Next generation maleimides enable the controlled assembly of antibody-drug conjugates via native disulfide bond bridging

Article abstract of DOI:10.1039/c4ob01550a

The advent of Adcetris and Kadcyla, two recently FDA-approved antibody-drug conjugates (ADCs), in the clinic has had a major impact on the treatment of lymphoma and breast cancer patients, respectively, worldwide. Despite these successes many new ADCs fail at various stages of development, often due to shortcomings in the methods used for their assembly. To address this problem we have developed next generation maleimides (NGMs), which specifically re-bridge reduced interchain disulfide bonds and allow the efficient conjugation of small molecules to antibodies, without the need for engineering of the target antibody. The method is site-specific and generates near homogeneous products in good yields. Moreover, adjustment of the reaction conditions allows control of the conjugation in terms of stoichiometry (drug-loading) and site selectivity. Using this method we prepared a series of ADCs from trastuzumab and doxorubicin (DOX) with a controlled drug-to-antibody ratio (DAR) of 1, 2, 3 and 4. All of these constructs were fully active by ELISA and had more than 90% of re-bridged disulfide bonds by CE-SDS when compared to clinical grade antibody. Furthermore, digest experiments of the DAR 2 material revealed that almost all of the drug had been targeted to the Fab arms of the antibody. Thus, NGMs offer a flexible and simple platform for the controlled assembly of ADCs from an antibody. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob01550a

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Biomolecular Chemistry
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Next generation maleimides enable the controlled
assembly of antibody–drug conjugates via native
disulfide bond bridging†
Cite this: DOI: 10.1039/c4ob01550a
Felix F. Schumacher,‡a João P. M. Nunes,‡a Antoine Maruani,a Vijay Chudasama,a
Mark E. B. Smith,a Kerry A. Chester,b James R. Baker*a and Stephen Caddick*a
The advent of Adcetris™ and Kadcyla™, two recently FDA-approved antibody–drug conjugates (ADCs), in
the clinic has had a major impact on the treatment of lymphoma and breast cancer patients, respectively,
worldwide. Despite these successes many new ADCs fail at various stages of development, often due to
shortcomings in the methods used for their assembly. To address this problem we have developed next
generation maleimides (NGMs), which specifically re-bridge reduced interchain disulfide bonds and allow
the efficient conjugation of small molecules to antibodies, without the need for engineering of the target
antibody. The method is site-specific and generates near homogeneous products in good yields. More-
over, adjustment of the reaction conditions allows control of the conjugation in terms of stoichiometry
(drug-loading) and site selectivity. Using this method we prepared a series of ADCs from trastuzumab and
doxorubicin (DOX) with a controlled drug-to-antibody ratio (DAR) of 1, 2, 3 and 4. All of these constructs
were fully active by ELISA and had more than 90% of re-bridged disulfide bonds by CE-SDS when com-
pared to clinical grade antibody. Furthermore, digest experiments of the DAR 2 material revealed that
almost all of the drug had been targeted to the Fab arms of the antibody. Thus, NGMs offer a flexible and
simple platform for the controlled assembly of ADCs from an antibody.
Received 22nd July 2014,
Accepted 23rd July 2014
DOI: 10.1039/c4ob01550a
www.rsc.org/obc
consequence ADCs typically incorporate very potent toxins,
such as tubulin inhibitors monomethyl auristatins E5–8 and
Introduction
From all drug classes currently under development, antibody– F9,10 or the novel DNA intercalators pyrrolobenzodiazepines.11
drug conjugates (ADCs) come closest to Paul Ehrlich’s late 19th The nature of these toxins necessitates the use of robust lin-
century vision of the perfect medicine, the “magic bullet”.1 kages to the antibody, which is inherently reliant on the under-
The specific binding capability of an antibody enables delivery lying conjugation strategy. Of particular importance is the
of an attached cytotoxic drug (payload) to a specific site, which selectivity of the conjugation method employed – non-selective
in principle restricts undesirable off-site toxicity effects.2 methods will lead to a heterogeneous mixture of ADC species
Despite being a relatively simple hypothesis, the development which12 will have different binding activities, potencies, thera-
of ADCs is marked with many failures.3 Only three molecules peutic indexes, clearance rates and other properties.13–16
of this drug class have received regulatory approval to date, Homogeneous ADCs are also desirable from a production
one of which has been voluntarily withdrawn from the market point of view, as heterogeneity places a large burden on purifi-
due to a lack of efficacy.3
cation, analytics and quality control17 to ensure patient safety,
One of the major issues for ADCs is the limited amount of which may become an important consideration for regulatory
cytotoxic compound that ultimately reaches the tumor.4 As a stakeholders.18
Developing conjugation methods to deliver homogeneous
products can in principle be achieved if the reaction is selec-
tive for particular conjugation sites and progresses to com-
aDepartment of Chemistry, University College London, 20 Gordon Street, London,
WC1H 0AJ, UK. E-mail: s.caddick@ucl.ac.uk, j.r.baker@ucl.ac.uk;
pletion. In addition, it is important that the conjugation
linkage is stable for storage and use in vivo. The conjugation
methods currently used to assemble ADCs are sub-optimal.
Kadcyla™ is produced via lysine conjugation,19,20 which, with
as many as 20 solvent accessible lysines in a typical antibody,
is not site-specific and thus cannot be allowed to go to
Tel: +44 (0)20 3108 7538
bUCL Cancer Institute, 72 Huntley Street, London, WC1E 6BT, UK
†Electronic supplementary information (ESI) available: Synthetic procedures, 1H
and 13C NMR spectra for synthesised compounds, additional SDS-PAGE gels,
LCMS chromatograms, Ellman’s analysis and other supplementary results. See
DOI: 10.1039/c4ob01550a
‡These authors contributed equally to this work.
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completion. An alternative strategy used for the synthesis of
Adcetris™ relies on partial reduction of interchain disulfide
bonds followed by alkylation.6,21 In this case, the number of
conjugation sites differs due to the non-selective nature of the
reduction step, thus resulting in a variety of drug-to-antibody
(DAR) conjugation ratios. Furthermore the loss of the disulfide
bonds introduces stability issues in vivo, as does the use of tra-
ditional maleimide reagents for conjugation.22–25
A number of innovative solutions have been developed to
enable the synthesis of highly homogeneous products. “Thio-
mabs”, which yield ADCs with a DAR close to 2, are based on
the insertion of additional free cysteines into the amino acid
sequence of the antibody.15,24 In a similar fashion, the
expression of antibodies containing non-natural amino acids
that can be targeted to install small molecules via “click chem-
istry” has been established.26 Another alternative involves
insertion of small amino acid sequences into the antibody,
which can then be used for conjugation using enzymatic
methods.27 Common to all of these methods is their depen-
dence on protein-engineering to insert additional reactive
groups for conjugation. Whilst technically feasible this adds
an additional layer of complexity to the production process of
ADCs, requiring detailed optimisation for determining suit-
able insertion sites and introducing potential challenges for
scale-up and reproducibility.
To avoid these issues we have developed reagents (next
generation maleimides, NGMs) for site-selective conjugation
in a controlled manner, using a feature native to all antibody
sub-classes: their interchain disulfide bonds. NGMs are a new
class of maleimides, which are substituted in the 3- and
4-position with good leaving groups enabling reaction with
two nucleophilic thiol groups such as the two cysteines of a
reduced cystine.28 This chemical process inserts a 2-carbon
bridge into a disulfide bond keeping this structural feature
intact whilst functionalizing the protein of interest. The reac-
tion is fast, efficient, high yielding and generates stable pro-
ducts that retain their full biological activity.29 Therefore,
NGMs fulfill the theoretical criteria for the synthesis of homo-
geneous conjugation products. Herein we show how this class
of compound can be used in the production of near homo-
geneous ADCs without the need for antibody-engineering
(Fig. 1a).
Fig. 1 Bridging of native antibody disulfide bonds with NGMs. (a)
Sequential and in situ functionalisation of antibodies with NGMs (b)
Initial sequential modification. (M) Molecular marker. (1) Untreated anti-
body. (2) Reduced antibody. (3) Antibody reduced in the presence of
DMF. (4) Reduction with 20 eq. TCEP, followed by addition of 25 eq.
dibromomaleimide. (c) Optimized sequential modification. (M) Molecular
marker. (1) Untreated antibody. (2) Reduced antibody. (3) Antibody
reduced in the presence of DMF. (4) Reduction with 6 eq. TCEP in pH
8.0 for 2 h at 37 °C followed by addition of 5 eq. dibromomaleimide in
10% final DMF for 1 h. (d) Selenol-based in situ modification. (M) Mole-
cular marker. (1) Untreated antibody. (2) Bridging with 10 eq. benzenese-
lenol and 20 eq. dithiophenolmaleimide in pH 8.0, 15% DMF on ice for
2 h. (e) TCEP-based in situ modification. (M) Molecular marker. (3)
Untreated antibody. (4) Bridging with 7 eq. TCEP and 7 eq. dithiophenol-
maleimide in pH 8.0, 10% DMF at 37 °C for 2 h.
mab were reduced in PBS buffer with a large excess of reducing
agent (20 eq. TCEP, Fig. 1b, lane 3) followed by the addition of
dibromomaleimide 1 (25 eq.) without any payload attached.
Our initial results were promising as SDS-PAGE revealed for-
mation of fully bridged antibody (Fig. 1b, lane 4), however, sig-
nificant fragmentation was also observed. It should be noted
that this was visible only when samples were heat-treated
before the run to ensure full dissociation of heavy and light
chains (HCs and LCs) not connected by an intact interchain
bridge. No free thiol group was detected by an Ellman’s test in
the purified product, indicating that NGMs do react very
efficiently. However, the method clearly needed further optimi-
sation. To this end, a screen of conditions was carried out,
Results and discussion
Bridging the interchain disulfide bonds of trastuzumab
To explore the suitability of NGMs in the context of the prepa-
ration of ADCs we first needed to establish if efficient re-brid-
ging of the multiple disulfide bonds of a full-length antibody
was feasible. We selected clinically validated drug trastuzumab
(Herceptin™), a monoclonal IgG1 antibody that targets the
internalising HER2 receptor, which is in wide use for the treat-
ment of HER2 positive breast cancer.30 It is also the antibody
component of ADC trastuzumab emtansine (Kadcyla™).19
Initially, all four solvent accessible disulfide bonds of trastuzu-
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focusing on the following parameters: pH, time, temperature, Most notably, we found that in situ modification of trastuzu-
organic solvent and concentration (Fig. S1, ESI†). Combining mab with the selenol reagent was best performed at 0–4 °C
the findings of these experiments in an improved re-bridging whilst the ideal reaction temperature when using TCEP was
protocol yielded promising results (Fig. 1c).
37 °C.
Using a revised protocol, the dissociation of the peptide
chains was greatly reduced leading to the isolation of two pro-
ducts, completely re-bridged antibody (i.e. LHHL) and half-
antibody (i.e. HL).
We speculate that the formation of this second, half-anti-
body species can be explained by formation of intrachain
bridges. This is possible because the cysteines that form the
two disulfide bonds connecting the HCs in the so-called hinge
region are separated by only two amino acids. When comple-
tely reduced and then re-bridged these can either form the
original interchain connections giving full antibody, or make
intrachain bridges to produce half-antibody by prohibiting
covalent connection of the HCs permanently.
In order to overcome the intrinsic challenge associated with
improving hinge region connectivity, we explored the use of an
in situ method, which we reasoned would avoid disulfide bond
shuffling by facilitating immediate re-connection of any
opened cystines.28 After some experimentation we discovered
that using dithiophenolmaleimide 2 in tandem with either
TCEP or benzeneselenol (Fig. S2, ESI†) could help mitigate
fragment formation as confirmed by SDS-PAGE (Fig. 1d and e).
Synthesis of NGM-doxorubicin
We then explored the possibility of using NGMs for conju-
gation of full antibodies with cytotoxic drugs. We selected anti-
cancer drug doxorubicin (DOX) as a warhead, given its prior
use as a model payload in ADCs and its convenient UV-vis
absorption profile that would facilitate DAR analysis.12,31,32 We
prepared NGM-DOX compounds 3 and 5, bearing a C6 alkyl
chain. Compound 5 also possesses the valine-citrulline linker
for drug release by lysosomal cathepsin B cleavage, which is
used in several ADCs.6,15,31,33–35 Compound 4 was synthesized
to allow evaluation of maleamic acid conjugates.36 The prepa-
ration of M-DOX 6 enabled comparison with existing techno-
logies (Fig. 2).
The preparation of these compounds started with conden-
sation of 3,4-dibromomaleic anhydride and the chosen amine
in acetic acid in accordance with the literature.37 This was fol-
lowed by addition of thiophenol to afford precursors 12 and 13
under efficient and mild conditions.28 DOX was attached using
HBTU promoted amide bond formation to afford NGM-DOX
3 and 4.
Fig. 2 Synthesis of NGM-DOX 3–5 and M-DOX 6.
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It is notable that whilst dibromomaleimides can be prone unreduced trastuzumab (Fig. 3c, lane 9); the absence of sul-
to side reactions in couplings with amines, reaction was facile phydryls following conjugation as determined via Ellman’s
and high yielding for dithiomaleimides. Since dithiophenol- analysis (Table S1, ESI†); and no further reaction observed
maleimides are just as effective as dibromomaleimides for with increased equivalents of NGMs.
cysteine conjugation and allow for in situ methods, we focused
The NGM-DOX reagents demonstrated less fragmentation
exclusively on NGM-DOX compounds 3–5 herein. M-DOX 6 (Fig. 3c, lanes 5, 6 and 7) compared to the use of simple male-
was also prepared by the described methods. The valine- imide compound 6, which afforded all possible fragments
citrulline p-aminobenzyl alcohol linker (Val-Cit-PABOH) was (Fig. 3c, lane 3). However, consistent with our expectations, we
synthesised as reported31 and coupled to compound 12 in observed formation of half-antibody (HL) in all cases of conju-
high yield via HBTU promoted amide bond formation. gation with NGMs 3–5. Nonetheless, ADCs prepared with
This was followed by reaction with p-nitrophenyl chloro- NGM-DOX 3–5 did still retain full binding activity to the HER2
formate and subsequent addition of DOX to afford NGM-DOX antigen, as determined by ELISA (Fig. 3d).
5 (Fig. 2).
We next explored the use of in situ protocols to avoid the
formation of half antibody using NGMs 3–5 and successfully
achieved moderate to good DAR values (Fig. S4, ESI†) in high
yields (Fig. 4b) and with negligible free sulphydryl groups in
the product (Table S2, ESI†). The quantity of half-antibody was
dramatically reduced, as indicated by SDS-PAGE and this was
confirmed by quantitative capillary gel electrophoresis
(CE-SDS) analysis (Fig. 4c and d). The use of NGMs 3 and 5 led
to decreased levels of half-antibody and with good DARs. The
use of NGM-DOX 4 under in situ conditions was optimal, gen-
erating conjugates with only minor by-products resulting from
fragmentation. This shows that the in situ method is capable
of affording a full antibody ADC with high selectivity and in
good yields. As anticipated, ADCs prepared by the in situ proto-
col displayed excellent retention of binding activity to HER2
(Fig. 4e).
Assembly of trastuzumab–doxorubicin conjugates with NGMs
With NGMs 3–5 in hand we attempted to prepare ADCs with a
DAR of 4 by targeting all 4 solvent accessible interchain disul-
fides in trastuzumab, initially via the sequential protocol
(Fig. 3a) as described above. Despite the propensity of this pro-
tocol to generate some half-antibody, it allowed for direct com-
parison with alternative contemporary methods of conjugation
(e.g. with compound 6). The sequential method using only a
slight excess of NGM-DOX reagents 3, 4 and 5 (5 eq., 1.25 eq.
per disulfide bond) provided DARs measured by UV-vis of 3.8,
3.0 and 3.5, respectively (Fig. S3, ESI†), with very high yields of
the conjugates obtained after purification (Fig. 3b). Complete
cysteine selectivity was confirmed by virtue of the following
observations: no conjugation observed between the NGMs and
Fig. 3 Bridging of native antibody disulfide bonds via optimised sequential method. (a) General outline of conjugation with NGM-DOX 3–5, com-
pared to conjugation with M-DOX 6. (b) Isolated yields and DAR from conjugation reactions. (c) Analysis by SDS-PAGE: (M) Molecular marker. (1)
Untreated antibody. (2) Reduced antibody with 2 eq. TCEP. (3) Conjugation with 5 eq. M-DOX 6. (4) Reduced antibody with 6 eq. TCEP. (5) Conju-
gation with 5 eq. NGM-DOX 3. (6) Conjugation with 5 eq. NGM-DOX 4. (7) Conjugation with 5 eq. NGM-DOX 5. (8) Reduced antibody with 6 eq.
TCEP in the presence of DMF 10% without NGM. (9) Unreduced antibody with DMF 10% and NGM-DOX 3 (DAR 0). (d) Binding activity of trastuzumab
ADCs with NGM-DOX 3–5 to HER2 compared to unmodified trastuzumab.
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Fig. 4 Bridging of native antibody disulfide bonds via in situ method. (a) General outline of conjugation with NGM-DOX 3–5. (b) Isolated yields and
DAR from conjugation reactions. (c) Analysis by SDS-PAGE: (M) Molecular marker. (1) Untreated antibody. (2) Conjugation with 5 eq. NGM-DOX 3. (3)
Conjugation with 5 eq. NGM-DOX 4. (4) Conjugation with 5 eq. NGM-DOX 5. (d) CGE of conjugation with NGM-DOX 4 by different methods; peak
area given as percentage of total peak area: (1) sequential method with 5 eq. of NGM-DOX 4. (2) in situ method with 5 eq. of NGM-DOX 4.
(e) Binding activity of trastuzumab ADCs with NGM-DOX 3–5 to HER2 compared to unmodified trastuzumab.
NGM-DOX 4 consistently afforded the lowest DARs, whether
Modification of the target antibody with compound 15 via
generated using the sequential (3.0) or in situ (1.8) protocols, TCEP and benzeneselenol protocols proceeded as expected
and we suspected that this might be a consequence of hydro- (Fig. S7, ESI†) and yielded the desired intermediate in more
lysis of the conjugate. NGM-DOX 4 was designed to undergo than 80% yield (Fig. 5c). Copper-catalysed Huisgen 1,3-dipolar
rapid hydrolysis to generate maleamic acid bridges following cycloaddition (click reaction) was carried out in the presence
conjugation and this was verified by SDS-PAGE (Fig. S5, of 3[tris(3-hydroxypropyltriazolylmethyl) amine] (THPTA) and
ESI†).36 To explore this further, we modified trastuzumab Fab aminoguanidine hydrochloride using a seven fold excess of 16
with NGM-DOX 4 and confirmed full hydrolysis of maleimide per disulfide bond, which had negligible impact on antibody
to maleamic acid using mass spectrometry (Fig. S6, ESI†). Sig- fragmentation (Fig. S7, ESI†).
nificantly, mass spectrometry also revealed partial hydrolysis
The products were purified by size exclusion chromato-
of the acetal moiety in DOX (Table S3, ESI†). The instability of graphy (SEC) and we isolated ADCs in 52–69% yield (Fig. 5c).
DOX has been documented38 and we have previously reported We observed that TCEP-based protocols gave DARs between 3
similar observations for another N-aryl maleamic acid DOX and 4, whilst methods employing benzeneselenol yielded
construct.39 Thus, we believe the lower DAR observed with DARs between 1 and 2 (Fig. S8, ESI†). We explored this obser-
NGM-DOX 4 is more likely to be due to DOX acetal hydrolysis vation further in an attempt to develop protocols that would
over the extended conjugation time of 2 h rather than a limit- deliver a series of trastuzumab–DOX conjugates with distinct
ation of the conjugation chemistry.
DARs. Careful variation of the reaction conditions, including
repeated additions of benzeneselenol, adjusting TCEP
stoichiometry and moderating reaction times enabled us to
develop protocols for the synthesis of ADCs with DARs of 1.1,
2, 3.1 and 4 (Fig. 5b and c). Furthermore, all molecules iso-
lated contained more than 90% of the connected interchain
bridges, at least 74% of intact antibody (Fig. 5d), and retained
full binding activity as shown by ELISA (Fig. 5e).
The 4 solvent accessible cystines in IgG1 antibodies can be
divided into 2 groups: 2 of them in the hinge region and 2 in
the switch/Fab region. The fact that the selenol-based in situ
protocol did not yield ADCs with a DAR higher than 2
prompted us to explore the possibility that the selenol-based
protocol was reacting specifically with the Fab arms.
Controlling drug loading and localization
To avoid problems based on the model payload and to assess
the true potential of NGMs in the context of ADC synthesis we
decided to examine an alternative approach in which the brid-
ging reaction and the drug-conjugation step could be carried
out separately. To this end, we prepared N-alkyne-
dithiophenolmaleimide 15,40 which would be inserted into the
trastuzumab disulfide bonds to provide a bioconjugate ready
for further elaboration. We also prepared a DOX-azide species
16 containing a polyethylene glycol (PEG) chain that we envi-
saged could lead to conjugation via a copper catalysed 3 + 2
cycloaddition (Fig. 5a).41
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Fig. 5 Controlled synthesis of trastuzumab–DOX conjugates and localization of the payload. (a) General outline of the two-step process using
in situ disulfide bond modification with NGMs followed by copper-catalysed Huisgen 1,3-dipolar cycloaddition. (b) Trastuzumab–DOX DAR series.
(M) Molecular marker. (1) Untreated antibody. (2) Reduced antibody. (3) Fragmentation control of trastuzumab prepared by treatment with 2 eq.
TCEP (4) Trastuzumab–DOX conjugate prepared with compound 6 (compare Fig. 3c, lane 3). (5) DAR 1.1: reaction on ice for 1 h followed by 90 min
click reaction. (6) DAR 2.0: as DAR 1.1 but with addition of 10 eq. benzeneselenol after 30 min and 16 h click reaction. (7) DAR 3.1: reaction at 37 °C
for 2 h followed by 90 min click reaction. (8) DAR 4.0: reaction at 37 °C for 2 h followed by 16 h click reaction. (c) Isolated yields and DAR from con-
jugation reactions, overall yield after SEC purification. (d) CE-SDS analysis of the trastuzumab–DOX DAR series; peak area given as percentage of
total peak area. It was assumed for the calculation of correctly bridged disulphide bonds (in contrast to the population of the fragments) that the H
and L chains contained 0, the HL fragment 1, the HH fragment 2 and the HHL fragment 3 correct bridges. DAR 0 material was generated by treat-
ment of trastuzumab as outlined for DAR 3.1 material but without addition of reducing agent. (e) Binding activity of the trastuzumab DAR 1 to 4
series to HER2 compared to unmodified trastuzumab. (f) Localization of DOX in DAR 2.0 material. (M) Molecular marker. (1) Untreated antibody. (2)
Trastuzumab bridged with compound 15. (3) Trastuzumab–DOX with a DAR of 2.0 after the click reaction. (4) DAR 2.0 material after pepsin digest.
(5) DAR 2.0 material after papain digest. (6) Control digest of unmodified trastuzumab with pepsin. (7) Control digest of pepsin digested trastuzumab
with papain.
Proteolytic digestion of the trastuzumab–DOX DAR 2 conju- fraction obtained from digestion was found to contain more
gate derived from the selenol protocol allowed us to isolate the than 95% of the originally conjugated DOX after enzymatic
corresponding Fab fragments (Fig. 5f) with a DAR of 0.79 cleavage. This observation suggests that the combination of
(Fig. S9, ESI†). As direct readout from this process would be NGMs with benzeneselenol can be used to target small mole-
misleading due to issues of DOX stability under the reaction cules exclusively to the Fab arms of antibodies. Additional
conditions, we required an appropriate control/correction experimentation will be necessary to understand the chemical
factor. To this end, we synthesized a Fab–DOX conjugate via and structural basis of this effect.
the same protocol as established for the full antibody, and this
species was subjected to the digest conditions. The loss of
20% of the DOX payload under these conditions, as observed
by mass spectrometry and UV-vis spectroscopy (Fig. S10 and
Conclusions
S11, ESI†), were used to adjust the results from the full anti-
ADCs offer a tantalizing possibility for the development of the
body digestion experiment. Taking this into account, the Fab
next generation of therapeutics, but careful consideration
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needs to be given to each component, including the conju- UV-vis (ε280 = 215 380 cm−1 M−1) and dilution of the antibody
gation method. The methods described in this paper show the to 30 µM, samples were treated with 30 eq. compound 16 in
possibility for delivering high yields of homogeneous and the presence of 150 µM CuSO4, 750 µM THPTA, 5 mM amino-
stable29 conjugates using site-specific reactions of native di- guanidine HCl and 5 mM sodium ascorbate. Reactions were
sulfide bonds. It is also possible to offer control over the drug incubated at 22 °C for 90 min or 16 h. ADCs were purified
loading in a range between 0 and 4 whilst maintaining the by size exclusion chromatography (on a HiLoad Sephadex 75
structure and activity of the antibody. The possibility of using 16/60 column, GE Healthcare, equilibrated in PBS) and the
the native disulfide bonds of antibodies for bioconjugation DAR calculated by UV-vis.
offers potential practical advantages over antibody engineer-
ing. Moreover the possibility for targeting the Fab arms has
CE-SDS and data analysis
not, to the best of our knowledge, been described and may be CE-SDS analysis was carried out on a PEREGRINE I machine
useful for spatial separation of hydrophobic payloads attached (deltaDOT). Samples were diluted to 1 mg mL−1 in SDS-MW
to antibodies. The synthetic methods described herein are sample buffer (Proteome Lab) and heated to 65 °C for 20 min.
practical, efficient and have the potential for further modifi- 50 μL were transferred into sample vials and loaded into the
cation and optimisation depending on the antibody and machine. Separations were performed in a 50 μm diameter
payload under investigation. For these reasons we anticipate fused silica capillary at 22 °C. Separation length was 20.2 cm,
that the NGM platform will find considerable utility for the run time 45 min and antibody fragments detected at a wave-
development of future ADCs.
length of 214 nm. The capillary was flushed with 0.1 M HCl,
water and run buffer before sample loading at 5 psi/16 kV.
Noise was recorded for 3 min from the run buffer. To verify
comparison-based fragment identification a protein sizing
standard (Beckman Coulter) was used. Data analysis was
carried out with the EVA software (version 3.1.7, deltaDOT).
Run files were loaded and analysed with the GST algorithm at
a frequency of 40 and a sensitivity of 1. GST peak search was
performed between 13 and 32 min (8000 to 20 000 scans)
based on the peak identification by mass and comparison
between unmodified, partially and fully reduced antibody
samples. Peaks corresponding to the LHHL, HHL, HH, HL, H
and L antibody species were added manually where necessary
and peak area boundaries adjusted for all signals. As the peak
area (absorbance) varies depending on the size a correction
factor between the absorbance of the full antibody and the
completely disassembled antibody (only H and L fragments)
was calculated. This factor was adjusted for the area correction
of the remaining fragments (HHL, HH, HL) depending on
their disulfide bond status, e.g. only 25% of the correction
factor was applied to the peak area of the HHL fragment as
75% of the disulfide bonds were assumed to be intact. The
normalization was established based on the samples of a
reduction series and transferred to the samples with varying
DARs. For the calculation of the correctly bridged disulfide
bonds it was assumed that the H and L fragments contained
none, the HL one, the HH two, the HHL three and the full
antibody (LHHL) four correct disulfide bonds.
Materials and methods
General sequential bridging protocol
To trastuzumab (22.9 µM, ∼300 µL) in borate buffer (25 mM
sodium borate, 25 mM NaCl, 1 mM EDTA, pH 8.0) was added
TCEP (min. 5 eq., in the same buffer) from a 400–1000× stock
solution and the reaction incubated at 37 °C for 2 h under
mild agitation. NGMs were prepared in dry DMF (purchased
from Sigma Aldrich) as 400–1000× stock solutions and added
to reduced trastuzumab (minimum 5 eq.) to achieve a final
DMF concentration of 10%. After 1 h at 37 °C excess reagents
were removed by ultrafiltration (10 kDa MWCO).
General in situ bridging protocol with TCEP
To trastuzumab (22.9 µM, ∼300 µL) in borate buffer were
added NGMs (min. 5 eq. in dry DMF) from 400–1000× stock
solutions to achieve a final DMF concentration of 10%. Then
TCEP (min. 6 eq.) in borate buffer was added from a
400–1000× stock solution and the reaction incubated at 37 °C
for 2 h under mild agitation after which the reaction was puri-
fied by ultrafiltration (10 kDa MWCO).
General in situ bridging protocol with benzeneselenol
To trastuzumab (22.9 µM, ∼300 µL) in borate buffer was added
DMF to a final concentration of 12% and the mixture put on
ice. Next benzeneselenol (min. 10 eq. in dry DMF) was added
from a freshly prepared 1000× stock solution followed by
addition of NGMs (min. 10 eq. in dry DMF) from 400–1000×
stock solutions to achieve a final DMF concentration of 15%.
The reaction was kept on ice for 1 h and purified by ultrafiltra-
tion (10 kDa MWCO).
Digest of DAR 2 material
A Herceptin–DOX conjugate with a DAR of 2.0 was prepared
and the pH of the sample lowered via a buffer exchange (into
20 mM sodium acetate, pH 3.1) by ultrafiltration (10 kDa
MWCO). Immobilized pepsin (0.15 mL) was washed 4× with
the same buffer and trastuzumab–DOX (0.45 mL, 3.19 mg
mL−1) was added. The mixture was incubated for 5 h at 37 °C
Copper-catalysed Huisgen 1,3-dipolar cycloaddition
Bridging reactions were stopped with 20 eq. of maleimide (in under constant agitation (1100 rpm). The resin was separated
dry DMF) and purified into PBS (pH 7.4) by ultrafiltration from the digest using a filter column, and washed 3× with
(MWCO 10 kDa). After determination of the concentration by digest buffer (50 mM sodium phosphate, 150 mM NaCl, 1 mM
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Organic & Biomolecular Chemistry
EDTA, pH 6.8). The digest was combined with the washes and 11 J. A. Hartley, Expert Opin. Invest. Drugs, 2011, 20,
the volume adjusted to 0.5 mL. Next, immobilised papain 733–744.
(0.5 mL, 0.25 mg mL−1) was activated with 10 mM DTT (in 12 D. Willner, P. A. Trail, S. J. Hofstead, H. D. King,
digest buffer) under an argon atmosphere and constant agita-
S. J. Lasch, G. R. Braslawsky, R. S. Greenfield, T. Kaneko
tion (1100 rpm) for 1 h at 25 °C in the dark. The resin was
and R. A. Firestone, Bioconjugate Chem., 1993, 4, 521–527.
washed 4× with digest buffer (without DTT) and the 0.5 mL of 13 K. J. Hamblett, P. D. Senter, D. F. Chace, M. M. C. Sun,
trastuzumab–DOX-Fab2 solution was added. The mixture was
incubated for 16 h at 37 °C under constant agitation (1100
rpm) in the dark. The resin was separated from the digest
J. Lenox, C. G. Cerveny, K. M. Kissler, S. X. Bernhardt,
A. K. Kopcha, R. F. Zabinski, D. L. Meyer and
J. A. Francisco, Clin. Cancer Res., 2004, 10, 7063–7070.
using a filter column, washed 3× with PBS (pH 7.0) and the 14 J. R. Junutula, K. M. Flagella, R. A. Graham, K. L. Parsons,
digest combined with the washes. The buffer was exchanged to
PBS by ultrafiltration (10 kDa MWCO) and the volume adjusted
to 0.3 mL. In parallel, a sample of unmodified Herceptin was
processed as a control. Sample and control were analysed by
SDS-PAGE and the control by MS as well. The drug loading of
the trastuzumab–Fab–DOX was assessed by UV-vis.
E. Ha, H. Raab, S. Bhakta, T. Nguyen, D. L. Dugger, G. Li,
E. Mai, G. D. Lewis Phillips, H. Hiraragi, R. N. Fuji,
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Acknowledgements
We gratefully acknowledge L. Castañeda for kindly providing
compound 15, and S. Harris, N. Stokes and M.-A. Sung from
deltaDot for support with the CE-SDS analysis. This work was
supported by the BBSRC, the Wellcome Trust, and UCL 16 C. F. McDonagh, E. Turcott, L. Westendorf, J. B. Webster,
Business.
S. C. Alley, K. Kim, J. Andreyka, I. Stone, K. J. Hamblett,
J. A. Francisco and P. Carter, Protein Eng., Des. Sel., 2006,
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17 A. Wakankar, Y. Chen, Y. Gokarn and F. S. Jacobson, MAbs,
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