopropylethylamine; THF,
tetrahydrofuran;
dimethylacetmide; NHS, N-hydroxysuccinimide ester; EDC, 1-
Ethyl-3-(3-dimethylaminopropyl)carbodiimide; SEC, size-
DMSO,
dimethylsulfoxide;
DMA,
exclusion chromatography; DAR, drug-to-antibody ratio; MTD,
maximum tolerated dose; MED, minimum effective dose; CR,
complete regression.
REFERENCES
(1) Chari, R. V.; Miller, M. L.; Widdison, W. C. Antibody-drug
conjugates: an emerging concept in cancer therapy. Angew. Chem. Int.
Ed. Engl. 2014, 53, 3796-827.
(2) Beck, A.; Goetsch, L.; Dumontet, C.; Corvaia, N. Strategies and
challenges for the next generation of antibody-drug conjugates. Nat.
Rev. Drug Discov. 2017, 16, 315-337.
(3) Polakis, P. Antibody Drug Conjugates for Cancer Therapy.
Pharmacol. Rev. 2016, 68, 3-19.
(4) Coats, S; Williams, M.; Kebble, B.; Dixit, R.; Tseng, L.; Yao,
N.; Tice, D.A.; Soria, J-C. Antibody Drug Conjugates: Future
Directions in Clinical and Translational Strategies to Improve the
Therapeutic Index. Clin. Cancer Res. 2019, doi: 10.1158/1078-
0432.CCR-19-0272.
(5) Chari, R. V. Expanding the Reach of Antibody-Drug Conjugates.
ACS Med. Chem. Lett. 2016, 7, 974-976.
(6) Sievers, E. L., Senter, P. D. Antibody-drug conjugates in cancer
therapy. Annu. Rev. Med. 2013, 64, 15-29.
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Corcoran, K. E.; Brooks, N. A.; Hartley, J. A.; Jenkins, T. C.; Patel, S.;
Guille, M. J.; Thurston, D. E. Linker Length Modulates DNA Cross-
Linking Reactivity and Cytotoxic Potency of C8/C8‘ Ether-Linked C2-
exo-Unsaturated Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) Dimers. J.
Med. Chem. 2004 , 47, 1161-1174.
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for immunohistochemical semiquantitation: validation for oestrogen
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HN
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N
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OMe
MeO
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13c
14c
(Ab = anti-FR)
(Ab = anti-EGFR)
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