Highly homogeneous antibody modification through optimisation of the synthesis and conjugation of functionalised dibromopyridazinediones

Article abstract of DOI:10.1039/c7ob03138f

Due to their exquisite cysteine-selectivity, excellent stability, and ability to functionally rebridge disulfide bonds, dibromopyridazinediones are emerging as an exciting new class of bioconjugation reagents, particularly in the field of antibody conjugation. Despite this, relatively little work has been performed on the optimisation of their synthesis and subsequent reaction with immunoglobulins. Herein we present a novel synthetic route towards functionalised dibromopyridazinediones, proceeding via an isolatable dibromopyridazinedione-NHS ester. Reaction of this activated intermediate with a variety of amines produces functional dibromopyridazinediones in good to excellent yields. The disulfide rebridging capacity of these reagents was optimised on the clinically relevant IgG1 trastuzumab, resulting in a general method which allows for the generation of site-selectively modified native trastuzumab with over 90% homogeneity (no disulfide scrambling) without the need for protein engineering or enzymatic conjugation.

Full text of DOI:10.1039/c7ob03138f

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Highly Homogeneous Antibody Modification through Optimisation of the
Synthesis and Conjugation of Functionalised Dibromopyridazinediones
Calise Bahou1,å, Daniel A. Richards,1,å , Antoine Maruani,1 Elizabeth A. Love,2 Faiza Javaid,1 Stephen Caddick, 1
James R. Baker, 1,* and Vijay Chudasama1,3,
*
1 Department of Chemistry, University College London, London, UK;
2 LifeArc, Accelerator Building, Open Innovation Campus, Stevenage, UK
3 Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal
å These authors contributed equally to this article
* E-mails: j.r.baker@ucl.ac.uk, v.chudasama@ucl.ac.uk

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Abstract
Due to their exquisite cysteine-selectivity, excellent stability, and ability to functionally rebridge disulfide bonds,
dibromopyridazinediones are emerging as an exciting new class of bioconjugation reagents, particularly in the field
of antibody conjugation. Despite this, relatively little work has been performed on the optimisation of their
synthesis and subsequent reaction with immunoglobulins. Herein we present a novel synthetic route towards
functionalised dibromopyridazinediones, proceeding via an isolatable dibromopyridazinedione-NHS ester. Reaction
of this activated intermediate with a variety of amines produces functional dibromopyridazinediones in good to
excellent yields. The disulfide rebridging capacity of these reagents was optimised on the clinically relevant IgG1
trastuzumab, resulting in a general method which allows for the generation of site-selectively modified native
trastuzumab with over 90% homogeneity (no disulfide scrambling) without the need for protein engineering or
enzymatic conjugation.
Introduction
Over the last few decades biologists, biochemists, and chemical biologists have put forth a concerted effort to
explore and harness the unique attributes of antibodies, particularly their exquisite targeting specificity, excellent
in vivo stability, and ability to initiate antibody-dependent cell-mediated cytotoxicity (ADCC).1 These properties
make antibodies excellent biomolecules for a plethora of tasks, especially as therapeutic agents. Their therapeutic
utility has been bolstered by advancements in hybridoma and phage display technology, which enable the rapid
generation of high-affinity monoclonal antibodies against emerging diseases.2,3 Despite the many advantages of
antibodies, their chemical functionality is limited to the reactivities of the 20 natural amino acids. To fully exploit
antibodies for biomedical applications it is desirable to expand their reactivity beyond the capacity of the natural
amino acids. To this end, significant research effort has been devoted to developing new methods for
functionalising antibody scaffolds through chemical conjugation.4t7 The chemical modification of antibodies has led
to the development of a novel class of biologics; antibodytdrug conjugates (ADCs).8
Despite their utility, many conjugation strategies for synthesising ADCs suffer from several drawbacks. The
targeting of amino acids frequently provides no regioselectivity over the modification when trying to aim for drug
loadings of 2-4 (commonly tolerated drug loadings in terms of pharmacokinetics), leading to heterogeneous
product mixtures. This is particularly true when targeting lysine residues, which are highly abundant on the solvent
accessible surface of antibodies.8 Despite this, lysine conjugation is employed for the generation of clinically
approved ADCs Kadcyla® and Besponsa®.9,10 To overcome the issue of lysine over-abundance, cysteine-selective
reagents are frequently employed. Although native antibodies contain no solvent accessible reactive cysteine
residues, liberation of cysteine residues from reduction of the inter-chain disulfide bonds provides suitable targets.
Unfortunately, this reduction results in a loss of the stabilising disulfide bridges and, in the instance of IgG1
antibodies, generates up to 8 reactive cysteine residues t factors which can contribute to heterogeneity when
targeting typically tolerated loadings of 2-4 drugs per antibody.8 Conjugation to liberated cysteine residues was
employed for the generation of the ADC Adcetris®.11 The issues of uncontrolled conjugation are particularly
problematic for therapeutic applications, where sub-optimal drug loading can lead to poor pharmacokinetic
properties and a narrow therapeutic window.12 As such, research focus has shifted to developing technologies
which allow for site-specific conjugation. This is commonly achieved using protein engineering to install a site-
specific cysteine residue or unnatural amino acid, or through enzymatic processes.8 Whilst these methods work
well, their accessibility is limited as the process must be optimised for each individual antibody; a time-consuming
and expensive process.
One promising alternative approach utilises reagents that are capable of functionally rebridging the cysteine
residues liberated from reduction of the disulfide bonds of native antibodies. This provides site-selectivity, a
generally tolerated drug loading of 4 (as IgG1s contain only 4 solvent accessible disulfide bonds), and allows for
retention of the covalent stabilising bridge. The most commonly employed disulfide bridging reagents include
next-generation maleimides and bis-sulfones.13t21 Whilst these reagents represent important steps towards
developing homogeneous antibody constructs without the need for protein engineering, improvements are still

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is also indicative of how capricious the reaction is; for each new amine a different activating reagent was
employed and specific reaction conditions developed. This precludes the use of a generalised synthetic
pathway to these useful reagents (Scheme 1a).
3) A general protocol for homogeneous antibody conjugation without having to use bespoke reagents. We
have previously reported a pyridazinedione-based reagent with built-in reductive capacity that is capable
of both reducing and functionally rebridging the inter-chain disulfide bonds of an antibody.26 Whilst this
reagent grants antibody conjugates with unprecedented homogeneity, and a desirable drug-to-antibody
ratio of 4 (DAR), the synthetic difficulty and poor long-term stability of the reagent without storing in the
freezer under argon limits wide-spread adoption. Methods which allow for similar levels of homogeneity
(low levels of mis-bridged species t see Fig. 1) and DAR without the associated synthetic difficulty and
oxidation instability are sought after.
Herein we report a general and novel synthetic route to functionalised diBrPDs 10 proceeding via an isolatable
activated ester 9, incorporating a one-pot synthesis for the formation of the core scaffold. This procedure was
utilised to generate a small library of functional diBrPDs (Scheme 1b). The subsequent conjugation of these
reagents was then optimised on the clinically relevant antibody trastuzumab, and found to proceed with excellent
and unprecedented homogeneity.
Scheme 1 (a) Current synthesis of diBrPDs 4 from maleic anhydride 1 and hydrazines 3, followed by amide coupling. (b)
Proposed one-pot synthesis of diBrPDs 8 from dibromomaleic acid 6 and hydrazines 7, followed by generation of an isolatable
activated ester 9 to enable a general route towards functionalised diBrPDs 10.
Results and Discussion
Pyridazinedione (PD) core scaffold synthesis
Towards the development of a one-pot synthesis of the core diBrPD scaffold, initial focus was given to the in situ
formation of dibromomaleic anhydride 2 from dibromomaleic acid 6 (£18/g through Sigma Aldrich).
Dibromomaleic acid 6 has been successfully reacted with amines for the synthesis of dibromomaleimides; a
synthetic pathway similar to the generation of diBrPDs. We proposed that this reaction was proceeding via
dibromomaleic anhydride 2, formed through the ring-closing dehydration of dibromomaleic acid 6 under acid

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conditions. This theory was explored by refluxing dibromomaleic acid 6 in deuterated acetic acid (AcOH-D4).
Following the reaction using 13C NMR demonstrated almost full conversion to dibromomaleic anhydride 2 within
30 minutes (Fig. 2).
Figure 2 13C NMR spectra showing formation of dibromomaleic anhydride 2 from dibromomaleic acid 6 in boiling deuterated
acetic acid (AcOH-D4).
Similarly, analysis by thin-layer chromatography (TLC) indicated rapid deprotection of diethyl diboc hydrazine 11
within 10 minutes of boiling in acetic acid. A protocol was developed in which dibromomaleic acid 6 was refluxed
in acetic acid for 30 mins prior to addition of diethyl diboc hydrazine 11, followed by a further 4 hours of reflux
(Scheme 2a). This produced diBrPD 12 in an 82% yield, higher than the overall yield of 68% granted by the
previously reported protocol. To explore the importance of the preformation of dibromomaleic anhydride 2, the
reaction was performed with all reagents added at the start in a one-pot synthesis. A decreased yield of 65% was
observed, indicating that preformation of dibromomaleic anhydride 2 is key to achieving high yields in this instance
(Scheme 2b). This new route reduces the complexity of the synthesis of the diBrPD scaffold, increases overall yield
and step-economy, decreases the number of reagents required, and eliminates the dangerous and cumbersome
bromination reaction of maleic anhydride through the use of relatively cheap and readily commercially available
dibromomaleic acid 6.

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Scheme 2 (a) Synthesis of diethyl dibromo PD 12 using commercially available dibromomaleic acid 6 (b) Synthesis of diethyl
dibromo PD 12 using dibromomaleic acid 6 refluxed for 1 h prior to addition of diethyl diboc hydrazine 11.
Development of Pyridazinedione-N-hydroxysuccinamide activated ester
Methyl ethanoic acid diBrPD 13 has been previously employed to generate functionalised diBrPDs through amide
coupling reactions with amines. However, the reaction has been shown to be capricious and amine-dependent,
requiring the development of tailored conditions for each coupling reaction (see introduction). To create a more
generalised route towards functionalised diBrPDs we attempted to synthesise and isolate a reactive NHS-ester
intermediate of diBrPD 14. It was hoped this intermediate could be synthesised in large quantities, stored for
extended periods, and be used in a general protocol towards functionalised diBrPDs. To this end, we attempted to
synthesise the NHS ester of methyl ethanoic acid diBrPD 13 by reacting it with with DCC and NHS. Whilst this
resulted in complete consumption of starting material, isolation of the desired species 14 was not possible under
various conditions despite various attempts. This may go some way to explaining why the amide coupling reactions
using methyl ethanoic acid diBrPD 13 were so capricious. It was hypothesised that degradation of the intermediate
was occurring through intramolecular reaction of the activated species with the carbonyl of the pyridazinedione
ring (possibly via a 6-membered ring transition state). To appraise this, we synthesised methyl propionic acid
diBrPD 15, in which the acid is separated from the ring system by a 2-carbon alkyl chain, i.e. leading to what would
be an undesirable 7-membered ring transition state. Pleasingly, subjecting methyl propionic acid diBrPD 15 to
analogous NHS ester formation conditions led to the generation of activated diBrPD 16 in a good yield (scheme 3).
We were also pleased that we were able to synthesise methyl propionic acid diBrPD 15 using the core scaffold
synthesis outlined above (see SI Scheme S2).

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Scheme 3 (a) Attempted synthesis of NHS-activated methyl ethanoic acid diBrPD 14 (b) Synthesis of NHS activated methyl
propionic acid diBrPD 16.
With activated diBrPD 16 in hand, we examined its propensity to form amides upon reaction with a library of
diverse amines. The reactions proceeded with good to excellent yields with amines containing hydrophobic,
hydrophilic, terminal alkyne and strained alkyne functional groups all being tolerated. A secondary amine was also
reactive towards diBrPD 16 producing amide 17c in good yield; a feat not feasible for reaction with methyl
ethanoic acid diBrPD 13. Moreover, synthesis of diBrPD-amide 17f proceeded with a high yield of 78%; this
contrasts starkly with the 8% yield obtained for the same coupling reaction when using previous methyl ethanoic
acid diBrPD 13.24
Table 1 Reaction of activated NHS-activated diBrPD 16 with a library of amines
Product
Reaction
A
Solvent
Amine
Yield
Number
CH2Cl2
17a
78%
B
C
D
CH2Cl2
17b
17c
17d
71%
76%
87%
MeCN
MeCN
MeCN
E
17e
72%

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MeCN
F
17f
78%
These results demonstrate the utility of activated diBrPD 16, a reagent which enables a general route towards the
functionalisation of the core diBrPD scaffold. Furthermore, this compound was found to be stable at -20 °C for at
least 3 months and not prone to oxidation or intramolecular decomposition; increasing its applicability as a
building block for the synthesis of functionalised diBrPD reagents (SI Fig. S11).
Antibody bioconjugation
Optimisation
The monoclonal antibody trastuzumab 18, an FDA-approved therapeutic for HER2+ breast cancers,33 was chosen as
a model to appraise and optimise the conjugation of functional diBrPD reagents 17a-e. Trastuzumab 18 is also
employed within the clinically approved ADC Kadcyla®,9 and multiple pre-clinical therapeutics,20,34,35 and thus was
deemed an appropriate model system. As discussed above, typical disulfide bridging reagents yield some mis-
bridged species in addition to the desired fully re-bridged antibody (see introduction). The only protocol that does
not suffer from this issue requires intricate synthesis of a complex reagent that is prone to oxidation.26 We set out
to develop a protocol using diBrPD reagents which could minimise these undesirable mis-bridged species without
the use of complex reagents. A key objective was to hinder formation of the mis-ꢀŒ]ꢁPꢂꢁ ^half ꢃvš]ꢀ}ꢁÇ_ •‰ꢂꢄ]ꢂ•
(Fig. 1), in which the hinge disulfides become scrambled and the bridging reagent adds across the intra-chain
cysteines rather than inter-chain. Therefore, an optimisation study with these reagents to determine the best
conditions for obtaining high levels of homogeneity and a pyridazinedione-to-antibody ratio (PDAR) of 4 was
performed.
We first explored the relationship between the two reactions steps, reduction and rebridging, to determine how
the order of reagent addition would affect the composition of the final product. There are 3 generally accepted
functional rebridging strategies: 1) a step-wise protocol in which reduction is performed first, followed by removal
of the reducing agent and addition of the bridging reagent; 2) a sequential protocol in which reduction is followed
by addition of the bridging reagent without intermediate purification to remove the reducing reagent; and 3) an in-
situ protocol in which the bridging reagent is added before addition of the reducing agent. Due to cross-reactivity
with commonly employed reducing agents, many disulfide bridging reagents require a step-wise protocol. It has
been noted previously that diBrPDs are amenable to both sequential and in-situ protocols due to a lack of (or
minimal) cross-reactivity with the commonly employed reducing agent tris(2-carboxyethyl)phosphine (TCEP) under
the reaction conditions.26 These 3 strategies were compared for the modification of trastuzumab 18 using
synthesised strained alkyne diBrPD 17e. This reagent was chosen for this study due to its chemical complexity; we
wanted to appraise the platform with a chemical linker which contained both hydrophilic and hydrophobic
components, as well as reactive functionalities.
The step-wise protocol reduction was achieved by incubation of trastuzumab 18 with TCEP, followed by
purification via ultrafiltration and subsequent addition of diBrPD 17e. A similar approach was employed for the
sequential protocol but with no purification before addition of diBrPD 17e. Finally, the in-situ protocol involved
addition of diBrPD 17e prior to addition of TCEP. In each case, 10 equivalents of reducing agent (i.e. 2.5 eq per
disulfide) and 20 equivalents of diBrPD 17e (i.e. 4 eq per disulfide) were employed (Fig. 3c). From the results, it is
clear to see that whilst all 3 protocols granted a desired PDAR of 4, both the step-wise and sequential protocols
yielded significantly poorer functional rebridging; SDS-PAGE and densitometry analysis show greater levels of half
antibody species when compared to the in-situ protocol. We hypothesise that this result is due to the relative
kinetics of the two reactions steps (reduction and rebridging); reduction of the disulfide bond using TCEP may be
slower than the subsequent reaction of the liberated cystine residues with diBrPD 17e. The presence of a high
concentration of diBrPD 17e in situ allows the rebridging step to occur almost immediately after reduction whilst

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the antibody domains are still templated by various intermolecular forces. Conversely, both step-wise and
sequential protocols allow enough time for some loss of the antibody template, leading to significant mis-bridging
of the liberated cysteine residues upon addition of the bridging reagent. This result is in accordance with the
previously reported two-in-one pyridazinedione reagent26.
We next studied the effect of temperature on the bridging efficiency. Based on the previous results, an in situ
protocol was utilised, with 10 equivalents of TCEP and 20 equivalents of diBrPD 17e. Incubation over 16 hours at
4 °C, 10 °C, 25 °C and 37 °C highlighted a significant temperature dependence on the overall composition of the
final construct (Fig. 3d): In each case, a PDAR of 4 was observed, though increasing levels of heterogeneity were
observed as the temperature was increased. This could be due to the higher temperature increasing the rate at
which the antibody loses its template upon reduction, or by increasing the rate of TCEP reduction itself.
Regardless, these results clearly show that lower temperatures are beneficial to increasing the homogeneity of the
final constructs. To the best of our knowledge the effect of temperature on the reactions of disulfide bridging
reagents has not been reported. We anticipate that this discovery will be transferable to other reagents and thus
prove useful throughout the field in a general sense.
Finally, we looked at the effect of equivalents of diBrPD 17e on bridging efficiency. Employing an in-situ protocol at
4 °C, we reacted 15, 30, 40, and 50 equivalents of diBrPD 17e with trastuzumab 18. In each case the amount of
organic solvent (DMSO) included to solubilise the linker remained constant. These results show that increasing the
equivalents of diBrPD 17e does not grant greater levels of homogeneity; in fact, increasing the amount of diBrPD
17e beyond 30 equivalents granted lower PDAR values (Table 2). During these reactions a visible precipitate was
observed, attributed to precipitation of the linker, which may account for the observation. Whilst the SDS-PAGE
gels of these experiments show high homogeneity, the corresponding low PDAR values indicate this could be down
to re-oxidation of the disulfide bridges rather than functional rebridging. Gratifyingly, these results demonstrate
that as low as 15 equivalents of diBrPD 17e can be employed without sacrificing bridging efficiency and the
homogeneity of the final product.
Figure 3 Optimisation of the disulfide bridging of trastuzumab using diBrPD 17e. Unless otherwise specified, a concentration of
20 mM of trastzumab was employed. Unless other specified, 10 equivalents of TCEP·HCl and 20 equivalents of PD 17e were
employed. For full details of each protocol refer to supplementary information. (a) Schematic representation of the generation
of a PDAR 4 trastuzumab conjugate. (b) SDS-PAGE gel: M: Molecular weight protein marker in kDa; N: Native trastuzumab 18; 1:
Step-wise protocol; 2: Sequential protocol; 3: in situ protocol; 4: in situ protocol 4 °C; 5: in situ protocol 8 °C; 6: in situ protocol

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25 °C; 7: in situ protocol 37 °C; 8: in situ protocol 15 equivalents of diBrPD 17e; 9: in situ protocol 20 equivalents of PD 17e; 10:
in situ protocol 30 equivalents of PD 17e; 11: in situ protocol 40 equivalents of diBrPD 17e; 12: in situ protocol 50 equivalents of
diBrPD 17e. (c) Densitometry traces showing the effect of protocol on bridging efficiency: 1) step-wise; 2) sequential; 3) in situ.
(d) Densitometry traces showing the effect of temperature (4 °C, 8 °C, 25 °C, and 37 °C) on bridging efficiency. (e) Densitometry
traces showing the effect of equivalents of diBrPD 17e (15, 20, 30, 40, and 50 equivalents) on bridging efficiency.
Table 2 Optimisation of the disulfide rebridging of trastuzumab 18 with diBrPD 17e
Protocol
2
Temperature / °C
25 37
3.5 4.8 14.1 18.5 6.0 4.8 4.0 0c 1.7c
4.2 3.9 3.9 3.9 3.6 3.3
Equivalents of diBrPD x
1
3
4
8
15 20 30 40 50
% Half Antibodya 23 20
PDARb
8
4.1 4.1 3.9 4.2 3.8 3.9
a Calculated from densitometry traces in Fig. 4 using Image J. See SI for details.
b PDAR was calculated using UV-vis spectrophotometry. See SI Fig. S19-S21
c Low PDAR values indicate these values may not be representative. See discussion above.
Validation of an optimised protocol
The result of the optimisation study detailed above were combined to develop an optimised conjugation protocol
of diBrPD 17e with trastuzumab 18. Trastuzumab 18 was mixed with 20 equivalents of diBrPD 17e and allowed to
cool to 4 °C for 2 hours. After this time, 10 equivalents of TCEP were added and the reaction incubated at 4 °C for
16 hours. Pleasingly, this led to a construct with a PDAR of 4 and a high level of homogeneity, with densitometry
analysis indicating only 5% of the total antibody concentration existed as the mis-bridged half antibody. To
demonstrate scope, this protocol was applied to diBrPDs 17a-d. To our delight, in each case equally high levels of
homogeneity were observed with conjugates 19a-e along with optimal PDARs (Fig. 4). ELISA of conjugates 19a-e
demonstrated no loss of antigen binding when compared to native trastuzumab 18 (Fig. S24), providing evidence
that modification with diBrPDs 17a-e does not affect antigen binding. This study confirms that the protocol is
compatible with linkers of varying hydrophobicity and functionalities, an important factor for the development of
ADCs.

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Figure 4 (a) Schematic representation of the rebridging of trastuzumab 18 with diBrPDs 17a-e under optimised conditions. (b)
SDS-PAGE gel: M: Molecular weight protein marker in kDa; H: Native trastuzumab 18; 1: Bridging with diBrPD 17a; 2: Bridging
with diBrPD 17b; 3: Bridging with diBrPD 17c; 4: Bridging with diBrPD 17d; 5: Bridging with diBrPD 17e. (c) Densitometry traces
of SDS-PAGE lanes 1 t 5.
Table 3 Optimised reaction of diBrPD 17a-e with trastuzumab 18
diBrPD 17a-e
17a 17b
17c
1
17d
6.2
4.0
17e
4.7
% Half Antibodya 3.3
3.8
PDARb
4.2
4.1
4.0
4.0
a Calculated from densitometry traces in Fig. 4 using
Image J. See SI for details.
b PDAR was calculated using UV-vis
spectrophotometry. See SI Fig. S22
Conclusion
To conclude, we have developed several novel protocols to improve both the synthesis and subsequent application
of functionalised dibromopyridazinediones, a promising class of disulfide functional rebridging reagents. During
this work we have addressed two pressing challenges in their synthesis; the formation of the core scaffold, and
subsequent amine-coupling reaction of this scaffold to enable chemical functionalisation. A one-pot synthesis of
the core scaffold from readily available starting materials provides a safer and more efficient alternative to
reported routes, whereas a novel isolable NHS-ester intermediate enables a more general protocol for the

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derivatisation of this scaffold with a variety of amines. The capacity of these reagents to functional rebridge
disulfide bonds was optimised on the humanised IgG trastuzumab, and a protocol was developed which grants
high levels of homogeneity (>90%) and a pyridazinedione-to-antibody ratio of 4. Furthermore, the novel findings
on the temperature of bioconjugation being critical to outcome could be a significant general find to the field of
bioconjugation as a whole. Moreover, this procedure comes at minimal expense and complexity when compared
to previously published routes. Therefore, it is expected that the development of these protocols will vastly
increase the accessibility of this exciting class of reagents, and facilitate their use within the broader scientific
community.
Acknowledgements
We gratefully acknowledge the EPSRC (CASE Award with LifeArc, 173621) for funding CB. We also gratefully
acknowledge the EPSRC (EP/M01792X/1) and UCLB/Albion Ventures for funding AM and DAR, respectively. We
also acknowledge the Wellcome Trust for funding FJ and the EPSRC UK National Mass Spectrometry Facility at
Swansea University.
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Organic & Biomolecular Chemistry
Page 14 of 14
View Article Online
DOI: 10.1039/C7OB03138F
Herein we report novel protocols for the generation and application of dibromopyridazinediones, an
exciting class of disulfide bridging reagents.