Bridging disulfides for stable and defined antibody drug conjugates

Article abstract of DOI:10.1021/bc500148x

To improve both the homogeneity and the stability of ADCs, we have developed site-specific drug-conjugating reagents that covalently rebridge reduced disulfide bonds. The new reagents comprise a drug, a linker, and a bis-reactive conjugating moiety that is capable of undergoing reaction with both sulfur atoms derived from a reduced disulfide bond in antibodies and antibody fragments. A disulfide rebridging reagent comprising monomethyl auristatin E (MMAE) was prepared and conjugated to trastuzumab (TRA). A 78% conversion of antibody to ADC with a drug to antibody ratio (DAR) of 4 was achieved with no unconjugated antibody remaining. The MMAE rebridging reagent was also conjugated to the interchain disulfide of a Fab derived from proteolytic digestion of TRA, to give a homogeneous single drug conjugated product. The resulting conjugates retained antigen-binding, were stable in serum, and demonstrated potent and antigen-selective cell killing in in vitro and in vivo cancer models. Disulfide rebridging conjugation is a general approach to prepare stable ADCs, which does not require the antibody to be recombinantly re-engineered for site-specific conjugation.

Full text of DOI:10.1021/bc500148x

Article
pubs.acs.org/bc
Bridging Disulfides for Stable and Defined Antibody Drug
Conjugates
George Badescu, Penny Bryant, Matthew Bird, Korinna Henseleit, Julia Swierkosz, Vimal Parekh,
Rita Tommasi, Estera Pawlisz, Kosma Jurlewicz, Monika Farys, Nicolas Camper, XiaoBo Sheng,
Martin Fisher, Ruslan Grygorash, Andrew Kyle, Amrita Abhilash, Mark Frigerio, Jeff Edwards,
and Antony Godwin*
PolyTherics Ltd, The London Bioscience Innovation Centre, 2 Royal College Street, London NW1 0NH, United Kingdom
ABSTRACT: To improve both the homogeneity and the stability of ADCs, we have developed site-specific drug-conjugating
reagents that covalently rebridge reduced disulfide bonds. The new reagents comprise a drug, a linker, and a bis-reactive
conjugating moiety that is capable of undergoing reaction with both sulfur atoms derived from a reduced disulfide bond in
antibodies and antibody fragments. A disulfide rebridging reagent comprising monomethyl auristatin E (MMAE) was prepared
and conjugated to trastuzumab (TRA). A 78% conversion of antibody to ADC with a drug to antibody ratio (DAR) of 4 was
achieved with no unconjugated antibody remaining. The MMAE rebridging reagent was also conjugated to the interchain
disulfide of a Fab derived from proteolytic digestion of TRA, to give a homogeneous single drug conjugated product. The
resulting conjugates retained antigen-binding, were stable in serum, and demonstrated potent and antigen-selective cell killing in
in vitro and in vivo cancer models. Disulfide rebridging conjugation is a general approach to prepare stable ADCs, which does not
require the antibody to be recombinantly re-engineered for site-specific conjugation.
Cysteine residues are less common within a mAb and
therefore provide fewer sites for attachment and with higher
reactivity. An intact mAb IgG1 has four accessible disulfide
bonds that can be reduced to release eight free cysteine thiols
that can serve as sites for conjugation.3−5 This strategy is used
to attach MMAE to brentuximab to produce the other FDA
approved ADC known as Adcetris.5 The reaction between the
mAb and the maleimide-MMAE reagent yields an ADC
composed of a mixture of species with between 0 and 8 drug
molecules per antibody molecule.5,6 The formation of high drug
loaded species, e.g., greater than DAR 4, is undesirable as it has
been shown to lead to lower tolerability, higher plasma
clearance rates, and decreased efficacy in vivo.6
INTRODUCTION
■
Antibody drug conjugates (ADCs) are a growing class of
chemotherapeutic agents for the treatment of cancer,
combining the tumor targeting properties of antibodies with
the cell killing properties of potent cytotoxic drugs. Although
both monoclonal antibodies (mAbs) and cytotoxic agents can
be used independently to treat cancer, the covalent attachment
of a drug to a targeting antibody allows selective delivery and
minimizes systemic toxic side effects from exposure of the drug
to healthy tissue.
The clinically established approach to conjugating cytotoxic
compounds to antibodies is to use linkers that are reactive
toward either the amino side chains of lysine residues or the
thiol side chains of cysteines obtained from reducing interchain
disulfide bonds. Conjugation to lysine residues can be achieved
via reaction with reactive groups such as a succinimidyl moiety
to form a stable amide bond. This approach is used for the
covalent attachment of the maytansinoid derivative, DM1, to
TRA to produce the FDA approved Kadcyla.1 However, as a
mAb may have more than 80 solvent-accessible lysine residues,
heterogeneous mixtures with variable DAR species are
produced due to the numerous sites of conjugation available.2
Maleimide based conjugates have been shown to undergo
retro-addition reactions in the presence of competing thiols.7−9
In serum this can result in loss of the drug from the ADC by
exchange reaction with the free thiol group of circulating
albumin.7,9 Monoalkylation reagents also leave the original
Received: April 3, 2014
Revised: May 2, 2014
© XXXX American Chemical Society
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Figure 1. Conjugation of a payload to a disulfide bridge using a bis-alkylating conjugation approach involving a sequence of Michael addition and
elimination reactions.
Figure 2. Chemical structures of the bis-alkylating reagents 1, 2, and 3 loaded with either a cytotoxic payload (MMAE) or fluorescent dyes, Alexa
Fluor 488 and FITC, respectively. The fluorescent dyes are linked to the disulfide bridging linker via a noncleavable amide bond while MMAE is
linked by an enzyme cleavable valine citrulline dipeptide and self-immolative p-aminobenzyl moiety.
disulfides unbridged, potentially introducing instability to the
antibody.
specifically into TRA.14 Conjugation of a cytotoxic payload is
then achieved by using an alkoxy-amine derivatized drug to
form an oxime bond. Similar approaches are possible using
azide/alkyne “click” chemistry.15 Conjugation to unnatural
amino acids is a promising alternative to produce homogeneous
ADCs but has some limitations. Re-engineering mAbs to
incorporate unnatural amino acids is complex and costly. In
addition, the current methods reported for conjugation to form
an oxime use a large excess of up to 30 equiv of cytotoxic
reagent and have long reaction times of up to 4 days.14 When
an ADC is derived from mAbs that have previously undergone
clinical development, re-engineering may not be cost-effective
given that GMP manufacturing processes will already be in
place.
We have developed a novel way to prepare more
homogeneous and stable ADCs that does not require protein
re-engineering (Figure 1). This approach uses bis-sulfone
reagents that are selective for the cysteine sulfur atoms from a
native disulfide (Figure 2). These reagents undergo bis-
alkylation to conjugate both thiols derived from the two
cysteine residues of a reduced native disulfide bond16 such as
the interchain disulfide bonds of a mAb. The reaction results in
covalent rebridging of the disulfide bond via a three carbon
bridge leaving the protein structurally intact. This approach has
also been applied for the site-specific conjugation of poly-
(ethylene glycol) (PEG) to a wide range of therapeutic proteins
One alternative to overcome a wide DAR distribution is to
use mAbs with engineered cysteine residues. Antibodies known
as THIOMABs have been described with an engineered and
unpaired cysteine residue on each heavy or light chain designed
for conjugation of a cytotoxic payload.9−11 Each of the cysteine
residues provides a site for conjugation of a thiol-reactive drug
reagent. When conjugated to MMAE10 or DM111 using a
maleimide reagent, an ADC with a DAR close to 2 is produced.
While this approach can be used to produce more
homogeneous products it does not address issues associated
with the instability of maleimide conjugates in vivo. Engineered
cysteine thiols can also be capped during production such as by
a disulfide forming reaction with glutathione.10 Liberating a
capped thiol to make accessible for conjugation without
reducing interchain disulfides is nontrivial. In addition, the
engineered cysteine residues have been shown to form triple
light chain species during production.12,13
Another re-engineering approach is to incorporate unnatural
amino acids in the antibody as sites for conjugation and avoid
the use of maleimide chemistry. To improve specificity non-
natural amino acids can be introduced with functionalities that
enable conjugation chemistries to be used that are orthogonal
to those typically used for native amino acids. ADCs have been
synthesized by introducing a p-acetylphenylalanine residue site
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to enhance their pharmacokinetic properties while retaining
their activity.17,18
A cytotoxic drug molecule is attached to the bis-alkylating
linker via a PEG chain producing a reagent suitable for
generating ADCs. The reagent can undergo reaction at each of
the four interchain disulfides of a mAb if they are first reduced,
to produce DAR 4 ADCs. As the disulfides are reannealed the
resulting ADCs are more homogeneous and will have greater
stability compared to maleimide conjugates. Here we describe
the use of bis-alkylating conjugation of MMAE to TRA and a
TRA derived Fab (FabTRA) to produce biologically active model
drug conjugates.
RESULTS
■
Figure 3. SDS-PAGE analysis of a conjugation reaction mixture
between MMAE and FabTRA using the bis-alkylating reagent 1.
Unreduced FabTRA and the FabTRA-bisAlk-vc-MMAE conjugate each
run as a single band at 50 kDa, while the reduced protein runs as a
single band at around 25 kDa corresponding to the VHCH1 and VLCL1
chains.
Preparation of Drug Conjugates. Conjugation of both
cysteine thiols derived from a reduced disulfide bond using the
bis-alkylating approach is carried out in two steps and
analogous to a standard maleimide conjugation process,
namely, (1) disulfide reduction to release the two cysteine
thiols followed by (2) conjugation under mild conditions.
Conjugation by bis-alkylation leads to the formation of a three-
carbon bridge relinking the two cysteine residues to which the
drug payload is covalently attached.18 The three-carbon bridge
is small enough and sufficiently flexible not to perturb the
tertiary structure of the antibody.20
The MMAE bis-alkylating reagent 1 was prepared with a
Cathepsin B cleavable valine citrulline p-aminobenzyl ether
linker designed to allow release of MMAE intracellularly from
the ADC. A small ethylene glycol spacer of 24 repeat units and
discrete mass was also incorporated into the reagent to improve
the water solubility and enable more efficient conjugation. It
was not possible to perform the conjugation without PEG
present in the reagent due to low solubility in the conjugation
media. It was also hoped that the incorporation of PEG would
reduce the propensity of the resulting ADCs to aggregate and
potentially improve the pharmacokinetic profile of the
conjugates in the case of the Fab. A PEG size of 24 repeat
units was chosen as this was one of the largest sizes of PEG
available with a discrete mass.
TRA was selected as a model antibody to explore the
preparation of ADCs by bis-alkylation because of well-
established in vitro and in vivo models. In addition to the
TRA ADC, a Fab drug conjugate (FabTRA-bisAlk-vc-MMAE)
was prepared which, when analyzed by SDS-PAGE, gave
supporting evidence that the bis-sulfone functionality of
MMAE reagent 1 was able to rebridge an interchain disulfide.
Purified FabTRA was first treated with DTT to reduce the
accessible single interchain disulfide17 and conjugation was
performed after removal of residual DTT. Analysis by SDS-
PAGE showed that conjugation conversion was nearly
quantitative (95%) using only 2 equiv of reagent 1 with only
a trace amount of reduced FabTRA remaining in the reaction
mixture (Figure 3). When 1.5 equiv of reagent 1 was used, 88%
conversion was observed. Unreduced FabTRA ran as a single
band at 50 kDa (Lane 2), but the reduced FabTRA, after
treatment with DTT, shifted in apparent molecular weight
running as a single band at 25 kDa (Lane 3). This band
corresponded to the VHCH1 from the heavy chain and the VLCL
from the light chain which dissociate in the presence of SDS
after reduction of the disulfide and migrate together as they are
the same size. In contrast, the FabTRA-bisAlk-vc-MMAE
conjugate migrated as a single band at 50 kDa indicating that
the disulfide had been bridged by the bis-alkylating ADC
reagent (Lane 4).
The rebridging of the FabTRA interchain disulfide bond using
reagent 1 was further confirmed by running SDS-PAGE under
reducing conditions (Figure 4a) with no dissociation of the
heavy and light chain fragments observed as would be expected
for unconjugated FabTRA. This result also demonstrated that the
bis-thioether bonding to the FabTRA was more stable than the
original disulfide bond as the sample survived incubation with
10 mM DTT for 30 min prior to loading on the gel, which
might be expected to cause dissociation of the reagent from the
FabTRA leading to heavy and light chain dissociation. Analysis of
the FabTRA-bisAlk-vc-MMAE conjugate by SE-HPLC (Figure
4b) indicated high purity with no indication of any product
aggregation. RP-HPLC also showed that a single product was
formed with a purity greater than 95% (Figure 4c).
Conditions similar to the preparation of FabTRA-bisAlk-vc-
MMAE were used to conjugate reagent 1 to TRA forming
TRA-bisAlk-vc-MMAE. Although there is a total of 16 disulfide
bonds in an intact IgG1, the 12 intrachain disulfide bonds are
less readily reduced and are less affected even by high
concentrations of reducing agents.21 The four accessible
interchain disulfide bonds can therefore be used for conjugation
under moderate reducing conditions. Analytical HIC was used
to analyze the reaction mixtures, as conjugation with MMAE
gave sufficient separation of the different DAR species to
determine the overall DAR distribution. Varying the concen-
tration of reductant and the stoichiometries of reactants (mAb
to reagent 1), it was possible to affect the DAR distribution.
When 6 equiv of MMAE reagent 1 were added to fully reduced
TRA it was possible to achieve 78% of the DAR 4 variant and
no unconjugated TRA (Figure 5). Lower average DAR could
also be achieved if desired by adapting reaction conditions. A
sample was prepared with an average DAR of 2.8 and with 2−4
drug molecules conjugated accounting for greater than 95% of
the whole sample. A minimal amount of unconjugated mAb
remained (<1%).
Stability of Conjugation. It has been reported that
instability of maleimide conjugates in serum results in
deconjugation of the drug and subsequent cross conjugation
of the drug to other thiol containing species including serum
albumin.7,9 To determine whether similar cross conjugation
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Figure 4. Analytical characterization of purified FabTRA-bisAlk-vc-MMAE: (a) SDS-PAGE analysis demonstrating disulfide bridging. After reduction
the FabTRA-bisAlk-vc-MMAE conjugate remains as a single band at 50 kDa indicating that the disulfide was bridged by the ADC reagent as the
interchain disulfide is no longer reducible. (b) SE-HPLC analysis and (c) RP-HPLC analysis demonstrating the purity and homogeneity of the Fab
drug conjugate which elutes as single peak under both analytical techniques.
Figure 5. HIC analysis of TRA and bis-alkylating MMAE reagent 1 reaction mixture. (a) HIC chromatogram, (b) histogram showing DAR
distibution based on relative peak areas from HIC chromatogram.
occurred for conjugates prepared with the bis-alkylating linker,
a serum stability study was conducted using an Alexa Fluor 488
fluorescently labeled bis-alkylation reagent 2 conjugated to
TRA. A maleimide functionalized Alexa Fluor 488 reagent was
conjugated to TRA as a control. Both conjugates were
incubated in either 50% rat or undiluted human serum for up
to 96 h at 37 °C. Serum samples were analyzed by SE-HPLC
with fluorescence detection to monitor the liberation of free
reagent and/or formation of albumin-Alexa Fluor 488 adducts.
For the bis-alkyl TRA conjugate prepared using Alexa Fluor
488 labeled reagent 2, the SE-HPLC chromatogram remained
largely unchanged over 96 h incubation in both rat and human
serum indicating that the disulfide conjugation was extremely
stable to serum exposure (Figure 6). The main peak eluted at
11.0 min corresponding to the starting TRA-Alexa Fluor 488
conjugate. In contrast, incubation of the TRA maleimide Alexa
Fluor 488 conjugate in both rat and human serum resulted in
chromatograms with very different profiles after 96 h. The peak
corresponding to the TRA maleimide conjugate at 11.0 min
decreased in peak area by 47% (rat) or 24% (human) and
several new peaks were observed. New peaks eluting at 11.9
and 16.3 min corresponded to an albumin adduct and
deconjugated Alexa Fluor 488 dye, respectively. The albumin
adduct, visible in both species’ serum, indicated that
deconjugation and cross conjugation to albumin from the
maleimide derived TRA conjugate had occurred.
incubations indicated the formation of aggregates. A second
new peak observed eluting at 13.3 min was consistent with a
light chain-Alexa Fluor 488 conjugate based on comparison
with molecular weight standards. This suggests that there was
some dissociation of TRA-Alexa Fluor 488 conjugate over time
as the heavy and light chains were not covalently linked
through the interchain disulfide bonds. These signs of antibody
instability were not observed for the conjugate prepared using
the disulfide rebridging reagent 2.
Experiments were also conducted to monitor the change in
DAR over time for MMAE TRA conjugates incubated in an
artificial serum composed of 20 mg/mL HSA in PBS. Artificial
serum was used as endogenous IgGs in whole serum would
overlap with the conjugate peaks in the analytical HIC method
used for DAR analysis.
The drug was conjugated to TRA either using MMAE
reagent 1 or maleimide-val-cit-PAB-MMAE. The reactions
yielded conjugates with an average DAR of 2.3 for the bis-
alkylating reagent and 3.2 for the maleimide reagent. Each
conjugate at a final concentration of 1.0 mg/mL was incubated
at 37 °C for up to 120 h and loss of MMAE was monitored by
measuring the change over time of HIC peak areas
corresponding to each DAR species. The conjugate prepared
using reagent 1 showed no decrease in DAR after 120 h. In
contrast, analysis of the incubation mixtures of the maleimide
conjugate showed that there was a decrease in the average DAR
indicating loss of MMAE with time (Figure 7). It is likely that
the deconjugated MMAE cross-conjugated to the HSA present
In addition to linker deconjugation, some evidence of
antibody instability was also seen with the maleimide
conjugates. A new early peak eluting at 8.5 min for both sera
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Figure 6. Serum stability of TRA-Alexa Fluor 488 conjugates prepared using either bis-alkylating or maleimide conjugation; maleimide conjugate
incubated for (a) 0 h, (b) 96 h in rat serum, (c) 96 h in human serum; bis-alkylation conjugate incubated for (d) 0 h, (e) 96 h in rat serum, (f) 96 h
in human serum. The peaks were identified as follows: peak 1, Trastuzumab-Alexa Fluor 488 conjugate; peak 2, albumin-Alexa Fluor 488 conjugate;
peak 3, released Alexa Fluor 488; peak 4, aggregated conjugate; peak 5, light chain-Alexa Fluor 488 conjugate.
in the mixture, although this was not detected using this
method.
Both mAb and Fab conjugates were found to have retained
binding activity which was comparable to the unconjugated
parent molecule (Figure 8) demonstrating that the bis-
alkylating linker has a minimal impact on receptor binding.
Internalization of Conjugates. Confocal microscopy was
used to monitor internalization of both TRA and FabTRA
conjugates prepared using the FITC labeled bis-alkylating
reagent 3. Following incubation with SK-BR-3 cells at 37 °C for
4 h, visualization of the cells showed that both Fab and mAb
conjugates had been internalized as efficiently as the
unconjugated antibody (Figure 9). No internalization was
observed in the A549 cell line (data not shown).
Retained Binding Activity of Conjugates Prepared
Using Bis-Alkylating Reagents. The binding of TRA and
TRA MMAE conjugates to the extracellular domain of HER2
was measured by ELISA to determine whether antigen specific
binding was retained after conjugation.
In Vitro Cytotoxicity of Conjugates Prepared Using
the Bis-Alkylating Linker 1. The in vitro potency of TRA-
bisAlk-vc-MMAE (DAR 2.8) and FabTRA-bisAlk-vc-MMAE
(DAR 1) conjugates was determined using the HER2-positive
human breast cancer cell lines, SK-BR-3 and BT-474, and the
antigen-negative cell lines, A549 and MCF-7.
FabTRA-bisAlk-vc-MMAE was found to reduce viability of SK-
BR-3 cells and its activity was found to match that of free
MMAE (Figure 10). No antiproliferative activity was observed
for unconjugated FabTRA in the concentration range tested
(data not shown). When tested in an antigen-negative cell line,
Figure 7. Change in DAR over time for ADCs incubated with HSA as
measured by analytical HIC. The DAR of the maleimide based
■
conjugate (red - -) is seen to decrease over time while little change is
●
observed in the TRA-bisAlk-vc-MMAE DAR (blue - -).
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Figure 8. Antibody−drug conjugates obtained using bis-alkylating MMAE reagent 1 retained the specific antigen-binding of the naked antibody
(ELISA) in both Fab and mAb formats.
Figure 9. Antibody conjugates obtained using the disulfide bis-alkylating approach internalize into HER2-positive cells (SK-BR-3). (a) Trastuzumab
detection with an anti-human mAb conjugated to Alexa Fluor 488. (b) TRA-bisAlk-FITC conjugate. (c) FabTRA-bisAlk-FITC conjugate.
A549, the potency of FabTRA-bisAlk-vc-MMAE was about 3
orders of magnitude lower than that of free MMAE.
activity. FabTRA-bisAlk-vc-MMAE was well tolerated and
fluctuations in body weight were similar between groups.
In the TRA-bisAlk-vc-MMAE study, mice were treated with
10 mg/kg, 20 mg/kg, or 30 mg/kg based on TRA with dosing
every fourth day for up to 21 days. The treatment regimen was
chosen based on published BT-474 xenograft studies with
trastuzumab based ADCs and because the specific BT-474
model used was particularly fast-growing in comparison.
Dosing was stopped when tumor volume reached less than
10% of the day 1 mean volume so the number of doses received
were 4, 5, and 6 for the 30 mg/kg, 20 mg/kg, and 10 mg/kg
groups, respectively. Control groups were treated with either 20
mg/kg TRA, 0.4 mg/kg free MMAE, or vehicle on the same
dosing schedule.
TRA-bisAlk-vc-MMAE was well tolerated with no dose
related effect on body weight observed. Tumor growth was
delayed at all three doses and the conjugate demonstrated a
dose-related efficacy in the model (Figure 13). Apparent
curative activity was attained at the two higher doses which
each elicited a TGD of 30.2 days with 10 out of 10 tumor-free
survival. The 10 mg/kg dose also resulted in the maximum
TGD of 30.2 days with tumor regression but no tumor-free
survival. As expected, only a marginal tumor growth delay was
obtained for groups treated with TRA or free drug.
The TRA-bisAlk-vc-MMAE conjugate was found to have
potent antiproliferative activity in both antigen positive cell
lines (Figure 11). In the antigen negative cell lines, the
conjugate was found to decrease viability but the potency was
about 3 orders of magnitude lower than that of free drug
supporting antigen selective delivery of the MMAE.
In Vivo Efficacy of TRA and FabTRA - MMAE
Conjugates. The ability of the TRA-bisAlk-vc-MMAE (DAR
2.8) and FabTRA-bisAlk-vc-MMAE (DAR 1) conjugates to
inhibit tumor growth in vivo was evaluated in a BT-474 breast
cancer model. The conjugates were administered to female
CB.17 SCID mice bearing subcutaneous BT-474 tumor
xenografts.
In the FabTRA-bisAlk-vc-MMAE conjugate study, mice were
treated with 20 mg/kg of the Fab conjugate with dosing on
alternate days over 25 days. Control groups were treated with
either 20 mg/kg TRA (every fourth day), 0.3 mg/kg free
MMAE (alternate days), or vehicle (alternate days). For
FabTRA-bisAlk-vc-MMAE conjugate and free MMAE dosed
groups each dose was equivalent with respect to the amount of
MMAE administered.
FabTRA-bisAlk-vc-MMAE was found to be highly active
against the BT-474 xenografts resulting in a marked delay in
tumor growth (Figure 12). Treatment with FabTRA-bisAlk-vc-
MMAE resulted in a median time to end point (TTE) of 66.3
days, corresponding to a tumor growth delay (TGD) of 48.7
days. In contrast, TRA (TTE 24.6 days) and free drug (TTE
21.8 days) each elicited only marginal TGDs of 7.0 days and 4.2
days, respectively, which did not translate to meaningful
DISCUSSION
■
ADCs are a proven strategy for improving the therapeutic
window of cytotoxic drugs. The high levels of toxicity
associated with the administration of highly potent dolasta-
tin22,23 and maytansinoids24−26 has prevented their use as
therapies. Despite the limitation of these compounds as small
molecule cytotoxic agents, their derivatives MMAE and DM1
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Figure 10. In vitro potency of FabTRA-bisAlk-vc-MMAE. (a,b) Potent antiproliferative effect is seen for both the conjugate and free drug in HER2
positive cell lines SK-BR-3 and BT-474. (c,d) In antigen negative cell lines the conjugate was found to decrease viability but the potency was two to
three orders of magnitude lower than that of free drug depending on the cell line. (e) IC50 values for free drug and conjugate in both antigen positive
and negative cell lines.
have demonstrated clinical efficacy when incorporated into the
approved ADCs, Adcetris27,28 and Kadcyla,29 respectively.
Despite the clinical success of many ADCs in both
hematological and solid tumors, they are far from the promise
of a “magic bullet”. Most clinical-stage ADCs have very similar
maximum tolerated doses with dose limiting toxicities due to
off-target toxicity. Strategies to produce ADCs with a wider
therapeutic window need to focus on improving efficacy and
tolerability, which is linked to having better defined ADC
structures. ADCs with more than four drug molecules are more
toxic, clear faster, and are less effective.6 Although higher drug
loading has been found to increase potency in vitro5,30,31 this
does not translate to a marked improvement of efficacy in vivo.6
In experiments investigating the in vitro cytotoxicity of MMAE
conjugates of cAC10 (brentuximab), an anti-CD30 mAb, a
trend in potency was observed of DAR8 > DAR4 > DAR2.6
However, in vivo the higher loaded ADC was found to have
lower activity than the DAR 4 and DAR 2 species at an
equivalent drug dose. This unexpected difference in potency is
attributed to the different pharmacokinetic properties of the
conjugates. The ADC with eight drug molecules loaded was
found to clear faster than the lower DAR conjugates resulting in
reduced exposure.
drug molecules attached.5,6,31 A lower average DAR can be
achieved after partial reduction, but this still yields a
distribution of conjugates with up to 30% of the ADC bearing
over four drug molecules. Each of the individual DAR species
within this mixture is also likely to be a mixture of different
isomers with different conjugation sites and potentially different
pharmacological properties. Although problems with broad
DAR distribution can be overcome by engineering new sites for
conjugation (e.g., cysteine residues or non-native amino acids)
the re-engineering involved may lead to increased development
timelines and problems with manufacturability/scalability and
ultimately increased costs.
We have shown that disulfide bridging reagents can be used
as an alternative thiol conjugation method to mono-alkylation
that results in a narrower DAR distribution and can produce an
ADC with DAR 4 as the major product. The TRA-bisAlk-vc-
MMAE ADC prepared contained only low amounts (<1%) of
unconjugated antibody. Optimal ADCs will contain little or no
unconjugated parent antibody since this can act as a
competitive inhibitor, preventing binding of the ADC to the
target cell.
The conjugates formed using the bis-alkylating linker were
stable in serum and in the presence of high concentrations of
albumin over 5 days. Maleimide conjugates by comparison were
shown to be unstable with cross conjugation reactions to
albumin occurring. In a study comparing conjugates with
different sites of conjugation and associated differences in
stability, the more stable ADCs elicited better efficacy and
Current strategies for preparing ADCs via conjugation to
naturally occurring amino acids produce heterogeneous
mixtures with varying amounts of drug conjugated.2,6
Maleimide conjugation of mAbs after full reduction of the
interchain disulfide bonds generates ADCs with up to eight
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Figure 11. In vitro potency of TRA-bisAlk-vc-MMAE ADC (DAR 2.8). (a,b) Potent antiproliferative effect is seen for both the conjugate and free
drug in HER2 positive cell lines SK-BR-3 and BT-474. (c,d) In antigen negative cell lines the conjugate was found to decrease viability but the
potency was 2 to 3 orders of magnitude lower than that of free drug depending on the cell line. (e) IC50 values for free drug and conjugate in both
antigen positive and negative cell lines.
Figure 12. In vivo efficacy of FabTRA-bisAlk-vc-MMAE (DAR = 1).
survival in in vivo cancer models.9 Instability leading to
deconjugation disarms the ADC resulting in increasing
amounts of naked antibody which reduces efficacy, acts as a
competitive inhibitor, and is also likely to have a longer half-life
and thus may compromise subsequent treatment cycles.
Figure 13. In vivo efficacy of TRA-bisAlk-vc-MMAE ADC (DAR =
2.8).
Whole molecule IgGs may be limited in their tumor
penetration due to their large size restricting diffusion and
extravasion from the vasculature.32 One approach to improve
penetrability is to use smaller antibody fragments, such as
domains, scFvs, minibodies, and other engineered formats.33,34
A limitation of these smaller formats is that their circulation
half-lives are too short for sufficient tumor exposure and
accumulation. By contrast, whole IgGs have very long
circulation half-lives due to FcRn mediated recycling. However,
the longer an ADC is in circulation, the greater the chance it
will bind off-target sites and degrade, which may lead to
increased systemic toxicity. An optimal half-life must balance
the requirements for sufficient tumor exposure and accumu-
H
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lation while ensuring ultimate elimination pathways limit
toxicity. Many studies have shown the utility of PEG in
improving the biodistribution of fragment antibodies for drug
conjugate and tumor radioimaging applications, often with
relatively small PEG sizes of less than 5 kDa that increase the
apparent size of the fragment to above the renal filtration cutoff
threshold.35 Avoiding kidney exposure and directing clearance
toward the liver, which has greater capacity for detoxification,
may be beneficial in improving tolerability. With the disulfide
conjugation approach described here, modifying the length of
the PEG chain incorporated between the reactive bis-sulfone
and the drug would confer increased half-life to a Fab conjugate
as can be achieved by protein PEGylation,38,39 allowing the
optimization of the pharmacokinetic profile of the Fab for drug
conjugate use.
Monovalent antibody formats may also avoid the antigen
barrier which has been proposed as a factor contributing to the
limited penetration of mAbs in many solid tumors due to their
bivalent and high affinity binding.36 Also, the bivalent binding
of antibodies may cross-link antigens and lead to unwanted
activation of signaling pathways. One example is the agonistic
effect seen for DN-30 anti-Met bivalent antibody but not as a
Fab or single Fab arm antibody.37,40
mented with 10% fetal bovine serum (FBS), 100 U/mL
penicillin, and 100 μg/mL streptomycin. All media were
obtained from Life Technologies. Cell lines were grown at 37
°C and 5% CO2 in a CO2 air-jacket incubator (Binder).
The extracellular domain of the HER2 receptor (ECD/
HER2) was purchased from Stratech Scientific Ltd., UK. Nunc
Maxisorp microtiter plates were purchased from Thermo
Scientific. Anti 6xHis antibody conjugated to HRP was
purchased from Clontech Laboratories Inc.
Preparation of Bis-Sulfone-PEG(24)-COOH. Bis-sulfone-
PEG(24)-COOtBu was prepared from H2N-PEG(24)-COOt-
Bu according to the method described previously for methoxy
PEG bis-sulfone synthesis.18,19 Removal of the t-butyl
protecting group was achieved by stirring the bis-sulfone-
PEG(24)-COOtBu (0.98 g, 0.58 mmol) in 50% trifluoroacetic
acid in DCM (8 mL) at room temperature for 2 h. The solvents
were removed under reduced pressure, then the residue was
dissolved in acetone (30 mL). The solution was filtered
through a sintered glass filter to remove insoluble material, then
cooled in a dry ice bath to give a white precipitate. The
precipitate was collected by centrifugation (4566 g, 30 min).
The resulting off-white solid was dried under vacuum (0.82 g,
1
85%). H NMR (400 MHz, CDCl3) δ 2.49 (s, 6H, Ar−CH3),
The conjugation of a cytotoxic payload via a three carbon
disulfide bridge to produce a biologically active Fab drug
conjugate was exemplified using a Fab derived from TRA. A
Fab fragment has a single interchain disulfide that is readily
reduced. After reduction of FabTRA, MMAE was successfully
conjugated using the bis-alkylating reagent 1. The resulting
drug conjugate was homogeneous with respect to DAR with a
single drug molecule conjugated, and was structurally intact
with the disulfide bridged by the linker.
Conjugation of MMAE to a mAb or Fab using the bis-
alkylating reagent approach was shown to produce effective
ADCs. The conjugates retained binding and demonstrated
antigen-selective in vitro cytotoxicity. Both mAb and Fab
conjugates were also found to be well tolerated and efficacious
in vivo. We believe bis-alkylation is a general approach to
producing stable and more homogeneous ADCs that is efficient
and predictable and does not rely on reengineering of the
antibody for site-specific conjugation.
2.59 (t, 2H, PEG−CH2−COOH), 3.47−3.79 (m, PEG), 4.33
(m, 1H, SO2−CH2−CH−CH2−SO2-), 7.03 (s, 1H, PEG-NH-
carbonyl), 7.36 (d, 4H, SO2Ar), 7.66 (d, 2H, COAr), 7.69 (d,
4H, SO2Ar), 7.81 (d, 2H, COAr).
Synthesis of Bis-Sulfone-PEG(24)-MMAE (Reagent 1).
HATU (14 mg, 0.036 mmol) was added to a stirred suspension
containing Na2CO3 (4 mg, 0.036 mmol), H2N-val-cit-PAB-
MMAE (53 mg, 0.048 mmol), and bis-sulfone-PEG(24)-
COOH (40 mg, 0.024 mmol) in anhydrous DCM/DMF
(3:1, 1 mL) and stirred for 48 h under an argon atmosphere.
The crude mixture was purified using an anion exchange
column eluting with MeOH followed by column chromatog-
raphy eluting with MeOH/DCM (5:95 v/v). Compound 1 was
isolated as a colorless solid (46 mg, 71%). 1H NMR (400 MHz,
CDCl3) δ 0.60−0.99 (m, aliphatic side chains), 2.43 (s, Me-Ts),
3.36−3.66 (m, PEG), 7.15−7.28 (m, Ar), 7.31 (d, J = 8.3 Hz,
Ar), 7.54−7.62 (m, Ar), 7.79 (d, J = 8.3 Hz, Ar).
Preparation of Boc-NH-PEG(2 kDa)-FITC. Boc-NH-PEG(2
kDa)-NH2 (107 mg, 0.056 mmol) was dissolved in DCM (1
mL). Triethylamine (8.6 μL, 0.062 mmol) was then added
followed by FITC (100 mg, 0.25 mmol) in DMF (1 mL). The
resulting mixture was stirred at room temperature under an
argon atmosphere for 24 h. Volatile solvents were removed by
evaporation, then the product was precipitated by addition of
acetone (20 mL). The precipitate was collected by
centrifugation (4566 g, 30 min), then dried under vacuum to
EXPERIMENTAL PROCEDURES
■
Materials. TRA (Herceptin) was produced by Roche.
Maleimide-val-cit-PAB-MMAE and NH2-val-cit-PAB-MMAE
were purchased from Concortis Biosystems. Boc-NH-PEG(2
kDa)-NH2 was purchased from Creative PEGWorks. Mal-
eimide-C5 and all other PEG precursors were purchased from
IRIS Biotech Gmbh. Alexa Fluor 488 C5-maleimide and Alexa
Fluor 488 NHS ester were purchased from Invitrogen. All other
reagents and solvents were purchased from Acros Organics or
Sigma-Aldrich and used as received. 1H NMR data were
collected using a Bruker 400 or 600 MHz spectrometer in the
NMR solvent stated. Chemical shifts δ are given in ppm
downfield from tetramethylsilane.
1
afford a yellow solid (76 mg, 59%). H NMR (400 MHz,
CDCl3) δ 1.40 (s, 9H, t-Bu), 3.20−3.28 (m, 2H, PEG), 3.39−
3.44 (m, 1H), 3.47−3.51 (m, 2H), 3.52−3.72 (m, 183H, PEG),
3.73−3.87 (m, 2H), 5.20 (br s, 1H), 6.50 (dd, 2H, J = 8.6, 2.3
Hz), 6.58 (d, 2H, J = 8.6 Hz), 6.67 (d, 2H, J = 2.3 Hz), 7.03 (d,
1H, J = 8.3 Hz), 7.53−7.58 (m, 1H), 7.94 (dd, 1H, J = 8.3,1.7
Hz), 8.09 (d, 1H, J = 1.7 Hz), 9.22 (br s, 2H), 9.44 (s, 1H,
NH).
Preparation of H2N-PEG(2 kDa)-FITC. Boc-NH-PEG(2
kDa)-FITC (73 mg, 0.031 mmol) was dissolved in DCM
(3.6 mL), then trifluoroacetic acid (0.4 mL) was added and the
resulting mixture stirred at room temperature for 2 h. Volatiles
were removed by evaporation under reduced pressure. The
crude product was dissolved in acetone (20 mL). The solution
All cell lines were purchased from the American Type
Culture Collection (ATCC), except for A549 which was
purchased from the Health Protection Agency Culture
Collection (HPACC). Cells were maintained in McCoy’s 5a
(SK-BR-3), DMEM:F12 (BT-474), Dulbecco’s modified Eagle
medium (DMEM) supplemented with 200 mM glutamine
(A549), or Minimum Essential Medium (MEM) supplemented
with 10 μg/mL insulin (MCF-7). All media were comple-
I
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was filtered through a sintered filter to remove insoluble
material, then cooled in a dry ice bath to give a yellow
precipitate. The precipitate was collected by centrifugation
(4566 g, 30 min), then dried under vacuum to afford a yellow
3.58−3.68 (m, 46H, CH2OPEG + PhSO2CH2), 4.28−4.35 (m,
1H, CH), 7.54 (br, 1H, NH), 7.34, 7.64 (AB dd, SO2Ar, 8H, J =
8.4 Hz), and 7.61, 7.82 (AB dd, COAr, 4H, J = 8.5 Hz).
Preparation of Bis-Sulfone-PEG(12) Amine TFA Salt. The
bis-sulfone-PEG(12)-NH-Boc (0.147 g, 125 μmol) was
dissolved in formic acid (2 mL) and the resulting mixture
was stirred at room temperature. After 16 h, the reaction was
concentrated under vacuum and the residue was resuspended in
water (1.5 mL), and purified by reverse-phase (C-18)
chromatography (gradient elution H2O/MeCN buffers con-
tended 0.1% TFA, 0% to 100%) to afford the desired bis-
sulfone-PEG(12) amine as a TFA salt (0.097 g, 68%). 1H NMR
(600 MHz CDCl3,) δ 2.46 (s, 6H, PhCH3), 3.16 (m, 2H,
CH2O), 3.48−3.52 (m, 2H, CH2O), 3.59−3.73 (m, 46H,
CH2OPEG + PhSO2CH2), 3.80 (m, 2H, CH2O), 4.28−4.34
(m, 1H, CH), 7.53 (br, 1H, NH), 7.36, 7.66 (AB dd, SO2Ar,
8H, J = 8.4 Hz), and 7.64, 7.86 (AB dd, COAr, 4H, J = 8.5 Hz).
Preparation of Bis-Sulfone-PEG(12)-Alexa Fluor 488
(Reagent 2). To a mixture of bis-sulfone-PEG(12) amine
(0.005 g, 4.80 μmol), Alexa Fluor 488 NHS ester (0.0025 g,
3.88 μmol), and anhydrous DMF (0.5 mL) under an argon
atmosphere was added sodium bicarbonate (0.0010 g, 11.9
μmol), and the reaction mixture was stirred at room
temperature. After 56 h, the reaction mixture was neutralized
by adding acetic acid (50 μL), purified by reverse-phase (C-18)
chromatography (gradient elution H2O/MeCN buffers con-
tended 0.1% TFA, 0% to 100%) to afford the desired bis-
sulfone-PEG(12)-Alexa Fluor 488 (0.002 g, 33%) as a red solid.
Purity by HPLC at 488 nm 91.2%. ESI-MS, m/z: 1543.1 (10%)
[M + 1H]+; 772.1 (100%) [M + 2H]2+.
1
solid (47 mg, 76%). H NMR (400 MHz, CDCl3) δ 3.15 (s,
2H, PEG), 3.45−3.94 (m, 167H, PEG), 6.59 (dd, 2H, J = 8.6,
2.3 Hz), 6.66 (d, 2H, J = 8.6 Hz), 6.78 (d, 2H, J = 2.3 Hz), 7.07
(d, 1H, J = 8.3 Hz), 7.76 (br, 2H, NH), 7.94 (d, 1H, J = 8.3
Hz), 8.29 (s, 2H), 9.31 (br, 2H, PhOH).
Preparation of Bis-Sulfone-PEG(2 kDa)-FITC (Reagent 3).
4-[2,2-Bis[(p-tolylsulfonyl)methyl]acetyl) benzoic acid-NHS
ester (bis-sulfone NHS) was prepared according to a previously
described method.18 H2N-PEG(2 kDa)-FITC (23 mg, 0.010
mmol) was dissolved in DCM (6 mL) with the bis-sulfone
NHS ester (24.9 mg, 0.042 mmol) and pyridine (38 μL). The
resulting mixture was stirred at room temperature under argon
for 48 h. Volatiles were removed by evaporation under reduced
pressure. The crude product was dissolved in acetone (20 mL).
The solution was filtered through a sintered filter to remove
insoluble material, then cooled in a dry ice bath to give a yellow
precipitate. The precipitate was collected by centrifugation
(4566 g, 30 min), then dried under vacuum to afford a yellow
solid (15 mg, 60%). 1H NMR (400 Hz, CDCl3) δ 2.49 (s, 6H,
PhCH3), 3.11 (s, 1H, PEG), 3.45−3.94 (m, 167H, PEG), 4.35
(m, 1H, CH), 6.59 (dd, 2H, J = 8.7, 2.3 Hz), 6.67 (d, 2H, J =
8.6 Hz), 6.77 (d, 2H, J = 2.2 Hz), 7.07 (d, 1H, J = 8.3 Hz), 7.37
(d, 4H, J = 8.0 Hz), 7.66 (d, 2H, J = 8.4 Hz), 7.71 (d, 4H, J =
8.0 Hz), 7.75−7.81 (m, 2H), 7.84 (dd, 2H, J = 8.4 Hz), 7.87−
7.92 (m, 1H), 8.00−8.03 (m, 1H), 8.14−8.28 (m, 2H), 9.0 (br
s, 1H).
Preparation of 4-(2,2-Bis[(p-tolylsulfonyl)methyl]acetyl)
Benzoic Acid HOBt Ester. Under an argon atmosphere, a
stirred mixture of 4-(2,2-bis[(p-tolylsulfonyl)methyl]acetyl)
benzoic acid (1.00 g, 2.0 mmol), prepared as described
previously,16,18 1-hydroxybenzotriazole (0.27 g, 2.0 mmol),
and anhydrous tetrahydrofuran (20 mL) were cooled using an
ice bath. Neat 1,3-diisopropylcarbodiimide (313 μL, 2.0 mmol)
was then added dropwise at ∼0 °C. The reaction mixture was
stirred for 20 min, the ice-bath was then removed, and the
reaction mixture stirred at room temperature. After 16 h, the
resulting precipitate was isolated using a no. 3 sintered glass
funnel and washed with tetrahydrofuran (2 × 5 mL) and dried
in vacuo. The crude solid was suspended in methanol (15 mL)
and collected by filtration after stirring for 1 h to afford the
Preparation of FabTRA. Digestion buffer (20 mM NaH2PO4
pH 7.0, 10 mM EDTA, 50 mM cysteine, 50 mL) was warmed
to 37 °C. TRA (150 mg) was resuspended in ultrapure water
(7.5 mL). An aliquot (5 mL, 100 mg) of the resulting solution
was then diluted with the prewarmed digestion buffer (40 mL).
Papain (5 mL, 1 mg/mL) was then added to the TRA solution
to give a TRA:papain ratio of 20:1 (w/w). The mixture was
mixed gently then incubated at 37 °C for 30 min with gentle
mixing. Purification was carried out by affinity chromatography
on a protein L column (Pierce) followed by hydrophobic
interaction chromatography using a XK 16/10 column (GE
Healthcare) packed in-house with Toyopearl, Phenyl-650S
resin (Tosoh Bioscience LLC).
Conjugation to FabTRA Using Bis-Alkylating MMAE
Reagent 1. Conjugation of reagent 1 to FabTRA was carried
out using methods described previously for conjugation of PEG
to disulfide bonds using the bis-alkylating linker.17,18
Conjugation to TRA Using Bis-Alkylating Reagents.
Conjugation of bis-alkylating MMAE reagent 1, bis-alkylating
Alexa Fluor 488 reagent 2, or bis-alkylating FITC-reagent 3 to
TRA was carried out using methods described previously for
conjugation of PEG to disulfide bonds.17,18
Conjugation to TRA Using Maleimide-val-cit-PAB-MMAE.
TRA (10 mg/mL) in 20 mM sodium phosphate pH 8.0, 150
mM NaCl, 20 mM EDTA was treated with tris(2-
carboxyethyl)phosphine (TCEP, 2 mol equiv) at 40 °C for
60 min. The reductant was then removed by buffer exchange
into fresh pH 8.0 phosphate buffer by gel filtration (PD-10
column, GE Healthcare). Determination of free thiols using
5,5′-dithiobis(2-nitrobenzoic acid) showed that there were
approximately four SH groups per mAb. Maleimide-val-cit-
PAB-MMAE (1.5 equiv per thiol) in DMSO (final concen-
tration 5% v/v) was added and the resulting mixture incubated
1
desired active HOBt ester (0.84 g, 68%) as a white solid. H
NMR (600 MHz CDCl3,) δ 2.49 (s, 6H, PhCH3), 3.52 (m, 2H,
PhSO2CH2), 3.65 (m, 2H, PhSO2CH2), 4.49 (q, 1H, CH, J =
6.5 Hz), 7.39, 7.74 (AB dd, SO2Ar, 8H, J = 8.6 Hz), 7.49 (m,
2H, PhH, HOBt), 7.60 (m, 1H, PhH, HOBt), 7.93, 8.31 (AB
dd, COAr, 4H, J = 8.4 Hz), 8.14 (d, 1H, PhH, HOBt, J = 9.2
Hz).
Preparation of Bis-Sulfone-PEG(12)-NH-Boc. Under an
argon atmosphere, a mixture of 4-(2,2-bis[(p-tolylsulfonyl)-
methyl]acetyl) benzoic acid HOBt ester (0.12 g, 194 μmol), α-
amino-ω-(t-butyloxycarbonyl)-dodecae(ethylene glycol) (0.10
g, 155 μmol) and anhydrous chloroform (4 mL) was stirred at
room temperature. After 16 h, the reaction mixture was
concentrated down to 1 mL of total volume, and purified by
flash silica chromatography (gradient elution 100% DCM to
DCM:MeOH 10:90 v/v) to afford the desired bis-sulfone-
1
PEG(12)-NH-Boc (0.15 g, 87%) as a white solid. H NMR
(600 MHz CDCl3,) δ 1.42 (s, 9H, (CH3)3), 2.46 (s, 6H,
PhCH3), 3.28 (m, 2H, CH2O), 3.45−3.56 (m, 4H, CH2O),
J
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at 40 °C overnight. Unreacted reagent was removed by gel
filtration (PD-10, PBS).
buffer B (50 mM sodium phosphate pH 7.0, 20% isopropanol)
in 50 min. The flow rate was 1 mL/min and the temperature
was set at 30 °C. Detection was carried out by following UV
absorption at 214, 248, and 280 nm.
Conjugation to TRA Using Alexa Fluor 488 C5-Maleimide.
TRA (10 mg/mL) in 20 mM sodium phosphate pH 8.0, 150
mM NaCl, 20 mM EDTA was treated with dithiothreitol
(DTT, 3 mol equiv) at 40 °C for 60 min. The reductant was
then removed by buffer exchange into fresh pH 8.0 phosphate
buffer by gel filtration (PD-10 column, GE Healthcare).
Determination of the number of free thiols present using
5,5′-dithiobis(2-nitrobenzoic acid) showed that there were
approximately 3.5 SH groups per mAb. Alexa Fluor 488 C5-
maleimide (1.2 equiv per thiol) in MeCN (final concentration
5% v/v) was added and the resulting mixture incubated at 4 °C
overnight. Unreacted reagent was removed by gel filtration
(PD-10, PBS).
Analytical Hydrophobic Interaction Chromatography
(HIC). Analytical HIC of conjugates was carried out using a
TOSOH, TSKgel Butyl-NPR column (35 × 4.6 mm)
connected to a Dionex Ultimate 3000RS HPLC system. The
method consisted of a linear gradient from 80% buffer A (1.5 M
ammonium sulfate in 50 mM sodium phosphate, pH 7.0) to
86% buffer B (20% isopropanol (v/v) in 50 mM sodium
phosphate) over 20 min at a flow rate of 0.8 mL/min. The
temperature was maintained at 30 °C throughout the analysis
and UV detection was carried out at 214 nm and 280 nm. For
each analysis an injection of 10 μg of mAb or ADC was carried
out.
Analytical Size Exclusion Chromatography (SEC). An
Acquity UPLC BEH200 column (Waters) connected to a
Dionex HPLC system was used for SEC. The mobile phase was
PBS, pH 7.4, containing 10% (v/v) isopropanol or 15% (v/v)
acetonitrile and the flow rate was kept constant at 0.25 mL/
min. The column was maintained at 30 °C throughout the
analysis. The analysis was carried out in a 24 min isocratic
elution with UV detection at 214, 248, and 280 nm.
Alexa Fluor 488 Conjugate Serum Stability. Alexa Fluor
488-TRA conjugates were diluted with 50% rat or human
serum at a concentration of 0.1 mg/mL in sterile tubes. Sodium
azide was added (final concentration 1 mM), and then the
mixtures were split into 4 equal aliquots. One aliquot was
immediately frozen at −80 °C. The remaining samples were
transferred to a CO2 incubator and kept at 37 °C. Aliquots were
removed from the incubator after 24, 48, and 96 h and
transferred to a −80 °C freezer. After the final time point,
mixtures were analyzed by SE-HPLC using the method
described above including fluorescence detection with an
excitation wavelength of 495 nm and emission wavelength of
525 nm.
Anti-HER2 ELISA. Maxisorp microtiter plates (96-well) were
coated overnight at 4 °C with TRA, FabTRA, TRA-bisAlk-vc-
MMAE, and FabTRA-bisAlk-vc-MMAE (5 μg/mL in 0.05 M
carbonate/bicarbonate buffer, pH 9.6). The plates were washed
three times with PBS/0.05% Tween 20 (PBS-T) and blocked
with 3% BSA/PBS-T for 1 h at room temperature. Serial
dilutions of ECD/HER2 were subsequently added and the
plates incubated for 3 h at room temperature. After 3 washes in
PBS/T, HRP-conjugated anti-6xHis antibody was added for 1 h
at room temperature to detect bound ECD/HER2. Tetra-
methyl benzidine (TMB) substrate and stop reagents were then
added in sequence, and the absorbance read at 630 nm using a
Spectramax M3 microplate reader. GraphPad Prism 5 software
using one site-specific binding with hill slope fit was used for
analysis.
Cell Viability Assays (Cell Titer Glo). The effect of TRA-
bisAlk-vc-MMAE and FabTRA-bisAlk-vc-MMAE MMAE con-
jugates on the viability of HER2-positive breast cancer cell lines
was assessed by Cell Titer Glo luminescence assay (Promega).
HER2-negative cell lines were included in the assays to confirm
antigen selectivity. Cells were seeded in 96-well white opaque
plates (Corning) at a density of 5 × 103 to 1 × 104/well
dependent on the cell line. Cells were allowed to attach for 24 h
at 37 °C and 5% CO2 in a humidified atmosphere. Following
incubation, cell culture medium was removed and serial
dilutions of TRA MMAE conjugates, TRA, and free drug
were added. Cells were incubated at 37 °C and 5% CO2 for 96
h and viability in response to treatment was quantified
according to the manufacturer’s instructions. Briefly, Cell
Titer Glo reagent was added to the wells and plates were
incubated on a plate shaker for 3 min and 300 rpm. Cells were
incubated for 20 min at room temperature to allow stabilization
of the signal and luminescence was subsequently measured on a
SpectraMax3 micro plate reader (Molecular Devices). A four-
parameter curve fitting model was applied (GraphPad) to
generate a dose−response curve. Response was expressed as %
viability of untreated controls.
Internalization Assay. SK-BR-3 and A549 cells were seeded
on poly(D-lysine) coated coverslips in 24-well plates at a
density of 2 × 105/well or 3 × 104/well, respectively. Cells were
allowed to attach for 24 h at 37 °C and 5% CO2 in a humidified
atmosphere. Cells were subsequently surface labeled for 30 min
on ice by replacing the cell culture medium with 4 μg/mL TRA,
TRA-bisAlk-FITC, or FabTRA-bisAlk-FITC in serum-free
medium. Following incubation, control plates were washed
with PBS and coverslips were carefully mounted on microscope
slides in ProLong Gold antifade reagent (Life Technologies).
The remaining plates were washed three times with serum free
medium, transferred to a CO2 air-jacket incubator, and
incubated for 4 h at 37 °C and 5% CO2 to allow internalization.
Cells were subsequently washed three times with PBS and
acetate buffer (pH 2.5) to remove membrane bound TRA or
TRA-FITC conjugates, respectively. Samples were fixed with
4% paraformaldehyde in PBS, washed three times with PBS,
and either mounted on microscope slides as described above
(FITC conjugates) or permeabilized with 0.5% Triton-X100 in
PBS (TRA). Binding and internalization of unconjugated TRA
was detected by means of Alexa Fluor 488 conjugated goat anti-
ADC Stability in the Presence of Free Thiols. TRA-bisAlk-
vc-MMAE was diluted to a final concentration of 1 mg/mL
with PBS pH 7.4 containing human serum albumin (HSA) at a
concentration of 20 mg/mL. Sodium azide was added (final
concentration 1 mM), and then the mixtures were split into 4
equal aliquots. One aliquot was immediately frozen at −80 °C.
The remaining samples were transferred to a CO2 incubator
and kept at 37 °C. Aliquots were removed from the freezer after
24, 48, and 120 h and transferred to a −80 °C freezer. After the
final time point, mixtures were analyzed by analytical
hydrophobic interaction chromatography using a method
modified from the standard protocol with a ProPac 2.1 mm
× 100 mm HIC-10 column (Fisher Scientific). The method
consisted of a linear gradient from 100% buffer A (50 mM
sodium phosphate pH 7.0, 1.5 M ammonium sulfate) to 100%
K
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human IgG secondary antibody (Life Technologies). Cells were
imaged with a Leica SP5 laser scanning confocal microscope.
In Vivo Efficacy Studies. Xenografts were initiated in female
severe combined immunodeficient mice (Fox Chase SCID,
C.B-17/Icr-Prkdcscid, Charles River Laboratories) with BT-474
human breast carcinomas maintained by serial subcutaneous
transplantation in SCID mice. On the day of tumor implant,
each test mouse received a 1 mm3 BT-474 tumor fragment
implanted subcutaneously in the right flank, and tumor growth
was monitored as the average size approached the target range.
Tumors were measured in two dimensions using calipers, and
volume was calculated using the formula:
w2 × l
tumor volume(mm3) =
2
where w = width and l = length, in mm, of the tumor.
The animals were fed ad libitum water (reverse osmosis, 1
ppm of Cl), and NIH 31 Modified and Irradiated Lab Diet. The
mice were housed on irradiated Enrich-o’cobs Laboratory
Animal Bedding in static microisolators on a 12 h light cycle at
20−22 °C and 40−60% humidity. On day 1 of the study, mice
were randomized into treatment groups (n = 10), and dosing
was initiated. All agents were administered intravenously (i.v.)
into the tail vein in a dosing volume of 0.2 mL per 20 g of body
weight (10 mL/kg), scaled to the body weight of the individual
animal.
In Vivo Efficacy of Fab Conjugate. Mice were treated with
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In Vivo Efficacy of mAb Conjugate. Mice were treated with
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MMAE (based on TRA) with dosing every 5 days for up to 7
occasions. Dosing was stopped when the tumor volume
reached less than 10% of the day 1 mean volume. Control
groups were treated with either 20 mg/kg TRA, 0.4 mg/kg free
MMAE, or vehicle on the same dosing schedule.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: antony.godwin@polytherics.com.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank our PolyTherics colleagues for their help, Dr.
Maurizio Muroni for providing analytical support, and Prof.
Steve Brocchini and Dr. Matthew Baker for critical review of
the manuscript.
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