Linker Immolation Determines Cell Killing Activity of Disulfide-Linked Pyrrolobenzodiazepine Antibody-Drug Conjugates

Article abstract of DOI:10.1021/acsmedchemlett.6b00233

Disulfide bonds could be valuable linkers for a variety of therapeutic applications requiring tunable cleavage between two parts of a molecule (e.g., antibody-drug conjugates). The in vitro linker immolation of β-mercaptoethyl-carbamate disulfides and DNA alkylation properties of associated payloads were investigated to understand the determinant of cell killing potency of anti-CD22 linked pyrrolobenzodiazepine (PBD-dimer) conjugates. Efficient immolation and release of a PBD-dimer with strong DNA alkylation properties were observed following disulfide cleavage of methyl- and cyclobutyl-substituted disulfide linkers. However, the analogous cyclopropyl-containing linker did not immolate, and the associated thiol-containing product was a poor DNA alkylator. As predicted from these in vitro assessments, the related anti-CD22 ADCs showed different target-dependent cell killing activities in WSU-DLCL2 and BJAB cell lines. These results demonstrate how the in vitro immolation models can be used to help design efficacious ADCs.

Full text of DOI:10.1021/acsmedchemlett.6b00233

Letter
pubs.acs.org/acsmedchemlett
Linker Immolation Determines Cell Killing Activity of Disulfide-
Linked Pyrrolobenzodiazepine Antibody−Drug Conjugates
§
,
†
‡
†
§
Donglu Zhang,* Thomas H. Pillow, Yong Ma, Josefa dela Cruz-Chuh, Katherine R. Kozak,
∥
⊥
⊥
∥
†
#
Jack D. Sadowsky, Gail D. Lewis Phillips, Jun Guo, Martine Darwish, Peter Fan, Jingtian Chen,
#
#
#
#
#
#
‡
Changrong He, Tao Wang, Hui Yao, Zijin Xu, Jinhua Chen, John Wai, Zhonghua Pei,
†
†
,‡
Cornelis E. C. A. Hop, S. Cyrus Khojasteh, and Peter S. Dragovich*
†
‡
§
∥
Drug Metabolism & Pharmacokinetics, Discovery Chemistry, Biochemical and Cellular Pharmacology, Protein Chemistry, and
⊥
Molecular Oncology, Genentech, South San Francisco, California 94080, United States
#
Wuxi Apptec, 288 Fute Zhong Road, Waigaoqiao Free Trade Zone, Shanghai 200131, China
*
S Supporting Information
ABSTRACT: Disulfide bonds could be valuable linkers for a variety of therapeutic
applications requiring tunable cleavage between two parts of a molecule (e.g., antibody−
drug conjugates). The in vitro linker immolation of β-mercaptoethyl-carbamate disulfides
and DNA alkylation properties of associated payloads were investigated to understand the
determinant of cell killing potency of anti-CD22 linked pyrrolobenzodiazepine (PBD-
dimer) conjugates. Efficient immolation and release of a PBD-dimer with strong DNA
alkylation properties were observed following disulfide cleavage of methyl- and
cyclobutyl-substituted disulfide linkers. However, the analogous cyclopropyl-containing
linker did not immolate, and the associated thiol-containing product was a poor DNA
alkylator. As predicted from these in vitro assessments, the related anti-CD22 ADCs showed different target-dependent cell
killing activities in WSU-DLCL2 and BJAB cell lines. These results demonstrate how the in vitro immolation models can be used
to help design efficacious ADCs.
KEYWORDS: Antibody−drug conjugate, cancer, disulfide linker, linker immolation, pyrrolobenzodiazepine
1
5
ntibody−drug conjugate (ADC) technology can enable
release of associated disulfide linker ADCs in cells. This linker
A
the use of very toxic compounds as potential cancer
is traceless in that, following internalization and lysosomal mAb
16
therapeutics. Such potent cytotoxins include maytansinoid and
auristatin-based antimitotic agents along with DNA alkylating
entities derived from pyrrolo[2,1-c][1,4]benzodiazepine dimers
proteolysis, it releases the free drug (payload) in a two-step
process involving (1) disulfide cleavage of a thiol-cysteine
adduct and (2) subsequent immolation of the resulting thiolate
intermediate (Figure 1A). The thiolate could also capture a
proton to form a thiol that is also set up for immolation. For
payloads that require separation from the linker to properly
exert their biological effects, the rate and extent of immolation/
release will likely impact the efficacy of corresponding ADCs. In
order to further optimize the disulfide stability and release
kinetics through substitution adjacent to the linker sulfur, we
have studied immolation/release of model compounds
containing various β-mercaptoethyl-carbamate disulfide linkers
with substitution of proton, methyl, ethyl, dimethyl, cyclo-
propyl, cyclobutyl, cyclopentanyl, and cyclohexanyl groups. In
this report, we provide detailed in vitro studies of immolation/
release parameters associated with PBD-dimer-containing β-
mercaptoethyl-carbamate disulfide linkers with methyl-, cyclo-
propyl-, and cyclobutyl-substitutions that showed representa-
1
−6
(
PBD dimers).
Several challenges are frequently encoun-
tered in the design of ADCs including (1) preventing the
premature release of the cytotoxin during in vivo circulation
and (2) ensuring that a biologically active form of the cytotoxic
payload is released at the desired site of action at an adequate
rate. The complex structure of an ADC together with its
associated unique dispositional and functional properties
requires careful design of every component of the molecule
including antibody, conjugation site, linker structure, and a
7
−10
small molecule drug (payload).
The linker determines the
mechanism and rate of payload release, which both affect cell
10,11
killing activity, and is thus a critical part of an ADC.
Cleavable linkers commonly employed in ADC design include
the valine-citrulline para-aminobenzyl carbamate (Val-Cit-
12
PABC) present in brentuximab vedotin (Adcetris) and
disulfide moieties contained in multiple maytansinoid-derived
1
3
conjugates as well as in gemtuzumab ozogamicin (Mylo-
1
4
Special Issue: Antibody-Drug Conjugates and Bioconjugates
targ). Recently, Pillow et al. reported a self-immolating
disulfide linker (β-mercaptoethyl-carbamate, −SCH(R)-
Received: June 9, 2016
Accepted: August 22, 2016
CH OCO−) that can be directly attached to antibody cysteine
2
residues and therefore decouples in vivo stability from payload
©
XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.6b00233
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Figure 1. (A) Catabolism and payload release mechanism of a disulfide-linked ADC. (B) Disulfide cleavage and immolation of PBD disulfide linker
drugs in the presence of cysteine or GSH,. (C) Structures of other PBD analogues and ADCs used in this study. Note: Thiol-cysteine adducts 1a, 2a,
and 7a are also proposed intermediates following internalization and lysosomal proteolysis of their corresponding ADCs 13−1, 14−1, and 15−1 in
cells; the corresponding thiolates of 1d, 2d, or 7d are proposed intermediates of disulfides 1a, 2a, or 7a following disulfide cleavage; the thiolates 1c,
2
c, or 7c could immolate directly to liberate the payload 4 or capture a proton for form thiols 1d, 2d, or 7d; the mechanisms for disulfide cleavage
and immolation are similar between in cells and in vitro (dotted-line area). The immolation did not start until the disulfide cleavage therefore was
not affected by the leaving groups such as cysteine or nitro-pyridyl group; the corresponding thiol-GSH adducts 1b, 2b, and 7b were identified as
major components from incubations with 1, 2, and 7 in the presence of 30 μM GSH.
tive immolation patterns. In addition, we characterized the in
vitro oligonucleotide (DNA Oligo) binding/alkylation proper-
ties of several related PBD-containing payloads. We sub-
sequently correlated these in vitro measurements with the cell
killing potency of related ADCs.
have a pronounced negative effect on payload efficacy given
that the C11−N10 imine is required for DNA alkylation.
To simplify the studies, we initially investigated disulfide
cleavage and immolation using unconjugated small molecule
model systems. Accordingly, we performed the experiments
with para-nitropyridyl disulfide-containing linker drug ana-
logues 1 and 2 (that were subsequently used to prepare
antibody conjugates) (Figure 1B) in the presence of cysteine
(0.2 and 0.03 mM) and glutathione (GSH, 4 and 0.03 mM)
with the thiol concentrations mimicking those in plasma and
cancer cells (unpublished data). Under these conditions, the
para-nitropyridyl disulfide moieties in these molecules under-
went rapid disulfide cleavage with a half-life of minutes (Figure
2, Table S1). Analysis by LC/UV/MS showed that reduction of
1 and 2 gave different products: PBD dimer 4 was obtained as a
major product from methyl-containing 1, while the thiol 2d was
formed from cyclopropyl-containing 2 (Figure 2A1,A2,C1,C2;
Tables S1 and S2) when an excess level of a reducing thiol was
present (0.2 mM cysteine or 4 mM GSH). In contrast, when a
relatively low concentration of cysteine (0.03 mM) was used
(Figure 2B1,B2), thiol-cysteine adducts 1a and 2a were the
major and stable products in the 24 h incubations due to
consumption of the reducing thiol. Similarly, thiol-GSH
The pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers com-
prise an important class of DNA alkylators for ADCs and PBD-
derived payloads have been used recently in several ADCs
4
,6,17
entering clinic trials.
The PBD dimer molecular structure
can influence DNA sequence recognition given that such
compounds contain a chiral C11a(S)-center that forms an
appropriate conformation to fit in the minor groove of
23
1
8−22
DNA.
The C10−N11 imine moiety in PBDs forms a
covalent adduct with a guanine residue in the minor groove of
double-stranded DNA with preferred 5′-pu-GA(A/T)TC-Py-3′
sequences (bold = covalent modification site) to produce inter/
1
8,20
intrastrand cross-links or monoalkylated products.
One
particularly attractive way to connect PBD-dimer payloads to
antibodies involves attachment of a self-immolative linker to the
4
N10 nitrogen, thereby blocking the C11−N10 imine in the
intact conjugates, but generating the C11−N10 imine upon
linker cleavage (Figure 1B). In this new linker/payload design,
insufficient release of a self-immolative spacer is expected to
B
DOI: 10.1021/acsmedchemlett.6b00233
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ACS Medicinal Chemistry Letters
Letter
Figure 2. Relative abundance of disulfide cleavage and immolation products over time. Products were monitored by LC/MS from incubations of
linker drugs 1, 2, and 7 in the presence of cysteine (0.2 mM, A1, A2, A7; and 0.03 mM, B1, B2, B7) or glutathione (4 mM, C1, C2, C7; and 0.03
mM, D1, D2, D7) at pH 7.0 and 37 °C. P = starting linker drugs 1 (methyl-), 2 (cyclopropyl-), and 7 (cyclobutyl-). Payload = PBD dimer 4. Thiol-
GSH or -cysteine = 1a or 1b (methyl-), 2a or 2b (cyclopropyl-), or 7a or 7b (cyclobutyl). Thiol = 1d, 2d, or 7d.
adducts 1b and 2b were observed as the major products when 1
and 2 were exposed to a low concentration of GSH (0.03 mM)
for the same time period (Figure 2D1,D2). These cleavage
reactions also formed the para-nitropyridyl thiol 3 as a
companion byproduct (Figure 1B). Interestingly, reversible
disulfide exchange was observed between para-nitropyridyl
thiol 3 and cyclopropyl-containing thiol 2d with a comparable
cyclopropyl-containing thiol 2d produced from disulfide 2 did
not appreciably immolate and appeared to be stable.
Furthermore, the thiirane 5 was detected via LC/MS in the
cleavage experiment utilizing 1, while the corresponding
thiirane 6 was not identified from studies employing 2 (Figure
1B). Subsequently, the cyclobutyl analogue 7 was prepared.
Both 7 and its thiol-cysteine adduct 7a underwent rapid
disulfide cleavage followed by efficient immolation of its
thiolate 7c and thiol 7d to form thiirane 8 and PBD dimer 4
(Figure 2A7,B7, C7). The cyclobutyl-substituted disulfide in 7
is more stable than the methyl analogue in 1 since greater
amounts of starting material 7 were observed relative to 1 in the
incubation with a lower concentration of glutathione (0.03
mM) (Figure 2D1,D7). Immolation of cyclobutyl-containing
thiol 7d may also be slightly more efficient than the methyl-
containing thiol 1d as less 7d was detected than 1d under all
incubation conditions (Table S1). Nonimmolation of the
cyclopropyl linker compared to efficient immolation of the
cyclobutyl or methyl-containing linkers is likely due to multiple
factors. For example, the bond angles associated with the
cyclopropyl ring are likely to result in increased ring strain in
the fused dicyclopropyl thiirane 6 relative to thiiranes 5 and 8
pK value (4.7−4.8) at 0.03 mM GSH concentration (Figure
a
2D2). The observation of intermediates 1a and 1d (albeit at
low levels) was consistent with the expected mechanism for
formation of PBD dimer 4 from 1 that involves (1) cysteine
attack on the 2-mercaptoethyl-derived disulfide sulfur atom to
form 1a, (2) second cysteine attack on the less hindered sulfur
atom of the resulting thiol-cysteine disulfide to afford a thiolate
intermediate 1c, and (3) the thiolate 1c immolates to liberate
PBD dimer 4 or captures a proton to form the corresponding
thiol 1d (Figure 1B). The depicted regiochemistry of initial
cysteine attack on 1 is likely influenced by both disulfide
electronics and the acidic nitropyridyl thiolate pK (i.e., this
a
24,25
fragment is a good leaving group).
The thiol-cysteine
disulfide 1a is identical to the proposed intermediate produced
by intracellular catabolism of the corresponding ADC following
15
26
internalization and lysosomal proteolysis in cells. As shown in
Figure 2A1,C1 and Table S1, the methyl-containing thiol
intermediate 1d could be detected by LC/MS at low levels in
the initial phase of the incubations but immolated quickly to
afford the PBD dimer 4. However, the corresponding
and thereby impede the desired immolation reaction. The
cyclopropyl ring may also exert an electronic effect (p-orbital
character) on the thiolate intermediate, which reduces its
nucleophilicity and therefore its ability to cyclize. Consistent
with this possibility, a significantly lower pK value (more
a
C
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ACS Medicinal Chemistry Letters
Letter
acidic) was measured for the cyclopropyl-containing thiol 9
relative to that measured for the cyclobutyl-containing thiol 10
associated comments). However, the cyclopropyl-containing
conjugate 14-1 was significantly (>50-fold) weaker than 13-1
and 15-1 in these experiments and also exhibited almost no
potency differences from the corresponding nontarget control
conjugate (14-2). These results were consistent with the in
vitro data depicted in Figures 2 and S1, which illustrate the
inability of the cyclopropyl-containing thiol 2d to both
efficiently alkylate DNA Oligos and to release PBD dimer 4
via immolation. The ADC cell data were also consistent with
the efficient release of a potent DNA alkylating agent (di-imine
4) from in vitro model systems related to conjugates 13-1 and
15-1 (Figure 2, Table S1). The similarity in BJAB and WSU
EC50 values exhibited by these conjugates is in agreement with
the potent antiproliferation activity (approximately 20 pM)
displayed by the released PBD dimer 4 against both cell lines.
In the studies described above, the similar cell killing potency
exhibited by conjugates containing the cyclobutyl- and methyl-
substituted disulfide linkers closely paralleled the disulfide
stability, immolation, payload release, and DNA binding
characteristics observed in vitro with the unconjugated linker
drugs 1 and 7. More importantly, cyclopropyl substitution
prevented linker immolation in vitro following disulfide
cleavage, and the resulting thiol product 2d was also shown
to poorly alkylate designed DNA oligos. These in vitro results,
collectively, explained the poor cell killing activity exhibited by
an ADC containing the cyclopropyl-disulfide linker. These
correlations indicate that the described in vitro assessments can
be good predictors of ADC efficacy, and suggest their future
use in routine profiling of unconjugated payloads, model linker
drugs, and ADC conjugates prior to conducting in vivo studies.
Collectively, these data also illustrate the value of conducting
detailed in vitro experiments to understand the ability of a given
ADC linker to release an attached payload coupled with the
assessment of biological activity of such released entities to
support design of efficacious ADCs.
(
4.8 versus 9.6, respectively). Collectively, these results
demonstrate that minor structural modifications to the disulfide
linker can lead to vast differences in the corresponding
immolation and release efficiencies.
To complement the linker cleavage and immolation studies,
we used a simple in vitro model based on known PBD
interactions with DNA Oligos of various lengths and sequences
to assess and compare the potential for PBD-containing
20,27
payloads to alkylate DNA.
As shown in Figure S1, both
bis-imine-containing PBD dimer 4 and the corresponding PBD
20
monomer 11 extensively alkylated the tested DNA Oligos in
this in vitro assessment (Figures S1B,C). In contrast, the
cyclopropyl-containing molecule 2d alkylated the DNA Oligos
at a minimal level that was similar to that observed for the
negative control 12 (the absence of reactive imine moieties in
2
d prevents it from alkylating DNA). This result suggests that
the structural modification by the cyclopropyl-moiety in 7
prevented the PBD analogue to fit in the DNA minor groove
for alkylation although there is still one imine functionality in
2
d. Various adducts were formed between different alkylators
and the DNA Oligos. As expected, the PBD dimer 4 mainly
formed an interstrand cross-link in which the two imine
1
9
moieties present in 4 separately reacted with guanines in each
strand of the double-strand DNA Oligo (Figure S1B, insert). In
contrast, the PBD monomer 11 formed two types of adducts by
independently reacting with the guanine residue in each strand
of the DNA Oligo (Figure S1C, insert). The described adduct
1
formation is consistent with observations by others in which H
NMR methods were used to demonstrate similar binding of
27
PBD compounds in the DNA minor groove. The level of
DNA Oligo alkylation by PBD dimer 4 appears to correlate
with its potent cell-killing activity in BJAB and WSU-DLCL2
(
IC50 = ∼20 pM). The inability of compound 2d to alkylate
DNA Oligos predicted that ADCs containing the cyclopropyl
linker would likely not afford potent cell-killing activity.
ASSOCIATED CONTENT
Supporting Information
■
*
S
We next tested the effects of immolation on cell-killing
activities of related ADCs. Anti-CD22 conjugates (LC-K149C-
anti-CD22-PBD-dimer) 13-1 (methyl-), 14-1 (cyclopropyl-),
and 15-1 (cyclobutyl-) and the corresponding control
conjugates (LC-K149C-anti-NaPi2b-PBD-dimer) 13-2, 14-2,
and 15-2 were prepared from 1, 2, and 7. The CD22 antigen
was chosen for our ADC design because of its high expression
on cancers of B-cell origin and relatively low prevalence on
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.6b00233.
Two tables, two figures, materials and methods, and
synthesis of all compounds used in this study (PDF)
AUTHOR INFORMATION
Corresponding Authors
E-mail: zhang.donglu@gene.com.
E-mail: dragovich.peter@gene.com.
■
28
non-B cell-related normal cells and tissues. The methyl- and
cyclobutyl-containing conjugates 13-1 and 15-1 showed potent,
target-dependent cell-killing activities in two CD22-expressing
cell lines (WSU-DLCL2 and BJAB, Table 1, Figure S2, and
*
*
Notes
The authors declare no competing financial interest.
Table 1. Cell-Killing Activities of Methyl-, Cyclopropyl, and
Cyclobutyl-Containing Conjugates 13-1, 14-1, and 15-1 in
CD22-Expressing BJAB and WSU-DLCL2 Cell Cultures
ACKNOWLEDGMENTS
■
The authors would like to thank Drs. Philip Howard, Luke
Masterson, and Stephen Gregson from Spirogen, and Leanna
Staben and Drs. John Flygare and Rebecca Rowntree from
Genentech for their technical contributions. The research of the
manuscript was supported by Genentech.
IC50 (nM)
compd
BJAB
WSU-DLCL2
1
1
1
1
1
1
3-1, methyl-CD22
1.16 ± 0.04
6.13 ± 0.03
85.0 ± 0.06
87.8 ± 0.14
0.47 ± 0.04
3.84 ± 0.04
1.19 ± 0.11
13.9 ± 0.02
95.7 ± 0.06
125 ± 0.04
1.50 ± 0.08
8.95 ± 0.05
3-2, methyl-NaPi
4-1, cyclopropyl-CD22
4-2, cyclopropyl-NaPi
5-1, cyclobutyl-CD22
5-2, cyclobutyl-NaPi
ABBREVIATIONS
■
ADC, antibody−drug conjugate; DNA Oligo, deoxyribonucleic
acid oligonucleotide; GSH, glutathione; LC/MS, high perform-
ance liquid chromatography tandem mass spectrometry
D
DOI: 10.1021/acsmedchemlett.6b00233
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(
14) Ricart, A. D. Antibody-drug conjugates of calicheamicin
REFERENCES
■
derivative: Gemtuzumab ozogamicin and inotuzumab ozogamicin.
Clin. Cancer Res. 2011, 17, 6417−6427.
(
Xu, K.; He, J.; Bhakta, S.; Ohri, R.; Kozak, K. R.; Flygare, J. A.;
Junutula, J. R. Decoupling stability and release in disulfide bonds with
antibody-small molecule conjugates. Chem. Sci. 2016, DOI: 10.1039/
C6SC01831A.
(
1) LoRusso, P. M.; Weiss, D.; Guardino, E.; Girish, S.; Sliwkowski,
M. X. Trastuzumab emtansine: a unique antibody-drug conjugate in
development for human epidermal growth factor receptor 2-positive
cancer. Clin. Cancer Res. 2011, 17, 6437−6447.
15) Pillow, T.; Sadowsky, J.; Zhang, D.; Yu, S. F.; Del Rosario, G.;
(
2) Senter, P. D.; Sievers, E. L. The discovery and development of
brentuximab vedotin for use in relapsed Hodgkin lymphoma and
systemic anaplastic large cell lymphoma. Nat. Biotechnol. 2012, 30,
(
16) Erickson, H. K.; Park, P. U.; Widdison, W. C.; Kovtun, Y. V.;
6
(
31−637.
Garrett, L. M.; Hoffman, K.; Lutz, R. J.; Goldmacher, V. S.; Blattler, W.
̈
3) Chari, R. V. J.; Miller, M. L.; Widdison, W. C. Antibody-drug
A. Antibody-maytansinoid conjugates are activated in targeted cancer
cells by lysosomal degradation and linker-dependent intracellular
processing. Cancer Res. 2006, 66, 4426−4433.
conjugates: An emerging concept in cancer therapy. Angew. Chem., Int.
Ed. 2014, 53, 3796−3827.
4) Saunders, L. R.; Bankovich, A. J.; Anderson, W. C.; Aujay, M. A.;
(
(
17) Kung Sutherland, M. S.; Walter, R. B.; Jeffrey, S. C.; Burke, P. J.;
Bheddah, S.; Black, K.; Desai, R.; Escarpe, P. A.; Hampl, J.; Laysang,
A.; Liu, D.; Lopez-Molina, J.; Milton, M.; Park, A.; Pysz, M. A.; Shao,
H.; Slingerland, B.; Torgov, M.; Williams, S. A.; Foord, O.; Howard,
P.; Jassem, J.; Badzio, A.; Czapiewski, P.; Harpole, D. H.; Dowlati, A.;
Massion, P. P.; Travis, W. D.; Pietanza, M. C.; Poirier, J. T.; Rudin, C.
M.; Stull, R. A.; Dylla, S. J. A DLL3-targeted antibody-drug conjugate
eradicates high-grade pulmonary neuroendocrine tumor-initiating cells
in vivo. Sci. Transl. Med. 2015, 7, 302ra136.
Yu, C.; Kostner, H.; Stone, I.; Ryan, M. C.; Sussman, D.; Lyon, R. P.;
Zeng, W.; Harrington, K. H.; Klussman, K.; Westendorf, L.; Meyer, D.;
Bernstein, I. D.; Senter, P. D.; Benjamin, D. R.; Drachman, J. G.;
McEarchern, J. A. SGN-CD33A: a novel CD33-targeting antibody-
drug conjugate using a pyrrolobenzodiazepine dimer is active in
models of drug-resistant AML. Blood 2013, 122, 1455−1463.
(18) Jenkins, T. C.; Hurley, L. H.; Neidle, S.; Thurston, D. E.
Structure of a covalent DNA minor groove adduct with a
pyrrolobenzodiazepine dimer: evidence for sequence-specific inter-
strand cross-linking. J. Med. Chem. 1994, 37, 4529−4537.
(19) Rahman, K. M.; James, C. H.; Thurston, D. E. Effect of base
sequence on the DNA cross-linking properties of pyrrolobenzodiaze-
pine (PBD) dimers. Nucleic Acids Res. 2011, 39, 5800−5812.
(20) Rahman, K. M.; Thompson, A. S.; James, C. H.;
Narayanaswamy, M.; Thurston, D. E. The pyrrolobenzodiazepine
dimer SJG-136 forms sequence-dependent intrastrand DNA cross-
links and monoalkylated adducts in addition to interstrand cross-links.
J. Am. Chem. Soc. 2009, 131, 13756−13766.
(
5) Sutherland, M. S. K.; Sanderson, R. J.; Gordon, K. A.; Andreyka,
J.; Cerveny, C. G.; Yu, C.; Lewis, T. S.; Meyer, D. L.; Zabinski, R. F.;
Doronina, S. O.; Senter, P. D.; Law, C. L.; Wahl, A. F. Lysosomal
trafficking and cysteine protease metabolism confer target-specific
cytotoxicity by peptide-linked anti-CD30-auristatin conjugates. J. Biol.
Chem. 2006, 281, 10540−10547.
(
6) Jeffrey, S. C.; Burke, P. J.; Lyon, R. P.; Meyer, D. W.; Sussman,
D.; Anderson, M.; Hunter, J. H.; Leiske, C. I.; Miyamoto, J. B.;
Nicholas, N. D.; Okeley, N. M.; Sanderson, R. J.; Stone, I. J.; Zeng, W.;
Gregson, S. J.; Masterson, L.; Tiberghien, A. C.; Howard, P. W.;
Thurston, D. E.; Law, C. L.; Senter, P. D. A potent anti-CD70
antibody-drug conjugate combining a dimeric pyrrolobenzodiazepine
drug with site-specific conjugation technology. Bioconjugate Chem.
(21) Petrusek, R. L.; Anderson, G. L.; Garner, T. F.; Fannin, Q. L.;
Kaplan, D. J.; Zimmer, S. G.; Hurley, L. H. Pyrrol[1,4]benzodiazepine
antibiotics. Proposed structures and characteristics of the in vitro
deoxyribonucleic acid adducts of anthramycin, tomaymycin, sibir-
omycin, and neothramycins A and B. Biochemistry 1981, 20, 1111−
1119.
2
(
013, 24, 1256−1263.
7) Junutula, J. R.; Raab, H.; Clark, S.; Bhakta, S.; Leipold, D. D.;
Weir, S.; Chen, Y.; Simpson, M.; Tsai, S. P.; Dennis, M. S.; Lu, Y.;
Meng, Y. G.; Ng, C.; Yang, J.; Lee, C. C.; Duenas, E.; Gorrell, J.; Katta,
V.; Kim, A.; McDorman, K.; Flagella, K.; Venook, R.; Ross, S.;
Spencer, S. D.; Lee Wong, W.; Lowman, H. B.; Vandlen, R.;
Sliwkowski, M. X.; Scheller, R. H.; Polakis, P.; Mallet, W. Site-specific
conjugation of a cytotoxic drug to an antibody improves the
therapeutic index. Nat. Biotechnol. 2008, 26, 925−932.
(
22) Hartley, J. A. The development of pyrrolobenzodiazepines as
antitumour agents. Expert Opin. Invest. Drugs 2011, 20, 733−744.
23) Brulisauer, L.; Gauthier, M. A.; Leroux, J.-C. J. Disulfide-
(
̈
containing parenteral delivery systems and their redox-biological fate. J.
Controlled Release 2014, 195, 147−154.
(24) Singh, R.; Whitesides, G. M. Thiol-disulfide interchange. Suppl.
S. Chem. Sulfur-containing Funct. Groups 1993, 633−658.
(
8) Junutula, J. R.; Flagella, K. M.; Graham, R. A.; Parsons, K. L.;
(25) Freter, R.; Pohl, E. R.; Wilson, J. M.; Hupe, D. J. Role of the
Raab, H.; Bhakta, S.; Nguyen, T.; Dugger, D. L.; Li, G.; Phillips, G. D.
L.; Hiraragi, H.; Fuji, R. N.; Tibbitts, J.; Spencer, S. D.; Scheller, R. H.;
Polakis, P.; Sliwkowski, M. X.; Way, D. N. A.; Francisco, S. S.
Engineered thio-trastuzumab-DM1 conjugate with an improved
therapeutic index to target HER2-positive breast cancer. Clin. Cancer
Res. 2010, 16, 2−5.
central thiol in determining rates of the thiol-disulfide interchange
reaction. J. Org. Chem. 1979, 44, 1771−1774.
(26) Jung, M. E.; Piizzi, G. gem-disubstituent effect: theoretical basis
and synthetic applications. Chem. Rev. 2005, 105, 1735−1766.
(27) Thurston, D. E.; Vassoler, H.; Jackson, P. J.; James, C. H.;
Rahman, K. M. Effect of hairpin loop structure on reactivity, sequence
preference and adduct orientation of a DNA-interactive pyrrolo[2,1-
c][1,4]benzodiazepine (PBD) antitumour agent. Org. Biomol. Chem.
(
9) Erickson, H. K.; Lambert, J. M. ADME of antibody-maytansinoid
conjugates. AAPS J. 2012, 14, 799−805.
10) McCombs, J. R.; Owen, S. C. Antibody drug conjugates: design
and selection of linker, payload and conjugation chemistry. AAPS J.
015, 17, 339−351.
11) Jain, N.; Smith, S. W.; Ghone, S.; Tomczuk, B. Current ADC
Linker Chemistry. Pharm. Res. 2015, 32, 3526−3540.
12) Doronina, S. O.; Toki, B. E.; Torgov, M. Y.; Mendelsohn, B. A.;
(
2
(
009, 13, 4031−4040.
28) Polson, A. G.; Williams, M.; Gray, A. M.; Fuji, R. N.; Poon, K.
2
(
A.; McBride, J.; Raab, H.; Januario, T.; Go, M.; Lau, J.; Yu, S.-F.; Du,
C.; Fuh, F.; Tan, C.; Wu, Y.; Liang, W.-C.; Prabhu, S.; Stephan, J.-P.;
Hongo, J.; Dere, R. C.; Deng, R.; Cullen, M.; de Tute, R.; Bennett, F.;
Rawstron, A.; Jack, A.; Ebens, A. Anti-CD22-MCC-DM1: an antibody-
drug conjugate with a stable linker for the treatment of non-Hodgkin’s
lymphoma. Leukemia 2010, 24, 1566−1573.
(
Cerveny, C. G.; Chace, D. F.; DeBlanc, R. L.; Gearing, R. P.; Bovee, T.
D.; Siegall, C. B.; Francisco, J. A.; Wahl, A. F.; Meyer, D. L.; Senter, P.
D. Development of potent monoclonal antibody auristatin conjugates
for cancer therapy. Nat. Biotechnol. 2003, 21, 778−784.
(
13) Kellogg, B. A.; Garrett, L.; Kovtun, Y.; Lai, K. C.; Leece, B.;
Miller, M.; Payne, G.; Steeves, R.; Whiteman, K. R.; Widdison, W.;
Xie, H.; Singh, R.; Chari, R. V. J.; Lambert, J. M.; Lutz, R. J. Disulfide-
linked antibody-maytansinoid conjugates: Optimization of in vivo
activity by varying the steric hindrance at carbon atoms adjacent to the
disulfide linkage. Bioconjugate Chem. 2011, 22, 717−727.
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DOI: 10.1021/acsmedchemlett.6b00233
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX