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Antibody−Drug Conjugates (ADCs) Derived from Interchain Cysteine
Cross-Linking Demonstrate Improved Homogeneity and Other
Pharmacological Properties over Conventional Heterogeneous ADCs
Christopher R. Behrens, Edward H. Ha, Lawrence L. Chinn, Simeon Bowers, Gary Probst,
Maureen Fitch-Bruhns, Jorge Monteon, Amanda Valdiosera, Abel Bermudez, Sindy Liao-Chan,
Tiffany Wong, Jonathan Melnick, Jan-Willem Theunissen, Mark R. Flory, Derrick Houser,
Kristy Venstrom, Zoia Levashova, Paul Sauer, Thi-Sau Migone, Edward H. van der Horst,
Randall L. Halcomb, and David Y. Jackson*
Igenica Biotherapeutics, 863A Mitten Road, Suite 100B, Burlingame, California 94010, United States
S Supporting Information
ABSTRACT: Conventional antibody−drug conjugates (ADCs)
are heterogeneous mixtures of chemically distinct molecules that
vary in both drugs/antibody (DAR) and conjugation sites.
Suboptimal properties of heterogeneous ADCs have led to new
site-specific conjugation methods for improving ADC homogeneity. Most site-specific methods require extensive antibody
engineering to identify optimal conjugation sites and introduce
unique functional groups for conjugation with appropriately
modified linkers. Alternative nonrecombinant methods have
emerged in which bifunctional linkers are utilized to cross-link antibody interchain cysteines and afford ADCs containing
four drugs/antibody. Although these methods have been shown to improve ADC homogeneity and stability in vitro, their effect
on the pharmacological properties of ADCs in vivo is unknown. In order to determine the relative impact of interchain cysteine
cross-linking on the therapeutic window and other properties of ADCs in vivo, we synthesized a derivative of the known ADC
payload, MC-MMAF, that contains a bifunctional dibromomaleimide (DBM) linker instead of a conventional maleimide (MC)
linker. The DBM-MMAF derivative was conjugated to trastuzumab and a novel anti-CD98 antibody to afford ADCs containing
predominantly four drugs/antibody. The pharmacological properties of the resulting cross-linked ADCs were compared with
analogous heterogeneous ADCs derived from conventional linkers. The results demonstrate that DBM linkers can be applied
directly to native antibodies, without antibody engineering, to yield highly homogeneous ADCs via cysteine cross-linking. The
resulting ADCs demonstrate improved pharmacokinetics, superior efficacy, and reduced toxicity in vivo compared to analogous
conventional heterogeneous ADCs.
KEYWORDS: antibody−drug conjugate, ADC, homogeneous, bifunctional, linker, site-specific, conjugation, maleimide,
dibromomaleimide, MMAF, auristatin, interchain, disulfide, DAR, hinge, cysteine, trastuzumab, Her2, CD98
Antibody−drug conjugates (ADCs) are a promising new class
of targeted therapeutic agents for treatment of cancer.1,2 Most
ADCs are synthesized by conjugating a cytotoxic compound or
“payload” to a tumor specific monoclonal antibody. The
payloads are conjugated using amino or sulfhydryl specific
linkers that react selectively with lysines or cysteines on the
antibody surface. A typical antibody contains over 50 lysines
and eight interchain cysteines as potential conjugation sites.
The optimal DAR (drugs/antibody ratio) for most ADCs,
however, ranges from two to four drugs/antibody.3 ADCs with
suboptimal DARs are prone to aggregation, poor solubility, and
instability, which often lead to increased toxicity and/or
inadequate efficacy in vivo.4 The discrepancy between the
number of potential conjugation sites and the desired DAR,
combined with the use of linkers that lack site-specificity, results
© 2015 American Chemical Society
in heterogeneous ADCs that vary in both DAR and the
conjugation sites.5 Consequently, most of the ADCs in clinical
development for cancer indications contain dozens or more of
chemically distinct ADC molecules, each with unique
pharmacological properties.6,7
Conjugation through antibody cysteines minimizes ADC
heterogeneity relative to lysine conjugation because there are
fewer potential conjugation sites.8 The process typically
involves partial reduction of four antibody interchain disulfide
bonds to generate up to eight reactive cysteine thiol groups,
followed by conjugation of payloads containing thiol-specific
June 1, 2015
September 18, 2015
September 22, 2015
September 22, 2015
DOI: 10.1021/acs.molpharmaceut.5b00432
Mol. Pharmaceutics 2015, 12, 3986−3998
Molecular Pharmaceutics
maleimide linkers.9 The resulting ADCs are composed of
dozens of chemically distinct molecules with DARs ranging
from zero to eight payloads per antibody. The maleimide
linkers typically used for cysteine conjugation result in thiosuccinimide linkages between the payload and the antibody
known to undergo side reactions such as elimination or thiol
exchange, resulting in premature release of the payloads from
the ADCs.10
New site-specific conjugation methods have emerged in
order to reduce ADC heterogeneity and other undesirable
properties associated with conventional methods. Most are
recombinant methods focused on modification of the antibody
with unique functional groups to enable site-specific conjugation with orthogonally modified linkers.11 For example,
cysteine mutations have been introduced into different
antibodies to provide free thiol groups for conjugation with
payloads containing conventional thiol-specific maleimide
linkers.12,13 The process affords homogeneous ADCs containing approximately two drugs/antibody, but additional antibody
reduction/oxidation steps are required to obtain mutants
suitable for conjugation. Later studies revealed that subtle
differences in the ADC microenvironments significantly affect
linker stability, which correlates with improved efficacy.14 The
combined results indicate that ADC activity is highly
dependent upon the conjugation sites and suggest that optimal
conjugation sites are likely to be different for each antibody.
Recombinant methods using non-natural amino acids to
enable site-specific conjugation have also been reported. For
example, stop codon suppression methodology was used to
produce antibodies containing phenyl ketone side chains for
site-specific conjugation to hydroxylamine linkers.15 The
approach was later combined with cell-free antibody expression
technology to introduce unique functional groups into over one
hundred different conjugation sites in trastuzumab, an antiHer2 antibody approved for treatment of breast cancer.16 The
results were consistent with previous findings in that ADC
activity was highly dependent on the conjugation site. Antibody
expression and conjugation efficiency were also site-dependent,
suggesting that site optimization is required for each ADC.
Alternative semisynthetic methods for site-specific conjugation have been described in which recognition sequences were
engineered into different locations on antibodies for subsequent
enzymatic modification in order to create unique functional
groups for site-specific conjugation. For example, a microbial
transglutaminase recognition sequence was introduced into 90
different positions on an anti-EGFR antibody.17 Twelve
different sites were found to be suitable for conjugation based
on antibody expression and conjugation efficiencies. The
mutated antibodies were then enzymatically conjugated to an
appropriately modified payload to afford ADCs with approximately two drugs/antibody. Similar methods were used to
construct anti-CD30 ADCs with four drugs/antibody which
demonstrated an improved therapeutic index in rodent
models.18 An alternative semisynthetic approach was used to
introduce a formyl-glycine generating enzyme (FGE) recognition sequence at different sites on a tumor-specific antibody.
Treatment of the mutant antibodies with FGE resulted in sitespecific formation of aldehyde tags that were subsequently
conjugated to appropriately modified linkers.19
Nonrecombinant methods for reducing ADC heterogeneity
are being developed in order to enable existing antibodies to be
used for construction of homogeneous ADCs. For example,
Badescu and co-workers used a bis-sulfone linker designed to
react with two antibody cysteines to conjugate an auristatin
payload (MMAE) to trastuzumab and its Fab fragments.20 The
linker contained a protease cleavable self-immolative dipeptide
to facilitate payload release and a PEG spacer to improve water
solubility and reduce potential aggregation. The conjugation
process results in formation of a three-carbon bridge across
interchain cysteines to afford an ADC enriched in the fraction
containing four drugs/antibody (up to 78%). The ADCs
inhibited BT474 tumor growth in vivo at relatively high doses
(>10 mg/kg), but conventional heterogeneous ADCs were not
included in the study and the contribution of the parent
antibody (trastuzumab) to the observed efficacy was not
reported. In a follow-up study by Godwin and co-workers,
preparative hydrophobic interaction chromatography (HIC)
was used to isolate cysteine bridged ADCs with defined DARs
ranging from one to four drugs/antibody.21 ADC activity
correlated with drug loading, but the potency relative to
conventional heterogeneous ADCs was not reported. Nonetheless, the results suggested that appropriately designed
bifunctional linkers could be used to improve ADC
homogeneity and other pharmacological properties without
antibody engineering.
Disubstituted maleimides were previously shown to be highly
efficient thiol cross-linking reagents and have been used in a
variety of protein conjugation applications including the
synthesis of bispecific antibodies and homogeneous
ADCs. 22−25 For example, Schumacher and co-workers
synthesized doxorubicin analogues containing dibromomaleimide linkers and conjugated them to trastuzumab, a commonly
used benchmark for evaluating new ADC technology.22 The
linkers were designed to cross-link two interchain cysteines on
the antibody forming a two-carbon bridge to afford ADCs with
approximately four drugs/antibody. The resulting ADCs
demonstrated comparable Her2 binding affinity to trastuzumab,
but their potency against antigen expressing cells and in vivo
pharmacological properties were not reported. A similar
approach was used by Maruani et al., who used dibromopyridazinedione linkers to conjugate doxorubicin to trastuzumab
via a dual click strategy.23 The resulting ADCs demonstrated
improved homogeneity and were rapidly internalized by
antigen expressing cells in vitro, but their pharmacological
properties in vivo were not reported.
In order to determine the relative effects of interchain
cysteine cross-linking on the pharmacological properties of
ADCs in vivo, we synthesized a novel derivative of monomethyl
auristatin F (MMAF) that contains a bifunctional dibromomaleimide (DBM) linker designed to react with two adjacent
cysteine thiol groups on the antibody. The DBM-MMAF
derivative is analogous to a well-known auristatin linker-payload
(MC-MMAF) except that the conventional maleimide is
replaced with a bifunctional dibromomaleimide. ADCs were
synthesized via conjugation of the DBM-MMAF derivative with
two different antibodies, and their properties were compared
with those of analogous ADCs synthesized using the conventional MC-MMAF payload. The results demonstrate that
conjugation via interchain cysteine cross-linking with dibromomaleimide (DBM) linkers yields highly homogeneous ADCs
containing four drugs per antibody. The ADCs demonstrate
improved pharmacokinetic properties, superior efficacy, and
reduced toxicity compared to conventional heterogeneous
ADCs. Moreover, the methods described here are scalable
and can be broadly applied to most antibodies without
recombinant engineering.
DOI: 10.1021/acs.molpharmaceut.5b00432
Mol. Pharmaceutics 2015, 12, 3986−3998
Molecular Pharmaceutics
Materials. MMAF and MC-MMAF were purchased from
Concortis Biosystems (San Diego, CA). All other chemical
reagents were purchased from VWR, AK Scientific, or SigmaAldrich and used as received. The anti-MMAF antibody was
prepared at Genscript (NJ). All human cell lines were
purchased from the American Type Culture Collection
(Manassas, VA, USA) or the Japanese Collection of Research
Bioresources Cell Bank (JCRB; Osaka, Japan) and were
maintained as recommended. Other materials and sources are
described below individually for each section.
Synthesis of Dibromomaleimide (DBM) Linker. 6Aminohexanoic acid (0.512 mg, 3.91 mmol) was added to a
solution of 3,4-dibromofuran-2,5-dione (1 g, 3.91 mmol) in
acetic acid (20 mL), and the solution was stirred at room
temperature for 10 min until all the solids dissolved. The
reaction mixture was heated to 100 °C for 18 h, after which
LC/MS indicated the reaction to be complete. The solution
was concentrated under vacuum and purified by silica gel
chromatography (eluent DCM/EtOAc 0−40%). Concentration
of pure fractions afforded 1.15 g (3.12 mmol, 80% yield) of
dibromomaleimide derivative, 6-(3,4-dibromo-2,5-dioxo-2,5dihydro-1H-pyrrol-1-yl)hexanoic acid. 1H NMR (400 MHz,
CDCl3): δ 3.62 (t, J = 7.2 Hz, 2H), 2.36 (t, J = 7.6 Hz, 2H),
1.68−1.62 (m, 4H) 1.41−1.30 (m, 2H). LC/MS: retention
time 3.172 min; acetonitrile:water gradient 5−95% acetonitrile
over 5 min at 0.8 mL/min. m/z: 391.9, 389.9, 393.9 [M + Na]+.
Synthesis of DBM-MMAF Payload. DIPC (34 mg, 0.271
mmol) and DIPEA (35 mg, 0.271 mmol) were added to a
solution of the DBM linker from above (250 mg, 0.677 mmol)
in DCM (5 mL), and the resulting solution was stirred for 1 h
at room temperature. MMAF (208 mg, 0.271 mmol) was added
in 50 mg portions over a 4 h period, and the resulting solution
was stirred for a further 16 h. The DCM was removed under
vacuum, and the residue was purified by preparative HPLC on
an Agilent 250 mm × 30 mm C18 column. The product was
eluted with a gradient of 50−90% CH3CN in water over 40 min
at a flow rate of 40 mL/min. Lyophilization of the combined
pure fractions afforded 170 mg of DBM-MMAF (2, Figure 1)
in 58% yield. 1H NMR (500 MHz, CDCl3): δ 7.15−7.26 (m,
5H), 4.60−4.92 (m, 4H), 3.70−4.20 (m, 4H), 3.59−3.63 (m,
2H), 3.39−3.42 (m, 1H), 3.26−3.35 (m, 6H), 2.93−3.09 (m,
6H), 2.20−2.60 (m, 6H), 1.70−2.15 (m, 4H), 1.61−1.69 (m,
8H), 1.25−1.37 (m, 3H), 1.15 (dd, J = 18.5, 7.5 Hz, 2H), 0.81−
1.05 (m, 20H). HPLC: 4.297 min (5−95% acetonitrile in water
over 5 min). m/z: calcd 1082.38 (100.0%), 1083.38 (53.0%),
1080.38 (51.4%), 1084.37 (48.6%), 1085.38 (25.8%), 1081.38
(16.7%), 1084.38 (11.1%); obsd [M + H] 1082.1 (15%),
1083.1 (8%), 1084.1 (52%), 1085.0 (25%), 1086.0 (26%),
1087.0 (17%) [M + H] .
Synthesis of ADCs. The conventional maleimide ADCs 3
and 4 (Figure 2) were synthesized using protocols similar to
methods described previously.24 Briefly, the purified antibody
(trastuzumab or IGNX) was buffer exchanged into PBS, pH
7.4. The antibody was diluted to a final concentration of 5 mg/
mL in PBS and warmed to 37 °C in a heat block. A stock
solution of TCEP (50 mM) was freshly prepared in water, and
2.5 molar equiv (relative to the antibody concentration) was
added. After 2 h the partially reduced antibody was removed
from the heat block and cooled to room temperature. A stock
solution of the MC-MMAF payload (2 mM in DMSO) was
freshly prepared, and 5 molar equiv was added to the antibody.
Figure 1. (A) Chemical structures of MMAF payloads containing
conventional MC (1) or bifunctional DBM (2) linkers. (B) Synthesis
of DBM-MMAF payload (2) used for synthesis of homogeneous
After 1 h the reaction mixture was buffer exchanged into PBS
using PD10 spin columns to remove small MW reagents and
stored at 4 °C until needed.
The DBM ADCs 5 and 6 were synthesized as follows. 60 mg
of purified antibody (trastuzumab or IGNX) was buffer
exchanged into PBS, pH 7.4. The antibody was diluted to a
final concentration of 5 mg/mL in PBS and warmed to 37 °C in
a heat block. A stock solution of TCEP (50 mM in water) was
freshly prepared, and 8 molar equiv (relative to antibody
concentration) was added to the antibody. The reaction
mixture was incubated at 37 °C in a sealed 15 mL conical
tube. After 2 h the reaction was removed from the heat block
and allowed to cool to room temperature. Five molar
equivalents of the DBM-MMAF derivative 2 (Figure 1) was
added from a freshly prepared 2 mM stock solution in DMSO.
The reaction was mixed gently at room temperature for 1 h.
The crude ADC was buffer exchanged into PBS at pH 7.4 to
remove excess small MW reagents and stored at 4 °C.
SEC Analysis of ADCs. Size exclusion chromatography was
performed on all ADCs to determine monomeric purity on an
Acquity H class UPLC (Waters) with a BEH200 SEC 1.7 mM,
4.6 × 150 mm column with a BEH SEC Guard column 4.6 ×
30 mM (Waters). The mobile phase consisted of 100 mM
sodium phosphate, 150 mM sodium chloride, pH 6.8. 5 μg of
sample was injected and run at a flow rate of 0.3 mL/min for 10
min at room temperature. Samples were analyzed by Empower
software (Waters). The SEC results are available in the
Supporting Information (see Table 1).
Hydrophobic Interaction Chromatography (HIC)
Analysis of ADCs. HIC analysis was performed on a Waters
Acquity UPLC system on a Tosoh TSKgel Butyl-NPR, 4.6 mm
× 10 cm column, 2.5 μm particle size; Tosoh (Cat. No. 42168).
The mobile phase consisted of buffer A (1.5 M ammonium
sulfate in 25 mM sodium phosphate, pH7) and buffer B (25
mM sodium phosphate pH 7, 25% isopropanol). A gradient of
25−100% buffer B was run at 0.5 mL/min over 60 min.
Native LC/MS Analysis of ADCs. Native LC/MS analysis
was performed on a Waters Acquity UPLC with TUV detector
(at 280 nM) and G2-S Q-ToF mass spectrometer (m/z range
DOI: 10.1021/acs.molpharmaceut.5b00432
Mol. Pharmaceutics 2015, 12, 3986−3998
Molecular Pharmaceutics
Figure 2. Synthesis of ADCs via conjugation of trastuzumab or IGNX with payloads containing conventional MC (1) or bifunctional DBM (2)
linkers. The conjugation protocols are identical except that excess TCEP is used to ensure full reduction of interchain disulfide bonds prior to
conjugation with DBM-MMAF (2).
of 1000−8000). A 4.6 × 300 mm BEH 200 column (1.7 μm
pore size) was used at a flow rate of 0.3 mL/min. 120 μg of
sample was deglycosylated for 5 h at 37 °C with PNGase F
prior to analysis.
Denaturing LC/MS of Analysis of ADCs. Denaturing
LC/MS analysis was performed on a Waters Acquity UPLC
with TUV detector (at 280 nm) and G2-S Q-ToF mass
spectrometer (scan m/z range of 1000−4000). The column
used was a 2.1 × 100 mm BEH 130 (Waters, C18, 1.7 μM pore
size) with a mobile phase gradient of water/acetonitrile + 0.5%
formic acid.
Denaturing SEC Analysis of ADCs. To resolve noncovalently bound antibody chains, a denaturing SEC method
was developed. The method consisted of a Zenix-C SEC-300
column (4.6 × 300 mm, 3 μm particle size, 300 Å pore size)
from Sepax Technologies with an isocratic mobile phase of 35%
acetonitrile + 0.1% trifluoroacetic acid + 0.1% formic acid. The
flow rate was 0.2 mL/min, and the run time was 25 min. 10 μg
of antibody or ADC was injected as a 2 mg/mL aqueous
solution. To confirm the identity of different isoforms present
in trastuzumab−DBM-MMAF, the isoforms were separated
using a semipreparative method similar to the analytical
method described above except that the column size used
was 10 × 300 mm, the flow rate was 0.87 mL/min, and 65 μL
of 12 mg/mL ADC was injected. The relevant fractions were
then frozen at −80 °C until denaturing LC/MS analysis was
performed as described above.
Antigen Binding via Surface Plasmon Resonance
(SPR). ADC binding to purified antigens, Her2 and CD98,
was performed via surface plasmon resonance analysis
conducted on a Biacore 1000 instrument. Each ADC sample
was diluted to a concentration of 100 nM and captured onto a
goat anti-human Fc surface (Invitrogen). The running buffer
included 10 mM HEPES pH 7.4, 150 mM NaCl, 0.005%
Tween-20, and 0.1 mg/mL BSA. All data were collected at 25
°C. Each mAb was captured 5 times and placed within each of
the 5 different flow cells. Capture levels were between 100 and
200 RU and averaged ∼150 RU. Surfaces were regenerated
with 2 × 18 s pulses of 1/50 dilution of phosphoric acid.
Recombinant human ErbB2 (Sino Cat 10004-H08H, lot
LC06AP0603) or CD98 prepared at 50, 16.6, 5.55, 1.85, 0.61
nM and a buffer blank were injected at 100 μL/min over the
captured mAb surfaces. The dissociation phase was monitored
for 1800 s. Data were processed and fit in Scrubber-Pro6
(Biological Software Pty Ltd.). Responses were referenced
using the reference channel as well as the buffer blank injection.
Data were fit to a 1:1 interaction model, and a summary of the
binding constants is shown in Table 1.
Table 1. Affinity of ADCs 3−6 for Their Purified Antigens,
ErbB2 and CD98, Determined via SPR
antibody or ADC
ka (M−1
kd (s−1)
Antigen Binding via Cell-Based ELISA. ADC binding to
antigen expressing cells was measured using a cell based ELISA.
CD98 transfected F279 sarcoma cells or BT474 cells expressing
Her2 were plated at 5000 cells per well in a 384-well plate. The
following day, antibodies were serially diluted in a separate
plate and transferred to the plate containing the cells. After a 2
h incubation at room temperature, the plate was washed with
buffer (DPBS pH 7.4 with 0.1% BSA), and 25 μL of goat antimouse HRP-labeled secondary antibody (Pierce Cat. No.
37069) diluted in cell culture medium was added and incubated
for 30 min at room temperature. The plate was washed, 15 μL
of a goat anti-mouse chemiluminescent substrate (Pierce Cat.
No. 37069) was added, and the plate was read in a plate based
luminescence reader (Spectramax).
In Vitro Cytotoxicity Assays. Tumor cell lines were
routinely passaged in RPMI medium (LifeTech) supplemented
with 10% fetal calf serum (LifeTech). To assay toxicity, cells
were plated in 384-well plates, black well/clear bottom
(Greiner), at 5000 cells per well in 40 μL of medium. ADCs
or antibodies were serially diluted (5×) from 100 nM in RPMI,
and 10 μL of 5× solution in complete medium was added to
appropriate wells in duplicate using an iPipette liquid handler
(Apricot Designs). Cell plates were then incubated for 3 days,
followed by lysis in 30 μL of Cell-Titer Glo assay reagent
(Promega). Luminescence was then quantified on a Synergy
HT plate reader (BioTek) and graphed. IC50 values were
calculated by fitting to a four-parameter sigmoidal fit using
GraphPad Prism software.
Efficacy in Xenograft Tumor Models. All procedures in
animals described were in compliance with the Animal Welfare
Act, the Guide for the Care and Use of Laboratory Animals,
and the Office of Laboratory Animal Welfare. Protocols were
reviewed by the Institutional Animal Care and Use Committees
of either Igenica Biotherapeutics (Burlingame, CA) or WIL
Research (Ashland, OH), respectively. Methods: Immunocompromised female NOD/SCID mice were used for the H446
tumor model (Charles River, Wilmington, MA) and female
DOI: 10.1021/acs.molpharmaceut.5b00432
Mol. Pharmaceutics 2015, 12, 3986−3998
Molecular Pharmaceutics
Dawley (SD) rats (ICON Laboratory Services, LLC, Whitesboro, NY). Briefly, after a seven-day acclimation period, 12
male rats were assigned to one of three treatment groups (4 per
group). Animals in each group received a single intravenous
bolus administration of the test article (trastuzumab, conventional ADC 3, or DBM ADC 5) on day 0. Blood samples were
collected 0.083 h, 1 h, 4 h, 8 h, 24 h, 48 h, 72 h, 120 h, 168 h,
240 h, 336 h, 408 h, 504 h, 576 h, and 672 h post
administration of the test articles. The PK parameters were
calculated in Phoenix WinNonlin (version 6.3, Pharsight Corp.,
St. Louis, MO) from the concentration time data obtained from
the ELISA protocols described above using a noncompartmental analysis (NCA) method with intravenous bolus input. Halflife (t1/2) was estimated by log−linear regression of the terminal
phase of the concentration versus time profiles. At least 3
points clearly visible in the terminal phase, an r2 value of at least
0.8, and time interval of at least 2 half-lives were required to
characterize half-life. Detailed protocols and results for PK
analysis are available in the Supporting Information.
Toxicology Study of ADCs in rats. The relative toxicity
and TK profiles of trastuzumab ADCs 3 and 5 were evaluated
in a repeat-dose study in male Sprague−Dawley (SD) rats
(WIL Research, Ashland, Ohio). Four rats were assigned per
dose group for PK analysis. Briefly, rats were dosed
intravenously (iv) weekly for a total of three doses on days 0,
7, and 14. Doses ranged from 14.4 to 14.9, 21.5 to 22.3, and
28.7 to 29.8 mg/kg of ADCs 5 and 3 respectively in order to
compensate for slight variations in ADC drug loading. Body
weights were recorded twice weekly, and food consumption
was recorded weekly. Blood samples were taken on day 3, and a
necropsy was conducted on day 17 (3 days after the third
dose). Dose-proportionality of the test articles was assessed,
and time to reach steady state and effective half-life were
estimated. The plasma accumulation of total antibody and
ADCs 3 and 5 was evaluated following repeat iv injections. The
parameters with notable differences measured between the
ADCs are discussed in Results, and complete results with all
measured parameters for the toxicology study are available
online as Supporting Information.
NOG mice for the SKOV3 tumor model (Taconic, Hudson,
NY), respectively. H446 (HTB-171) and SKOV3 (HTB-77)
were obtained from ATCC. Mice were subcutaneously injected
on the right flank with at least 1 × 107 viable cells. When the
tumor reached a size between 65 and 300 mm3, mice were
randomized to treatment. Antibodies were administered weekly
and tumors measured twice weekly. Tumor volume was
calculated using the modified ellipsoid formula (π/6)(length
× width2). All experiments were performed on groups of 8
animals per group. Animal experiments were performed in
accordance with protocols approved by the Igenica Biotherapeutics Institutional Review BoardAnimal Care and Use
Committee. Data are expressed as the mean ± standard error of
the mean (SEM). Group means were compared using Student’s
2-tailed, unpaired t test. Probability (p) values of <0.05 were
interpreted as significantly different. All statistical analyses were
performed using Microsoft EXCEL (Microsoft, Redmond,
WA) and GraphPad Prism v.5.0f (GraphPad Software, Inc., La
Jolla, CA).
Total Antibody and ADC ELISA. The anti-MMAF (ADC)
ELISA was designed to detect all ADC species with DARs > 0
via capture with an anti-MMAF monoclonal antibody prepared
at GenScript (New Jersey) using a KLH−MMAF conjugate for
immunization. The MMAF was conjugated to KLH through
the N-terminus using a C6 linker identical to those used for
ADC synthesis. Eight different hybridomas were screened for
binding vs two heterogeneous MC-MMAF ADCs, one with low
drug loading (DAR < 2) and one with high drug loading (DAR
> 4). A single hybridoma with equal affinity for both ADCs was
selected to use as a capture antibody for the ADC PK analysis.
A mouse anti-human IgG (Fc) CH2 domain-peroxidaseconjugate was utilized for detection (Southern Biotech). The
total antibody (total Ab) ELISA was designed to measure all
antibody components including unconjugated and conjugated
ADC in plasma. For trastuzumab and ADCs, an anti-human
herceptin antibody was used as the capture reagent and a
mouse anti-human IgG (Fc) CH2 domain-peroxidase-conjugate was utilized for detection (Southern Biotech). The assay
range for each analyte was 39.1 to 2500 ng/mL in plasma. The
lower limit of assay quantitation (LLOQ) was 39.1 ng/mL in
rat plasma. Briefly, anti-MMAF or anti-herceptin antibody
(Serotech) was diluted in coating buffer (PBS, pH 7.4) and
immobilized onto a 96-well microtiter sample plate. The plate
was washed, and all unabsorbed sites were blocked with the
addition of Block Buffer (PBS with 0.05% Tween-20, pH 7.4;
1% BSA) for no less than 1 h and no more than 3 h. After
washing the plate, analytes were diluted 1:1000 with Block
Buffer, dispensed onto the sample plate, and then incubated for
approximately 1 h. After the final wash step, a tetramethylbenzidine (TMB) peroxidase substrate solution was added and
incubated for approximately 13−15 min with nonvigorous
shaking. The reaction was stopped with a phosphoric acid stop
solution (1 M). Color developed in proportion to the amount
of herceptin and herceptin conjugate, respectively, present in
the sample. Plates were read on a plate reader (Bio-Tek Power
Wave HT) at two wavelengths (450 nm for detection and 620
nm for background). Analyte concentrations were determined
on a standard curve obtained by plotting optical density (OD)
versus concentration using a four parameter logistic curvefitting program (Gen5 Secure Software ver. 1.08 or higher).
Pharmacokinetic Analysis of ADCs. This was a nonclinical laboratory, open-label, randomized, single-dose study of
trastuzumab antibody−drug conjugates in adult Sprague−
Synthesis of DBM-MMAF Payload 2. In order to evaluate
the pharmacological properties of ADCs synthesized via
interchain cysteine cross-linking, we selected a clinically
validated auristatin payload (MMAF) containing a conventional
maleimide (MC) linker as a benchmark (1, Figure 1A).25 An
analogous auristatin derivative containing a bifunctional DBM
linker, 2 (Figure 1A), was synthesized in two steps from
commercially available reagents as shown in Figure 1B. The
DBM-MMAF derivative 2 is designed to react with two
adjacent interchain cysteines to form a dithiomaleimide linkage
with the antibodies. Derivatives 1 and 2 both contain stable six
carbon spacers to minimize potential bystander effects
sometimes observed with protease cleavable linkers and differ
only in their cross-linking capabilities.26 As a result, subtle
differences in activity between homogeneous and heterogeneous ADCs can be accurately measured.
Synthesis of ADCs. Two different antibodies were selected
to serve as models for ADC synthesis via conjugation with
MMAF payloads 1 and 2. Trastuzumab, an anti-Her2 antibody
approved for treatment of breast cancer in 2011, was selected
based on its known pharmacological properties and its frequent
use as a benchmark antibody for evaluating new ADC linker/
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payload technologies.27 A second antibody, IGNX, was selected
from our in-house library of tumor specific antibodies in order
to demonstrate the versatility of DBM linkers for constructing
homogeneous ADCs from native antibodies. IGNX binds
specifically to CD98, a proprietary tumor associated antigen
expressed on a number of hematological tumor types.28 The
conjugation protocols used for synthesis of ADCs with DBM
derivative 2 are comparable to previously reported methods for
making conventional ADCs, except that excess reducing agent
(TCEP) is used to ensure complete reduction of antibody
interchain disulfide bonds prior to conjugation (Figure 2).29
The reduction/conjugation process is carried out in PBS at
room temperature, and purification of the reduced antibody is
not required prior to conjugation. A slight excess (5 equiv) of
DBM-MMAF derivative 2 was required to obtain DARs of four
drugs/antibody. The resulting ADCs, 3−6 (Figure 2), were
purified via buffer exchange to remove any remaining low MW
reagents. The overall yield for the two-step process typically
ranges from 70 to 90% based on recovered protein. Size
exclusion chromatography (SEC) analysis indicated that ADCs
3−6 were >98% monomer, suggesting that minimal intermolecular cross-linking of different antibody molecules
occurred during the conjugation process (see Supporting
Information Table 1).
Homogeneity and DAR Analysis of ADCs. The relative
homogeneity of ADCs 3−6 were determined by hydrophobic
interaction chromatography (HIC) and native LC/MS
analysis.30 ADC fractions containing different DARs are
resolved by HIC based on differences in hydrophobicity, with
higher DAR species eluting progressively later than the
unconjugated parent antibodies. The conventional methods
for conjugation with maleimide linkers typically involve partial
reduction of antibody interchain disulfides and yield ADCs
containing predominantly even numbered DARs. ADCs 3 and
4 were synthesized using conventional conjugation protocols
optimized to obtain DARs of exactly four drugs/antibody in
order to minimize variability and maintain consistency with
previously reported results.19 HIC analysis of the conventional
ADCs afforded typical heterogeneous profiles with even
numbered DARs ranging from 0 to 8 drugs/antibody. Average
DARs of 3.9 and 4.0 were calculated for ADCs 3 and 4
respectively based on peak areas shown in Figure 3. In contrast,
the DBM cross-linked ADCs 5 and 6 eluted as single peaks that
had retention times consistent with DARs equal to 4 drugs/
antibody. The results demonstrate that conjugation via cysteine
cross-linking with DBM linkers yields ADCs with significantly
improved homogeneity compared to analogous ADCs synthesized from conventional maleimide linkers.
LC/MS Analysis of ADCs. Native LC/MS analysis of ADCs
3−6 confirmed the HIC results and provided accurate MWs for
the DAR components present in the ADCs (Figure 4).31 The
DAR compositions determined by native LC/MS analysis are
comparable to the DARs estimated by HIC, and the observed
MWs are consistent with calculated MWs (±5 AMUs) for the
cross-linked ADCs. Minor quantities of DAR3 and/or DAR5
fractions were observed by LC/MS that were not resolved via
HIC analysis due to the superior sensitivity and accuracy of
native LC/MS analysis. Nonetheless, the results indicate that
the cross-linked DBM ADCs 5 and 6 have average DARs of 4.0
and 4.1 drugs/antibody and have significantly improved
homogeneity (>85% DAR4) over the analogous conventional
ADCs. No other higher MW adducts were observed in ADCs 5
and 6. However, fragments with MWs equal to one-half the
Figure 3. Hydrophobic interaction chromatography (HIC) analysis of
ADCs 3−6. Cross-linked (DBM) ADCs are shown in blue, and
conventional (MC) ADCs are shown in red. Average DAR values were
calculated based on peak areas. (A) HIC overlays comparing
trastuzumab ADCs 3 and 5. (B) HIC overlays comparing IGNX
ADCs 4 and 6.
calculated MW of the cross-linked ADCs were detected when
denaturing LC/MS conditions were employed. Similar fragments were observed in the conventional ADCs 3 and 4, in
addition to other heavy and light chain fragments commonly
observed in conventional ADCs.30
Denaturing SEC Analysis of ADCs. Denaturing LC/MS
analysis of the DBM-MMAF ADCs 5 and 6 indicated the
presence of an ADC fragment with half the expected mass of
the fully cross-linked ADCs (data not shown). We hypothesized that this half ADC fragment potentially results from
intrachain cross-linking of two hinge cysteines on the same
heavy chain rather than interchain cross-linking of cysteines on
two different heavy chains (see Figure 5E). Since antibody
hinge region cysteines are in close proximity to each other
(separated by two prolines in IgG1), the possibility of
intrachain cross-linking could not be ruled out. In fact,
Schumacher et al. recently reported formation of similar
fragments based on SDS−PAGE analysis of ADCs synthesized
using substituted maleimide linkers.26 Both inter- and intrachain cross-linking afford in ADCs with DARs of 4 drugs/
antibody, but only the interchain cross-linked isoform contains
covalent bonds between the two heavy chains. Intrachain cross3991
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Figure 4. Native LC/MS analysis comparing DAR compositions of conventional ADCs, 3 and 4, with cross-linked DBM ADCs, 5 and 6. The data
shown are the deconvoluted MS profiles, and observed MWs are consistent with calculated MWs within ±2 AMUs. Average DAR values shown for
each ADC were calculated based on peak intensities.
DAR = 4 drugs/antibody. To further characterize and quantify
the relative amounts of inter- vs intrachain cross-linking in
ADCs 5 and 6, we developed a denaturing size exclusion
chromatography (dSEC) method that enables separation of
noncovalently linked antibody chains or fragments. In contrast
to the previously reported SDS−PAGE methods for analyzing
ADC fragments, the dSEC method described here does not
require the use of reducing agents or high temperatures known
to cause degradation of maleimide linkers.20,22
The results shown in Figure 5 demonstrate that ADC 5
(TRA−DBM-MMAF) is composed of two different isoforms in
a 7:3 ratio (Figure 5B). The major isoform elutes earlier and
accounts for ∼70% of the total ADC based on peak areas.
Moreover, the retention time is similar to that of intact
trastuzumab containing 2 heavy and 2 light chains (Figure 5A),
which is consistent with interchain cysteine cross-linking
(Figure 5E). The smaller, later eluting peak represents ∼30%
of the total ADC and has a retention time consistent with a
fragment containing single heavy and light chains.
To support this observation, a trastuzumab double mutant
(C225A/C228A) was prepared as a standard for comparison.
The double mutant lacks both hinge region disulfide bonds
between the two heavy chains and therefore elutes as a half
antibody (HL) fragment under denaturing conditions (Figure
5C). To provide further confirmation for the proposed
structural isoforms (Figure 5E), ADC 5 was purified via
preparative dSEC and the individual isoforms were isolated and
analyzed by LC/MS (see Supporting Information Figures 2 and
Figure 5. Denaturing SEC analysis of ADC 5 (TRA−DBM-MMAF)
showing the presence of two ADC isoforms resulting from inter- or
intrachain cross-linking of hinge cysteines on the heavy chains: (A)
native trastuzumab with intact interchain disulfide bonds; (B) ADC 5
(TRA−DBM-MMAF); (C) trastuzumab C225A/C228A double
mutant which lacks hinge disulfide bonds; (D) fully reduced
trastuzumab lacking interchain disulfides; (E) proposed structures of
interand intrachain ADC isoforms. LC/MS analysis of the separated
isoforms is consistent with the proposed structures (see Supporting
linking of two hinge cysteines on the same heavy chain would
result in an ADC fragment that lacks covalent bonds between
heavy chains with a MW equal to one-half of an ADC with
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3). The observed MWs for the isolated isoforms are consistent
with calculated MWs for the structures depicted in Figure 5E.
Similar results were obtained with ADC 6 (see Supporting
Information Figure 1), which indicates that isoform formation
is not antibody dependent. The relative contributions of the
two isoforms to ADC activity remain uncertain, but recent
studies by Lyon et al. suggest that the absence of covalent
bonds between ADC heavy chains has little or no impact on the
pharmacological properties ADCs in vivo.32
Antigen Binding Affinity of ADCs. The affinity and
specificity of ADCs 3−6 for their respective antigens were
determined using surface plasmon resonance (SPR) and cell
based ELISA methods. The parent antibodies were used as
benchmarks for comparison with the ADCs. The binding
kinetics of the ADCs to immobilized purified antigens, Her2 or
CD98, was determined on a Biacore 1000 SPR instrument. The
results shown in Table 1 indicate that ADCs 3−6 have
comparable affinities to their respective parent antibodies; and
the observed KDs for trastuzumab ADCs 3 and 5 are consistent
with previously reported values for trastuzumab.
Binding of ADCs 3−6 to antigen expressing cell lines was
determined using a cell based ELISA. A murine sarcoma cell
line, F279, which lacks expression of both human target
antigens, was transfected with human CD98 and used for
testing the IGNX ADCs, 4 and 6. Human breast carcinoma
(BT474) cells that express high levels of Her2 were used for
testing the trastuzumab ADCs 3 and 5. Cells were incubated
with serial dilutions of the ADCs, and the relative fraction of
bound antigen was measured via luminescence after addition of
an HRP labeled secondary antibody. The data shown (Figure
6) indicates that the DBM ADCs 5 and 6 bind antigenexpressing cells with low nM affinities comparable to those of
the parent antibodies, but they do not bind to cells lacking
expression of their relevant target antigens. The results indicate
that interchain cross-linking with bifunctional DBM linkers
does not compromise antigen binding affinity or specificity of
the ADCs, and suggests that ADCs containing bifunctional
DBM linkers maintain the structural integrity and functionality
of their parent antibodies.
Potency of ADCs in Vitro. Although the antigen binding
properties of ADCs 3−6 remained comparable to those of the
parent antibodies, changes in ADC stability resulting from
conjugation with DBM linkers could potentially impact their
potency against antigen expressing cells in vitro. To address this
possibility, the relative potencies of ADCs 3−6 against tumor
cell lines with and without antigen expression were determined.
Three cell lines were selected based on their antigen expression
profiles. The human breast carcinoma cell line, BT474,
expresses high levels of Her2 but does not express CD98.
The human lung carcinoma cell line, H446, expresses CD98,
but not Her2. A third cell line, SKOV3 (human ovarian tumor)
was selected because it expresses both antigens. Two control
ADCs (7 and 8) were synthesized via conjugation of MMAF
derivatives 1 and 2 (see Figure 1) using methods previously
described for ADCs 3−6 with an anti-IgE isotype control
antibody (C1.18) that does not bind either of the antigen
targets. Cells were incubated with serial dilutions of the ADCs
for 3 days, and cell growth was determined using a standard
luminescence assay for measuring ATP concentrations. The
results shown (Figure 7) indicate that DBM ADCs 5 and 6
inhibit growth of antigen expressing cells with comparable
potency to the conventional ADCs 3 and 4. Moreover, ADCs 5
and 6 do not inhibit growth of cells lacking antigen expression
Figure 6. Binding affinity for antigen expressing cells: (A) binding of
trastuzumab ADCs to BT474 (human breast carcinoma) cells that
express Her2 but not CD98; (B) binding of IGNX ADCs to murine
sarcoma cells transfected with human CD98, but lacking Her2
and no tumor growth inhibition was observed for the control
ADCs 7 and 8 against any of the cell lines, indicating that
cytotoxicity is antigen dependent. Overall, the results
demonstrate that DBM ADCs 5 and 6 are highly potent and
selective against antigen expressing cells in vitro (Figure 7).
Efficacy of ADCs in Xenograft Tumor Models. The
efficacy of the ADCs was evaluated in two different xenograft
tumor models and compared with that of the unconjugated
parent antibodies. SKOV3 ovarian tumors express high levels of
Her2 and are sensitive to treatment with trastuzumab (Figure
8A), while H446 lung carcinoma tumors express the CD98
antigen at high levels (Figure 8B). The control ADCs, 7 and 8,
were included in the study as negative isotype controls to
evaluate potential target independent activity. Tumor cells were
implanted in mice, and when the tumors reached an
appropriate size (200−300 mm3), mice were randomized into
different treatment groups (10 per group) and treated twice
intravenously with the test ADCs (dosed at 3 mg/kg) on days 1
and 8. Tumor growth and weight gain for each mouse were
monitored at 3-day intervals for the duration of the study. Both
DBM ADCs, 5 and 6, demonstrated tumor growth inhibition
superior to that of the parent antibodies and resulted in
complete tumor remissions in both tumor models (Figures 8A
and 8B) demonstrating that cross-linked DBM ADCs are highly
efficacious in vivo. A third xenograft study was performed in
order to compare the relative activities of the cross-linked
trastuzumab ADC 5 with the conventional ADC 3. NOG mice
(which are insensitive to trastuzumab in this model) were
selected for this study to remove contributions of the parent
antibody to the observed efficacy. Any observed differences in
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Figure 7. Potency and selectivity of ADCs 3−6 in vitro: (A) growth inhibition of BT474 (human breast carcinoma) cells that express Her2, but not
CD98; (B) growth inhibition of H446 (human lung carcinoma) cells that express CD98, but not Her2; (C) growth inhibition of SKOV3 (ovarian
tumor cells that express both Her2 and CD98; (D) IC50s of ADCs 3−6 against three different cell lines.
Figure 8. (A) Tumor growth inhibition in Her2 positive SKOV3 xenograft mice treated twice (days 62 and 69) at 3 mg/kg. (B) Tumor growth
inhibition in CD98 positive H446 (lung carcinoma) xenograft mice treated twice (days 41 and 48) at 3 mg/kg. (C) SKOV3 xenograft tumor model
in NOG mice (insensitive to treatment with trastuzumab) treated once at 3 mg/kg with homogeneous (DBM) ADC 5 or with conventional (MC)
ADC 3.
accurate measurement of the relative amounts of total antibody
(total mAb) and conjugated antibody (ADC) throughout the
duration of the study. ADCs 3 and 5 were administered
intravenously (4 rats/group), and blood samples were taken at
increasing time points. The concentrations of total antibody
and conjugated antibody were determined for each time point
using previously described sandwich ELISA assay protocols.
Total antibody concentrations [total mAb] were measured
using a mouse anti-human IgG1 capture antibody, while the
concentration of conjugated antibody [ADC with DAR ≥ 1]
was determined using a murine anti-MMAF capture antibody.
The results shown (Figure 9) represent the average of
quadruple measurements; unconjugated trastuzumab was
included as a control. No significant differences in total
antibody half-life were observed for ADCs 3 and 5, suggesting
that the linker type has minimal impact on antibody stability in
vivo. The half-lives of the conjugated antibodies (ADCs with
tumor growth inhibition could be attributed to the ADC linkers
with greater confidence by removing all other potential
variables. NOG mice (10 per group) were implanted with
SKOV3 tumors and treated twice with conventional ADC 3 or
DBM ADC 5 at 3 mg/kg. SKOV3 tumor growth was
significantly delayed in mice treated with ADC 5, while
treatment with the conventional ADC 3 did not significantly
inhibit tumor growth relative to the control ADCs (Figure 8C).
The results demonstrate that DBM ADC 5 is more efficacious
than the conventional ADC 3 at equivalent doses and suggests
that improved ADC homogeneity results in superior tumor
growth inhibition in vivo.
Pharmacokinetics of ADCs in Rats. A rat pharmacokinetics study was conducted to compare the relative clearance
rates of ADCs 3 and 5 with unconjugated trastuzumab. Rats
were selected as the species for PK evaluation so multiple
samples could be taken from each animal, enabling more
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life and exposure observed for DBM ADC 5 over the
conventional ADC 3 likely contribute to the enhanced efficacy
of the DBM ADC 5 that was observed in the SKOV3 xenograft
tumor model (Figure 8C).
Toxicity of ADCs in Rats. The relative toxicity profiles of
trastuzumab ADCs 3 and 5 were compared in rats. Equivalent
doses of both ADCs were administered weekly on days 1, 8,
and 15 via intravenous injection. Statistically significant
differences were observed for the parameters shown in Figure
10. Decreases in absolute reticulocytes were observed in
animals treated with conventional ADC 3, but not in groups
treated with ADC 5 on study day 3 (Figure 10). On day 17 (3
days after the final dose) decreases in absolute reticulocytes
were observed in all ADC treatment groups, but to a lesser
extent in rats treated with the cross-linked ADC 5 (Figure
10A). Similarly, mid and high doses of the conventional ADC 3
induced a statistically significant increase in hepatic parameters,
ALT and AST, on study days 3 and 17 (Figures 10B and 10C).
In contrast, ALT and AST values were only elevated in the high
dose group treated with ADC 5 on day 3, and in the mid and
high groups on day 17. There were no test article-related effects
on survival, clinical observations, or food consumption.
However, gross macroscopic examinations of the testes revealed
that the ADC 3 treatment groups had abnormally small (2/10low, 3/10-mid, and 4/10-high) and/or soft (2/10-low, 2/10mid, 6/10-high) testes, suggesting reproductive organ toxicity;
while no unusual findings were observed in the ADC 5
treatment groups. Overall, the results suggest that ADC 5 has a
superior safety profile compared to the conventional ADC (3).
Detailed toxicology results are available in the Supporting
Information, Table 6.
Figure 9. Pharmacokinetics of trastuzumab ADCs 3 and 5 in rats after
single iv injection at 1 mg/kg. Total Ab was determined using rat antimouse Fc capture antibody. Total ADC (DAR > 0) was determined
using a murine anti-MMAF capture antibody. Detailed PK results are
available in the Supporting Information.
DAR ≥ 1) however, were significantly different for the
conventional heterogeneous ADC 3 (t1/2 = 130 h) compared
to the DBM cross-linked ADC 5 (t1/2 = 184 h), suggesting that
the MMAF payload is released more rapidly from the ADC
containing a conventional maleimide linker. The increased half-
Figure 10. ADC toxicity in rats treated with increasing doses of ADC 3 (red) or ADC 5 (blue) measured on days 3 and 17. Group averages (10 rats
per group) are shown as a green bar. (A) Dot plot showing absolute reticulocyte counts on days 3 and 17 in individual rats. (B) Dot plot showing
alanine aminotransferase (ALT) levels on days 3 and 17 in individual rats. (C) Dot plot showing aspartate aminotransferase (AST) levels on days 3
and 17 in individual rats.
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Versatility and Scalability of ADCs. In order to
determine the versatility of DBM linkers for constructing
ADCs from different antibodies, we selected 3 commercially
available antibodies (Erbitux, Avastin, and Rituxan) and
conjugated them with 2 (DBM-MMAF) using previously
described protocols for ADCs 5 and 6. HIC analysis of the
resulting ADCs afforded single peaks with retention times
consistent with DARs of 4 drugs/antibody (Figure 11A), and
heterogeneous ADCs suggests that they have not yet reached
their optimal therapeutic potential. The absence of efficient
methods for producing homogeneous ADCs, however, has
compelled pharmaceutical companies to develop heterogeneous ADCs despite their known limitations.
Numerous recombinant approaches for improving ADC
homogeneity have been developed to overcome the limitations
of heterogeneous ADCs, and significant progress has been
made toward the synthesis of ADCs with defined DARs and
conjugation sites. These recombinant approaches, however,
often require extensive antibody engineering to identify the
optimal conjugation sites where unique side chains can be
introduced for conjugation with payloads modified with
appropriate linkers. As a result, they are not suitable for
converting existing “off-the-shelf” antibodies directly into
ADCs. Many of these methods require complex linker and
payload modifications that have not been clinically validated
and therefore present a greater risk for development into
therapeutic agents.
With the limitations of current recombinant methods in
mind, nonrecombinant methods have been developed for
synthesizing more homogeneous ADCs that do not require reengineering of the antibody. Disubstituted maleimide linkers
have been used successfully for synthesizing homogeneous
ADCs via interchain cysteine cross-linking, but their impact on
the pharmacological properties of the resulting ADCs relative
to conventional heterogeneous ADCs was unknown.33,34,35
The results presented here have demonstrated that conventional maleimide linkers can be easily modified into bifunctional
DBM linkers to enable the synthesis of DAR 4 ADCs via
interchain cysteine cross-linking. The DBM linkers are
compatible with clinically validated ADC payloads, and they
can be applied to a broad range of antibodies to consistently
yield ADCs with improved homogeneity and other pharmacological properties over conventional heterogeneous ADCs. No
antibody engineering or conjugation site optimization is
required because the conjugation sites are conserved for most
antibodies. Moreover, antigen affinity and specificity are
maintained during the conjugation process, and the resulting
ADCs are highly potent against antigen positive cells in vitro. In
summary, these results demonstrate that interchain cysteine
cross-linking with DBM linkers yields ADCs with improved
pharmacokinetics, superior efficacy, and reduced toxicity in vivo
over conventional ADCs. These improvements should
ultimately result in a superior therapeutic window and lead to
more effective therapeutic agents in the future.
Figure 11. Versatility and scalability of DBM linkers. (A) HIC traces
of ADCs derived from conjugation of 2 (DBM-MMAF) with 3
different commercially available antibodies: Avastin, Rituxan, and
Erbitux. (B) HIC traces of ADCs resulting from conjugation of 2 with
IGNX at 3 different reaction scales (1 mg, 25 mg, and 1000 mg).
LC/MS data confirms the HIC results (see Supporting
Information). The results demonstrate that DBM linkers can
be applied to a variety of different antibodies and consistently
afford ADCs with DARs of 4 drugs/antibody. The scalability of
the conjugation reaction between DBM-MMAF 2 and IGNX
was investigated at three different reaction scales (1 mg, 25 mg,
and 1000 mg). The HIC profiles of the resulting ADCs (Figure
11B) indicate that there was no change in ADC homogeneity
up to a 1 g scale. Multiple conjugations were performed at the 1
and 25 mg scales (10 each) to demonstrate the reproducibility
of the conjugation reactions. Less than 10% variation was
observed in relative homogeneity, demonstrating that the
conjugation reaction is highly reproducible.
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.molpharmaceut.5b00432.
SEC results, dSEC purification and analysis, LC/MS
data, and PK and toxicology protocols and results (PDF)
ADCs represent a rapidly growing new class of targeted
chemotherapeutic agents for treatment of cancer. The recent
approvals of two new ADCs, Adcetris in 2011 and Kadcyla in
2013, has led to a marked increase in the number of ADCs
entering clinical trials. There are currently at least 40 different
ADCs in various phases of clinical evaluation, and the number
is increasing continuously. Although ADCs have demonstrated
promising antitumor activity in preclinical studies, the broad
range of pharmacological properties exhibited by conventional
Corresponding Author
*E-mail: [email protected] Phone: (650) 231-4327. Fax:
(650) 697-4900.
The authors declare no competing financial interest.
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Research reported in this publication was supported in part by
the National Cancer Institute of the National Institutes of
Health under Award No. R43CA171492. The content is solely
the responsibility of the authors and does not necessarily
represent the official views of the National Institutes of Health.
ADC, antibody−drug conjugate; DBM, dibromomaleimide;
MC, maleimidocaproyl; MMAF, monomethyl auristatin Phe;
PK, pharmacokinetics
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This paper was published ASAP on October 2, 2015. Three
additional references were added to the Discussion section and
the corrected version reposted on October 15, 2015.
DOI: 10.1021/acs.molpharmaceut.5b00432
Mol. Pharmaceutics 2015, 12, 3986−3998