Chemoselective Installation of Amine Bonds on Proteins through Aza-Michael Ligation

Article abstract of DOI:10.1021/jacs.7b10702

Chemical modification of proteins is essential for a variety of important diagnostic and therapeutic applications. Many strategies developed to date lack chemo- and regioselectivity as well as result in non-native linkages that may suffer from instability in vivo and adversely affect the protein's structure and function. We describe here the reaction of N-nucleophiles with the amino acid dehydroalanine (Dha) in a protein context. When Dha is chemically installed in proteins, the addition of a wide-range N-nucleophiles enables the rapid formation of amine linkages (secondary and tertiary) in a chemoselective manner under mild, biocompatible conditions. These new linkages are stable at a wide range of pH values (pH 2.8 to 12.8), under reducing conditions (biological thiols such as glutathione) and in human plasma. This method is demonstrated for three proteins and is shown to be fully compatible with disulfide bridges, as evidenced by the selective modification of recombinant albumin that displays 17 structurally relevant disulfides. The practicability and utility of our approach is further demonstrated by the construction of a chemically modified C2A domain of Synaptotagmin-I protein that retains its ability to preferentially bind to apoptotic cells at a level comparable to the native protein. Importantly, the method was useful for building a homogeneous antibody-drug conjugate with a precise drug-to-antibody ratio of 2. The kinase inhibitor crizotinib was directly conjugated to Dha through its piperidine motif, and its antibody-mediated intracellular delivery results in 10-fold improvement of its cancer cell-killing efficacy. The simplicity and exquisite site-selectivity of the aza-Michael ligation described herein allows the construction of stable secondary and tertiary amine-linked protein conjugates without affecting the structure and function of biologically relevant proteins.

Full text of DOI:10.1021/jacs.7b10702

Article
pubs.acs.org/JACS
Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Chemoselective Installation of Amine Bonds on Proteins through
Aza-Michael Ligation
Allyson M. Freedy,†,# Maria J. Matos,†,# Omar Boutureira,†,# Francisco Corzana,†,‡,# Ana Guerreiro,⊥,#
Padma Akkapeddi,⊥ Víctor J. Somovilla,†,‡ Tiago Rodrigues,⊥ Karl Nicholls,§ Bangwen Xie,∥
Gonzalo Jimenez-Oses,‡ Kevin M. Brindle,∥,‡ Andre A. Neves,∥ and Goncalo J. L. Bernardes*,†,⊥
̧
́
́
́
†Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge, U.K.
‡Departamento de Química, Centro de Investigacion
́
en Síntesis Química, Universidad de La Rioja, 26006 Logrono, Spain
̃
§Albumedix Ltd., Castle Court, 59 Castle Boulevard, NG7 1FD Nottingham, U.K.
∥Li Ka Shing Centre, Cancer Research UK Cambridge Institute, Robinson Way, CB2 0RE Cambridge, U.K.
⊥Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa,
Portugal
S
* Supporting Information
ABSTRACT: Chemical modification of proteins is essential
for a variety of important diagnostic and therapeutic
applications. Many strategies developed to date lack chemo-
and regioselectivity as well as result in non-native linkages that
may suffer from instability in vivo and adversely affect the
protein’s structure and function. We describe here the reaction
of N-nucleophiles with the amino acid dehydroalanine (Dha)
in a protein context. When Dha is chemically installed in
proteins, the addition of a wide-range N-nucleophiles enables
the rapid formation of amine linkages (secondary and tertiary)
in a chemoselective manner under mild, biocompatible
conditions. These new linkages are stable at a wide range of pH values (pH 2.8 to 12.8), under reducing conditions (biological
thiols such as glutathione) and in human plasma. This method is demonstrated for three proteins and is shown to be fully
compatible with disulfide bridges, as evidenced by the selective modification of recombinant albumin that displays 17 structurally
relevant disulfides. The practicability and utility of our approach is further demonstrated by the construction of a chemically
modified C2A domain of Synaptotagmin-I protein that retains its ability to preferentially bind to apoptotic cells at a level
comparable to the native protein. Importantly, the method was useful for building a homogeneous antibody-drug conjugate with
a precise drug-to-antibody ratio of 2. The kinase inhibitor crizotinib was directly conjugated to Dha through its piperidine motif,
and its antibody-mediated intracellular delivery results in 10-fold improvement of its cancer cell-killing efficacy. The simplicity
and exquisite site-selectivity of the aza-Michael ligation described herein allows the construction of stable secondary and tertiary
amine-linked protein conjugates without affecting the structure and function of biologically relevant proteins.
defined amino acids within a protein’s structure. For many
applications, particularly for the development of therapeutics,
introducing these modifications selectively at particular sites is
of paramount importance. In one example, it has been
demonstrated previously that a heterogeneously labeled ADC
is significantly less efficacious than a homogeneous, but
otherwise identical, conjugate.9 For this application as well as
many others, the development of a site-selective conjugation
methodology is essential.
One particularly powerful approach to achieve site-selective
chemical modification of proteins utilizes engineered non-
canonical amino acids.2 These amino acids can be incorporated
into a protein’s structure using triplet amber codon
INTRODUCTION
■
For a variety of important diagnostic and therapeutic
applications, there is considerable interest in the covalent
modification of proteins.1−6 This field has grown to include a
diverse range of reactions that modify a variety of different
proteins with unique functions. The modification of such
proteins enables the synthesis of protein conjugates suitable for
the study of post-translational modifications,3 the imaging of
biological processes6 and the construction of protein conjugates
for targeted therapeutics, such as antibody-drug conjugates
(ADCs).7,8
Despite the great interest in the field of covalent chemical
modification of proteins over the past decade, many of the
documented methods lack site-selectivity within the chemically
complex protein environment. For this reason, there is a
pressing need for the development of reactions that modify
Received: October 11, 2017
© XXXX American Chemical Society
A
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suppression10,11 or auxotrophic strains of bacteria.12 The
utilization of these techniques to include unnatural function-
alities, such as alkynes, azides, ketones, alkenes or tetrazines
into a protein has greatly accelerated the expansion of site-
selective protein modification.2,5 Although reactions that target
such functional groups are highly useful and often show a high
degree of chemoselectivity, even in living systems,2 the
installation of noncanonical amino acids requires a high degree
of protein engineering that can lead to low expression levels,
which can be detrimental for many diagnostic and therapeutic
applications. A complementary approach exploits the unique
reactivity of the N-terminal position of a given protein, for
instance, via imidazolidinone formation using 2-pyridinecarbox-
aldehyde.13 While N-terminal modification methods are
attractive because they do not require protein engineering,
they are limited to the N-terminal position as the site of
attachment. More recently, the introduction of a specific amino
acid sequence that enhances the reactivity of a particular amino
acid side chain, for example cysteine (Cys), has enabled site-
selective protein modification even if other cysteine residues
were present in the protein sequence.14 However, this method
requires extensive engineering of the native protein sequence
for efficient bioconjugation.
disease imaging or proteins that serve as vehicles for drug-
delivery purposes such as albumins or antibodies.7,22
Here, we report a simple and robust methodology for protein
site-selective bioconjugation based on the addition of N-
nucleophiles to engineered internal Dha residues in a protein
context. This reaction, which we refer to as aza-Michael
ligation, proceeds in a chemoselective fashion under mild
conditions (25 to 37 °C in buffered aqueous solution at pH
8.0) and results in a natural secondary amine linkage (truncated
Lys analogue) that is stable to degradation in biological
conditions. To date, generation of a secondary amine bond on a
protein has been limited to reductive amination strategies that
requires either the use of highly reactive reducing agents such
as NaBH4 or NaBH3CN,23 or the use of an iridium catalyst,
[Cp*Ir(4,4′-dimethoxy-2,2′-bipyridine)(H2O)]SO4, in the
presence of formate ions.24 Additionally, acrylamide-containing
noncanonical amino acids have been genetically encoded on
proteins and reacted with N-nucleophiles in a proximity-driven
reaction.25 We demonstrate the potential of the aza-Michael
ligation method for three different proteins that contain
naturally occurring or genetically engineered surface-exposed
Cys residues. We also show the high degree of disulfide
compatibility of this novel method, whose simplicity and
robustness is likely to find broad applicability for the
modification of proteins used in diagnostic and therapeutic
applications.
The most commonly used approach for the site-selective
modification of therapeutic proteins remains the Michael
addition of the sulfhydryl side-chain of Cys residues on the
protein’s surface with maleimide reagents.15 For example, a
recently Food and Drug Administration approved ADC,
brentuximab vedotin, used maleimide chemistry to conjugate
a cleavable linker bearing a cytotoxic drug to genetically
engineered Cys residues on the surface of an antibody against
RESULTS AND DISCUSSION
■
Amine Additions to Dha on Amino Acids. Our design of
a conjugation method to site-selectively install amine linked
modifications on a protein was inspired by the potential
reactivity of Dha toward N-nucleophiles (Figure 1a). There are
very few examples of the reaction of Dha with N-nucleophiles
on peptides,26−28 and to an even lesser extent on proteinsin a
single example, Dha has been used as a precursor for the
installation of an isomer of histidine through the reaction with
imidazole.29−32
CD-30, a marker used to target Hodgkin’s Lymphoma.16
A
potential drawback of this approach is that the thioether
succinimide linkage that results from this reaction can rapidly
undergo retro-Michael addition under physiological conditions,
resulting in early release of the cargo from the carrier antibody
leading to off-target toxicity.17 In a complementary approach to
direct Cys-based conjugation methods, Cys has been utilized as
a precursor to chemically install the amino acid dehydroalanine
(Dha) on proteins.18,19 Its unique electrophilic character, when
compared with the natural nucleophilic residues, enabled the
development of a robust and reliable method to form stable
thioether-linkages between the protein and modification (e.g.,
post-translational modifications (PTMs) such as phosphor-
ylation, acetylation, methylation or glycosylation) through the
Michael addition of suitable thiol reagents.20,21 Although this
methodology has found many applications in protein
modification, the small molecule sulfur nucleophiles used may
interfere with existing disulfide bonds present in the protein,
especially in cases where these are solvent exposed, forming
mixed disulfides that may disrupt the protein native structure
and function.
On the basis of these considerations, we postulated that the
development of a method for the chemical site-selective
modification of Dhaaccessed through ready chemical
conversion from Cysthat is disulfide compatible, not limited
to terminal positions and affords a native chemical linkage
between protein and the desired modification would find great
utility for the modification of proteins that are already available
through large-scale production and that have been engineered
with additional Cys residues. These include, for example,
several recombinant proteins that are used as biomarkers for
We first investigated the potential of Dha as an aza-Michael
acceptor within a small molecule context. This was performed
by examining the reaction between a Boc-Dha methyl ester 1
and a variety of small molecule nitrogen nucleophiles 2a−9a
with different N-hybridizations, which are representative of
common motifs found in drug fragments, spectroscopic probes,
linkers and PTMs (Figure 1b). These reactions were carried
out in aqueous conditions, specifically in a 1:1 mixture of DMF
and 50 mM sodium phosphate buffer at near neutral pH 8.0
and 37 °C. These reaction conditions fulfill the essential
requirements for a potentially useful protein modification
reaction.
Chemical yields were calculated after SiO2 flash chromatog-
raphy purification for each reaction and ranged between 25−
30%, with the exception of benzylamine 2a, for which the
reaction proceeded with 69% yield (Figure 1b). Poorly
nucleophilic amines, such as aniline 3a, proved to be unreactive
under the conditions tested. We also evaluated the chemo-
selectivity (N-sp3 vs N-sp2) of the aza-Michael addition by
reacting Boc-Dha methyl ester 1 with histamine 5a, which has
two possible reactive nitrogen atoms. Under the conditions
tested, 60% conversion in a 1.7:1 sp3/sp2 ratio was observed,
suggesting predominant addition of the primary amine to Dha
1 (Figure S1). Next, we studied the diastereoselectivity of the
addition reaction by reacting dipeptide Boc-Ala-Dha methyl
ester S1 with 2a. As expected, this afforded the corresponding
B
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Staudinger ligations.2 The superior reactivity of benzylamine
2a compared to imidazole 4a in the aza-Michael reaction,
especially in the protein context (vide infra), was examined
computationally using abbreviated models for the dehydro
amino acid (Figure 1e and 1f, Figure S4, Table S1). Using the
M06-2X33 DFT functional, which has been proved to give
accurate results for Michael addition reactions,34 the exper-
imental reactivity trends were reproduced, revealing the higher
nucleophilic character of the N-sp3 in 2a compared to the
reactive N-sp2 in 4a. The calculated relative reaction rates at 25
°C for benzylamine 2a (krel = 13) and imidazole 4a (krel = 1)
correlate well with the experimental values (krel = 11 and 1 for
2a and 4a, respectively) when the dehydro amino acid is in the
same local environment and peptide backbone conformation
(Figure 1e and 1f). Once the ability of the aza-Michael addition
to Dha derivative 1 was demonstrated with various amines, we
expanded our investigation to proteins with chemically
engineered, internal Dha residues on their surface.
Benzylamine Addition to Dha-Tagged Proteins. With
these encouraging small-molecule results in hand, we next
examined the reaction with a model protein to evaluate its
potential as a novel protein ligation methodology for the
construction of chemically defined secondary amine linked
protein conjugates. To this aim, we selected a single cysteine
mutant of the C2A domain of Synaptotagmin-I (C2Am).35 We
selected this protein due to its diagnostic relevance as an
apoptosis imaging agent, as well as the accessibility of the
engineered Cys residue on the protein surface. We successfully
generated Dha from the engineered Cys at position 95 on the
C2Am protein by the previously reported bis-alkylation-
elimination method.19 To achieve this we treated C2Am-
Cys95 with α,α′-dibromo-adipyl(bis)amide 10 (92.4 mM) and
complete conversion to C2Am-Dha95 was observed after 3 h at
room temperature and 1 h at 37 °C (Figure S10). Chemical
mutation of Cys to Dha proceeds with minimal perturbation of
the global structure of the protein as evidenced by Circular
Dichroism analysis (CD) (SI). Having obtained successfully the
Dha residue at position 95 of C2Am, we began our study with
the addition of benzylamine 2a (11 mM), the amine that
achieved the highest Dha conversion in the small molecule
experiments (Figure 2a). We obtained complete conversion
after 3 h at room temperature in sodium phosphate buffer pH
8.0, as confirmed by liquid-chromatography mass-spectrometry
(LC−MS) analysis (Figure 2b; see Figure S5 for a typical
conjugation analysis by LC−MS). The presence of the expected
secondary benzylamine linkage at position 95 was further
confirmed by tryptic digest and MS/MS analysis (Figure 2e).
Next, we sought to broaden the scope of this reaction to
other proteins. Thus, we investigated the addition of benzyl-
amine 2a to an engineered Dha residue in Annexin-V (Figure
2c). Annexin-V is another apoptosis imaging agent, functionally
similar to C2Am in that it binds to the phosphatidylserine (PS)
externalised to the outer leaflet of the plasma membrane of cells
undergoing apoptosis.36 It contains a single free Cys that is
positioned in a more hindered and challenging position when
compared to that on C2Am. We synthesized Dha from this
single Cys by incubating Annexin-V-Cys315 (27.8 μM) with
α,α′-dibromo-adipyl(bis)amide 10 (14 mM) for 4 h at 37 °C
(Figure S15). We observed full addition of benzylamine 2a
(165.7 mM) to Annexin-V-Dha315 (14.9 μM) after 5 h at 40
°C, as shown by LC−MS (Figure 2d). Although these
conditions are harsher when compared to the addition of 2a
to C2Am-Dha95, this is unsurprising due to the buried nature
Figure 1. Reaction between a protected Dha amino acid derivative and
amine nucleophiles. (a) Reaction of Boc-Dha methyl ester 1 with N-
nucleophiles. (b) Graphical representation of the isolated yields of the
reaction of 1 and N-nucleophiles 2a−9a. General conditions for amine
addition to Dha: Boc-Dha methyl ester 1 (1 equiv) and N-nucleophile
2a−9a (1.5 equiv) in a 1:1 mixture of DMF/sodium phosphate buffer
(50 mM, pH 8.0) at 37 °C for 4 h. All yields were calculated after SiO2
flash column chromatography purification with the exception of the
addition of 5a to 1 for which conversion using the crude mixture is
indicated* (1.7:1 N-sp3/N-sp2 ratio). (c,d) Experimental determination
of the second order rate constant for the addition of benzylamine 2a
and imidazole 4a to Boc-Dha methyl ester 1, respectively. (e,f)
Transition structures and associated activation free energies (ΔG‡) at
25 °C and relative reaction rates (krel) calculated with PCM(water)/
M06-2X/6-311+g(2d,p) for the aza-Michael ligation of model dehydro
amino acid Ac-Dha-NHMe with benzylamine 2a and imidazole 4a,
respectively. NaPi, sodium phosphate buffer; DMF, dimethylforma-
mide; Boc, tert-butyloxycarbonyl.
aza-Michael ligation product S2 in 40% yield as a 1:1 mixture of
diastereomers (Figure S2). Using the product of benzylamine
2a addition to the amino acid Dha 1, we also evaluated the
stability of the newly formed secondary amine linkage under a
variety of pH conditions. We found that the secondary amine
linkage was stable from pH 2.8 to pH 12.8, as confirmed by 1H
NMR. Peaks corresponding to the alkene group of Dha or
products resulting from peptide cleavage/BnNH2 release were
not observed in these spectra, indicating that there was no
detectable degradation of the product under this range of pH
conditions (Figure S3). These data show that although the
secondary amine may be protonated in slightly acidic biological
environments, it should remain stable, an important feature for
diagnostic and therapeutic applications of protein conjugates.
Kinetic Measurements and Theoretical Calculations.
Building upon these results, we then examined the kinetics of
the reaction between Dha 1 and two N-nucleophiles,
specifically, benzylamine 2a and imidazole 4a, by calculating
second order rate constants. By monitoring the reaction using
1H NMR, we determined the second order rate constants (k2)
to be 6.1 × 10−4 and 5.6 × 10−5 M−1 s−1, respectively (Figure
1c and 1d). In the case of benzylamine 2a this value is of the
same magnitude as the second order rate constants of common
protein modification reactions, such as the oxime and
C
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Figure 2. Chemical site-selective Dha modification with benzylamine
on proteins. (a) Addition of 2a to the engineered Dha residue at
position 95 of the C2Am. (b) ESI−MS spectrum of C2A-NHBn. (c)
Benzylamine 2a addition to the engineered Dha at position 315 on the
surface of Annexin-V. (d) ESI−MS spectrum of Annexin-V-NHBn. (e)
MS/MS spectrum of the m/z 519.78 doubly charged ion of the tryptic
peptide VPYCELGGK, containing the −NHBn− modification at the
original Cys95 residue. The fragment ions generated are consistent
with the mass of the modification. (f,g) Surface representation of
C2Am and Annexin-V, respectively, showing in purple the Cys residue
that is converted into Dha. While in C2Am the reactive Cys is exposed
to the solvent, in Annexin-V this residue is found in a more buried
position.
Figure 3. Site-selective modification of C2Am-Dha95 with a range of
amines. (a) The reaction of C2Am-Cys95 with a wide variety of N-
nucleophiles. The conversions listed here are the maximum
conversions that could be obtained by reacting the different N-
nucleophiles with C2Am-Dha95. Further experimental details on each
reaction may be found in Supporting Information. (b) The reaction of
compound 26 with C2Am-Dha95 went to completion as detected by
ESI−MS shown here. (c) Treatment of C2Am-Dha95 with compound
27 afforded a fluorescent protein conjugate, as detected by SDS-PAGE
gel shown here. Lanes 1 and 2, Coomassie staining. Lanes 3 and 4,
fluorescence.
of the free Cys of Annexin-V, as clearly observed in a surface
representation of the modification sites on both proteins
(Figure 2f and 2g).
Scope of N-Nucleophile Additions to Dha-Tagged
Proteins. Having demonstrated that it is possible to generate a
secondary amine bond at the internal position of Dha-tagged
proteins, we explored the scope of the aza-Michael addition
ligation to Dha on C2Am-Dha95 by exhaustively testing a wide
range of different N-nucleophile reagents, as shown in Figure
3a. Similar results to those obtained with benzylamine 2a were
obtained in the addition of cyclohexylmethylamine 6a with
complete addition observed after 3 h at room temperature. A
complete addition was also observed with 4-hydroxybenzyl-
amine 11, 4-(aminomethyl)benzoic acid 12 and 4-
(aminomethyl)cyclohexanecarboxylic acid 15 after 24 h at 37
°C. Interestingly, full conversion was also observed after 24 h at
37 °C with histamine 5a, an important neurotransmitter and
immunomodulatory small molecule.37 The addition of 4-
aminobutyl β-D-galactopyranoside 22, as an example of protein
glycosylation via direct attachment of sugar units, also
proceeded with complete conversion. Interestingly, we found
that the secondary amine piperidine 23 (example of N-sp3
heterocyclic motif found in many biologically active drugs−see
Table S4) adds efficiently to Dha to form a tertiary amine-
linked conjugate (Figure S39). Unlike other amines, complete
conversion was achieved with the more nucleophilic 23 using
only 300 equiv. Of note, we also observed full conversion by
addition of “softer” nucleophilic species such as hydroxylamine
24 and hydrazine 25 as shown by LC−MS (Figure S41 and
S42). The modification site for the hydrazine addition product
was also confirmed by peptide mapping and MS/MS analysis
(Figure S58). These results expand the scope of N-nucleophiles
that may be successfully reacted with Dha beyond simple amine
handles. These reactions also compare favorably in terms of
mildness and operational simplicity with standard reductive
amination-like protocols using hydroxylamines/hydrazines with
aldehyde/ketone handles. Among the other amines tested,
imidazole 4a (example of N-sp2 heterocyclic motif found in
many biologically active amines), N-Boc-1,6-hexadiamine 8a
(aliphatic linker), 4-bromobenzylamine 13 (handle for C−C
cross-couplings), phenylethylamine 14 (an important neuro-
modulator) showed the greatest conversions−up to 90% after
16−48 h at 37 °C. All other amines surveyed showed
conversions ranging between 40%−60% upon incubation at
37 °C for 24−48 h, with the aqueous solubility of each amine
nucleophile being a key factor in determining its reactivity. Yet,
the lower conversion could be mitigated by the addition of
variable amounts of DMF as a cosolvent (SI).
Protein Labeling Using Aminomethylbenzoic Acid
Derivatives. To explore the versatility of aza-Michael ligation
on proteins, particularly toward PEGylation and fluorescent
labeling,5,38 we investigated the addition of tetraethylene glycols
21 and 26 and fluorescein derivative 27 to C2Am-Dha95. p-
Aminomethylbenzoic derivatives 26 and 27 were readily
synthesized from accessible p-aminomethylbenzoic acid 12.
The basic structure of this compound is composed of a primary
amine for conjugate addition to Dha and a carboxylic acid for
D
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functionalization with a variety of different molecules and
proved to be one of the best p-substituted benzylamine
performers with C2Am-Dha95 (see SI for synthesis details).
Preliminary ligations with 21 afforded the corresponding
PEGylation product in 40% conversion (Figure 3a). In contrast,
treatment of 26 (62.5 mM) with C2Am (6.1 μM) for 48 h at 37
°C gave full conversion to the corresponding PEGylated
protein, as shown by LC−MS (Figure 3b). Similarly, when
C2Am-Dha95 was reacted with the fluorescein derivative 27, a
fluorescence conjugate was formed as evidenced by fluorescent
SDS-PAGE gel (Figure 3c). This result shows that through the
use of the appropriate primary amine handle, this reaction may
be generalized to connect a variety of important synthetic
compounds to biomolecules.
Aza-Michael Ligation Is Disulfide Compatible. To
verify the disulfide compatibility of this new amine addition
methodology, we first took advantage of the ready dimer
formation of C2Am through a disulfide bond between the
engineered free Cys residue at position 95. After leaving C2Am-
Cys95 open to the atmosphere for 1 h at 37 °C, we observed
complete conversion to the oxidized 32 kDa dimer from the
original 16 kDa protein (Figure S45). We then treated this
protein dimer with a model thiol, β-mercaptoethanol 28, and
benzylamine 2a to compare the compatibility of these
nucleophiles with disulfide bonds (Figure 4a). We were
encouraged that we observed maintenance of the dimer, and
thus preservation of the disulfide bond integrity, when the
C2Am-Cys95 dimer was treated with 2a (56.6 mM − the same
amine concentration required to afford complete conversion on
Dha) for 2 h at room temperature, as shown by LC−MS
analysis (Figure 4b). Instead, when this dimer was treated with
β-mercaptoethanol 28 (22.5 mM) under the same conditions, 2
h at room temperature, complete reduction of the disulfide
bond was readily achieved with no significant dimer observed in
the mass spectrum (Figure 4c).
To further demonstrate the chemoselectivity of the reaction,
we next evaluated amine addition to a particularly relevant
protein, albumin. Human serum albumin is the most abundant
protein found in plasma. It is comprised of 585 amino acids and
contains 17 structural disulfides plus one free Cys residue at
position 34. Plasma derived human albumin has been used in
the clinic for decades to expand plasma volume in order to
counter severe blood loss as well as in the formulation of active
pharmaceutical ingredients.39 In the interest of having a well-
defined single pure protein for these therapeutic applications,
recombinant human albumin has become available and gained
wide acceptance as a bespoke human albumin for formulation,
drug delivery and imaging applications.22 Thus, we investigated
the addition of benzylamine 2a to the single engineered Dha
residue in a recombinant human albuminRecombumin
(Albumedix Ltd.) (Figure 4d). We first confirmed the reactivity
of the free Cys34 residue by reaction with Ellman’s reagent,
which resulted in full conversion to the corresponding disulfide
adduct (Figure S53), showing the thiol of Cys34 to be in its
reduced and reactive form. Dha was then installed into albumin
by reaction of the single Cys residue at position 34 with α,α′-
dibromo-adipyl(bis)amide reagent 10 (22.5 mM). However,
although we found monoalkylation of the single Cys to proceed
rapidly to completion in 1.5 h at 37 °C, elimination proved to
be more difficult. Bis-alkylation and elimination to yield the
desired Dha residue were ultimately completed through the
addition of 3 M guanidinium hydrochloride to slightly
destabilize the protein structure and by temporarily raising
Figure 4. Addition of N-nucleophiles to Dha on proteins is compatible
with disulfides. (a) Comparative experiment where the readily formed
disulfide dimer of C2Am-Cys95 was exposed to a S-nucleophile, β-
mercaptoethanol 28, and to an N-nucleophile, benzylamine 2a. (b)
ESI−MS spectrum of the reaction of disulfide dimer of C2Am-Cys95
with benzylamine shows the unreacted dimer of C2Am-Cys95
indicating that benzylamine does not cross-react with the disulfide.
(c) ESI−MS spectrum of the reaction of disulfide dimer of C2Am-
Cys95 with β-mercaptoethanol 28 shows rapid disulfide reduction and
formation of the monomer C2Am-Cys95. (d) Conversion of the free
Cys34 of Recombumin to Dha and addition of benzylamine 2a. (e)
ESI−MS spectrum of the product of the reaction of Recombumin-
Cys34 with α,α′-dibromo-adipyl(bis)amide 10. (f) Reaction of
Recombumin-Dha34 with benzylamine 2a results in a homogeneous
conjugate Recombumin-NHBn34, as shown in the ESI−MS spectrum.
(g) Surface representation of Recombumin showing the Cys residue at
site 34 (in pink) that is first converted into Dha and then used as a
handle for aza-Michael ligation. A number of disulfides bridges that are
solvent exposed are shown in yellow.
the pH to 12.40 Under these conditions, the monoalkylated
Cys34 in albumin (Figure S54) yielded the Dha residue with
full conversion (Figure 4e). The reluctance of the elimination
reaction to occur could be a reflection of the distinct local
environment of the groove where Cys34 is found.41 This
residue was then reacted with benzylamine 2a to yield the
desired conjugate (Figure 4f) as shown by ESI−MS analysis.
Importantly, we conclude that the new method for installing
secondary amine bonds on proteins via aza-Michael ligation
with amine nucleophiles is fully compatible with disulfide
bonds, even in cases where these disulfides are solvent exposed
(Figure 4g).
Protein Conjugates Are Stable to Biological Thiols.
Upon obtaining the benzylamine conjugated C2Am protein,
C2Am-NHBn, we aimed to verify its potential value of the
secondary amine linkage. First, we sought out to demonstrate
the linker’s stability in human plasma. Linker stability is an
essential requirement for the development of useful protein
conjugates for therapeutic and diagnostic applications since
systemic, nontargeted release of the conjugated payload would
result in undesired side-toxicity and nonspecific imaging,
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respectively. Importantly, no detectable degradation of the
benzylamine conjugate was observed after incubation with
human plasma for 24 h at 37 °C, demonstrating the conjugate’s
stability under these conditions (Figures S48 and S49).
Complete stability in human plasma was also observed for
the conjugate formed after the reaction of C2Am with
piperidine 23 that features a tertiary amine bond (Figure
S40). Additionally, when C2Am-NHBn was incubated under
reducing conditions (1 mM glutathione for 24 h at 37 °C), the
conjugate remained intact, further demonstrating its stability in
biologically relevant conditions (Figures S50 and S51).
Protein Conjugates Retain Biological Function. Albu-
min has an extended serum half-life of approximately 3 weeks
due to its size and neonatal Fc receptor (FcRn) mediated
recycling that prevents intracellular degradation.42 Albumin
binds FcRn in a noncooperative and strictly pH dependent
manner, with strong binding at pH 6.0 that becomes
progressively weaker approaching physiological pH.43 Using
Surface Plasma Resonance (SPR), we confirmed that the
conjugate Recombumin-NHBn34 binds to FcRn in a reversible
and pH dependent manner (Figure 5a, Table S2). While the
rate of association was slower than the unmodified
Recombumin-Cys34 control, the binding event was comparable
(Figure 5a, Table S2). This result highlights the utility of our
method to provide functional protein conjugates regardless of
exposure and reactivity of the target Cys.
C2Am has been validated in vivo for detecting apoptotic
tissue, using a variety of methods, including magnetic resonance
imaging (MRI)44 and single photon emission computed
tomography (SPECT).45 C2Am specifically labels apoptotic
cells by binding to phosphatidylserine (PS) available on their
cell membrane. Therefore, we verified that C2Am modified
through a secondary amine linkage retained its natural high
affinity for PS and the ability to detect apoptotic cells in vitro.
Using affinity chromatography, we verified that the conjugate
C2Am-NHBn shows similar binding to PS as the original
unaltered C2Am protein (Figure 5b). Additionally, using flow
cytometry, we proved that C2Am-NHBn retained its ability to
preferentially bind to apoptotic cells over viable cells, at a level
comparable to the unmodified protein (Figure 5c). With these
results, we surmised that the benzylamine adduct retained the
original biological function of C2Am even upon modification,
highlighting the potential of our conjugation method for the
construction of functional modified proteins.
Figure 5. Assessment of the biological activity of Recombumin and
C2Am protein after chemical modification. (a) Biacore SPR analysis of
Recombumin-Cys34, blue, and Recombumin-NHBn34, red, n = 3
replicates. (b) Fast protein liquid chromatography analysis utilizing a
HiPrep S FF (GE Healthcare)−affinity column comparing C2Am-Cys,
black, and C2Am-NHBn, red. (c) Flow cytometry plots obtained by
fluorescence activated cell sorting (FACS) of C2Am-Cys (top) and
C2Am-NHBn (bottom) labeling viable (green), apoptotic (blue) and
necrotic (red) EL4 cells. Cell populations (%) for C2Am-Cys95;
C2Am-NHBn95:33
(apoptotic), 31
ratios (C2Am-Cys95; C2Am-NHBn95): 35
1%; 34
6%; 32
1% (viable), 36
1% (necrotic). Apoptotic/viable MFI
1%; 87 11%.
4%; 34
1%
Necrotic/viable MFI ratios (C2Am-Cys95; C2Am-NHBn95): 98
4%; 411 32%. MFI-median fluorescence intensity at 660 nm. Data is
mean s.d., n = 3 replicates, 2 independent experiments.
Construction of Homogeneous Antibody−Drug Con-
jugates. Tumour specific antibody-mediated delivery of
cytotoxic molecules holds great promise for cancer therapy.5,8,16
To further demonstrate the utility of our method, we sought to
build a homogeneous ADC through aza-Michael ligation at
Dha. We chose a Thiomab antibody targeting Her2 receptor.
This antibody has been engineered so that it possesses an
additional Cys residue at position 205 in each light-chain
(Thiomab LC-V205C).9 Most ADCs developed to date utilize
DNA alkylating agents or tubulin polymerization inhibitors as
payloads. These highly potent cytotoxic motifs are usually
released upon cleavage of a conditionally labile linker between
the antibody and the payload. While ADCs are finding
significant clinical use for cancer treatment, there are increasing
reports of side-effects and toxicity associated with ADC
therapy.46 Such off-target toxicity may result, for instance,
from premature drug release in the blood and/or the intrinsic
cytotoxicity of the payloads. Considering these issues, we chose
to develop a strategy based on our aza-Michael addition to Dha
to incorporate novel payloads with differentiated mechanisms
of action into antibodies. Our strategy relies on the formation
of a fully stable ADC where the drug is directly conjugated to
the antibody through an amine linkage. Upon internalization,
cellular processing and degradation in the endosome and
lysosome, the drug is released intracellularly.
We searched for terminal piperidine motifs that could serve
as amine handle for direct aza-Michael site-selective antibody
conjugation in approved/investigational anticancer drugs. The
search pooled several kinase inhibitors (Table S4) from which
we selected crizotinib 30 as a suitable drug to test our
hypothesis. Crizotinib is a known inhibitor of the MET, ALK
and ROS1 kinases47 that has been approved for the treatment
of ALK-rearranged nonsmall-cell lung carcinoma (NSCLC). It
has also been recently described to promote T cell interactions
with monocytes, as well as with cancer cells, through inhibition
of the receptor tyrosine kinase MSTR1 and subsequent up-
regulation of the expression of major histocompatibility
complex molecules.48 Chemically, the piperidine motif was
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Figure 6. Construction of a stable and chemically defined ADC through direct, aza-Michael conjugation of crizotinib to Dha-tagged Thiomab and its
biological evaluation. (a) Reaction scheme for the conversion of Thiomab LC-V205C to Thiomab-Dha using a bis-alkylation/elimination procedure.
(b) ESI−MS spectra of the light- and heavy-chains of Thiomab LC-V205C after reduction and reoxidation protocol. (c) ESI−MS spectra of the
light- and heavy-chains of the reaction of Thiomab LC-V205C with methyl 2,5-dibromopentanoate 29 shows the formation of two Dha residues per
light-chain. (d) KD constants derived from BLI experiments for Thiomab and Thiomab-Dha. For the BLI curves and fitting curves obtained for
Thiomab and Thiomab-30 with Her2 receptor see the SI. (e) Reaction scheme for the reaction of Thiomab-Dha with either piperidine 23 or
crizotinib 30. ESI-MS spectra of the light-chain shows the addition of two piperidine molecules and the addition of one crizotinib 30, respectively. (f)
SKBR3 cells viability after treatment with Thiomab-30 for 24 h. See SI for data with crizotinib and naked Thiomab (control). Results are shown as
percentage of control (medium + vehicle − PBS) and correspond to 3 biological replicates (mean s.d.). (g) IC50 of Thiomab-30 in SKBR3 cells.
(h) Thiomab-30 binding affinity to SKBR3 cells measured by flow cytometry.
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found to be highly reactive and to form a stable tertiary amine
linkage upon aza-Michael addition to Dha. Crizotinib also
features a 2-amino pyridine motif that we found to be highly
unreactive even when used in large excess (Figure S71). This is
expected since piperidine is a much better nucleophile than
anilines in the Mayr’s scale.
to the Her2 antigen in SKBR3 cells as demonstrated by Flow
Cytometry analysis (Figure 6h). With these experiments, we
provide evidence of the potential of this site-selective
conjugation method for the construction of stable and
homogeneous ADCs with a defined DAR. In addition, this
work highlights the potential of directly conjugating drugs that
are outside the traditional choices for ADC construction to
increase their cancer cell killing efficiency by antibody-mediated
intracellular delivery. Moreover, with the advent of ADCs for
treating infectious diseases49 our approach may have broader
applicability beyond cancer therapy.
We started by treating Thiomab LC-V205C with tris(2-
carboxyethyl)phosphine (TCEP) followed by dehydroascorbic
acid mediated disulfide reoxidation. This procedure is required
to ensure that the engineered Cys is in its free form and readily
available for chemical manipulation.9 Using typical Dha bis-
alkylation/elimination procedure with 10 on Thiomab LC-
V205C proved unsuccessful under several conditions (large
excess, high temperatureup to 40 °C, mild denaturing
conditions and high pH 10). In all cases not even the alkylation
product could be detected using LC−MS. Instead methyl 2,5
dibromo-pentanoate29 29 proved to be highly reactive with
Thiomab LC-V205C and enabled the formation of Thiomab-
Dha at 37 °C. Of note, we found that removal of the excess
reagent using size exclusion chromotography after 2 h at pH 8.0
followed by buffer exchange to pH 10.0 and further shaking for
an additional 22 h period to be necessary to facilitate
elimination to Dha (Figure 6a). Analysis by LC−MS showed
the presence of two Dha residues per light-chain while no
modifications were observed in the heavy-chain (Figure 6b).
This data suggests that the inter heavy-chain disulfides were
successfully reoxidized while the one between the Cys on the
light-chain and the hinge Cys on the heavy-chain was not.
Control experiments with thiol-specific Ellman’s reagent also
showed two modifications within the light-chain of Thiomab
LC-V205C supporting the presence of two reactive Cys
residues (Figure S65). Nevertheless, Thiomab-Dha was tested
for its binding affinity to Her2 using biolayer interferometry
and we found that the binding capability of Thiomab-Dha
remained comparable to the nonmodified antibody (Figure 6d).
Next, we tested the reactivity of Thiomab-Dha toward
piperidine. Using a small excess of 23 (3 mM, 100 equiv),
complete conversion to the tertiary-amine modified antibody
was observed after 2 h at 37 °C (Figure 6e). Two modifications
per light-chain were detected by LC−MS while no
modifications were found on the heavy-chain further high-
lighting the compatibility of the aza-Michael addition approach
with disulfides. When the same procedure was applied using
Crizotinib, a chemically defined ADC was obtained bearing
only one drug molecule per light-chain (Figure 6e). This may
be rationalized on the basis of steric hindrancethe engineered
Cys is within proximity of the Cys that is usually present as a
disulfide with the hinge Cys of the heavy-chain. In addition,
solvent accessible surface area (SASA) calculated through 100
ns molecular dynamic simulations in explicit water for Thiomab
(PDB id: 5d6c) indicated a greater SASA value for the 205
residue in comparison to the inner 194 residue. This data
suggests that Dha at position 205 is likely the one that
undergoes aza-Michael addition with 30 (Figure S70). This
homogeneous ADC bearing a defined drug-to-antibody ratio
(DAR) of 2 featured a tertiary amine bond that is fully stable in
human plasma (Figures S40 and S68) and showed an
important, 10-fold improvement in cell-killing activity when
compared to the free drug as assessed by CellTiter-Blue assay
in SKBR3 breast cancer cells that overexpress Her2 (Figures
6f,g). Naked Thiomab showed no effect on cell viability
(control) (Figure S73). Furthermore, we confirmed that the
modified antibody retained its specificity and capacity to bind
CONCLUSION
■
We have described a thorough evaluation of N-nucleophile
additions to internal Dha residues as a chemoselective and
biocompatible protein site-selective modification methodology
for the construction of homogeneous protein conjugates. We
first demonstrated its potential as a protein modification
reaction on a small molecule level both in terms of general
reactivity and kinetics. The second rate order constants were
similar to the rates of widely used protein modification
reactions including the Staudinger ligation50 and the oxime
reaction between noncanonical ketone amino acids and
aminooxy reagents, which are used currently for the assembly
of ADCs that are under evaluation in the clinic.51 We then
conducted a thorough evaluation of the addition of various
amine nucleophiles to the Dha engineered from single Cys
residue in the site-directed mutant of C2Am. From this study,
we demonstrated that some amines, in particular benzylamine,
cyclohexylamine and piperidine as well as their derivatives, and
histamine, are reactive toward the Dha residue at near neutral
pH. Hydroxylamines and hydrazines also reacted efficiently
with Dha, further expanding the scope of N-nucleophiles that
can be used to chemically site-selectively modify Dha on
proteins. In addition, we noticed that the aza-Michael addition
is not (or is poorly) diastereoselective. While it is very
challenging to calculate the diastereomeric ratio on intact
proteins, it is well documented that the ratio is dependent on
the amino acid sequence adjacent to the site of modification, at
least in peptide models.52 However, for most diagnostic and
therapeutic applications racemization of the α-carbon of Cys
will not be detrimental. Even synthetic histones either bearing
thioether-linked PTM mimics that were produced through
thiol-Michael addition at Dha20 or bearing PTMs installed
through carbon free radical chemistry at Dha53,54 were found to
be fully functional despite the likely generation of a
diastereomeric mixture. The same was observed for the
installation of mimics of phosphorylation on protein kinase
p38α and on a single single-domain antibody cAb-Lys3.21,55
However, and in one case, only the natural diasterioisomer of a
γ-thialysine mimic of Lys165 in the enzyme N-acetylneuraminic
acid lyase (NAL) was able to refold correctly and retain
enzymatic activity.56 Therefore, the lack of stereoselectivity of
amine addition to Dha should be of little relevance to most
applications. However, in those applications where stereo-
selectivity is needed, modulation of the local amino acid
environment could be investigated as a means to promote
stereoselective Dha additions.
The utility of the novel conjugation procedure was
demonstrated by extending the reaction to other proteins of
biological interest, namely Annexin-V as well as a recombinant
human albumin used clinically. Albumin provides an example of
a protein that although it displays 35 Cys, with 34 of those
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Healthcare). Post immobilization, the chip was left to stabilize with
a constant flow (5 μL/min) of running buffer. The chip surface was
conditioned by injecting 3× injections of running buffer followed by
3× injections of regeneration buffer. Surfaces were checked for activity
with an unmodified Recombumin-Cys34 albumin control. For
determination of binding kinetics, serial dilutions of Recombumin-
Cys34 and Recombumin-NHBn34 (10−0 μM) were injected over
immobilized receptor at a constant flow rate (30 μL/min) at 25 °C. In
all experiments, data were zero adjusted and the reference cell
subtracted. Data evaluations were performed using BIAevaluation 4.1
software (GE Healthcare).
Flow Cytometry of Cell Death Using C2Am-Cys95 or C2Am-
NHBn95. Murine lymphoma (EL4) cells (ATCC, Piscataway, NJ)
were propagated in RPMI 1640 media (Sigma) supplemented with
10% fetal calf serum (FCS) and 2 mM L-glutamine (Sigma). Cell
number and viability were monitored using the trypan blue dye
exclusion assay on a Vi-cell system (Beckman Coulter, Brea, CA). EL4
cell death was induced by addition of 10 μM etoposide (Teva, Leeds,
UK) for 18 h at 37 °C. In preparation for flow cytometry, cells (10
million) were then pelleted (600 g, 4 °C, 4 min), washed in ice-cold
HBS+ buffer (HBS, 2 mM CaCl2, 1% FCS), and resuspended in 100
μL of the same buffer containing either C2Am-Cys95 or C2Am-
NHBn95 (2 μM), in combination with 50 nM of the necrosis probe
Sytox green (Life Technologies, Grand Island, NY) for 15 min at 37
°C, with orbital shaking (300 r.p.m.). The resulting cell suspension was
washed twice with cold HBS+ buffer, kept briefly on ice, and analyzed
in a LSRII cytometer (BD Biosciences, Rockville, MD), equipped with
488 and 630 nm lasers and counting 20 000 cells per event.
Dha Formation on Thiomab LC-V205C. To a 100 μL aliquot of
reduced/reoxidized Thiomab (10.0 μM, 1.0 nmol), a freshly prepared
solution of methyl 2,5-dibromopentanoate (1.0 μL of 50.0 mM
solution, 0.05 μmol) was added and the resulting mixture was vortexed
for 30 s and then was shaken at 37 °C. The reaction progress was
monitored by LC−MS. After 2 h, small molecules were removed from
the reaction mixture by a buffer exchange column Viva 500 (10 kDa).
The sample was eluted via centrifugation (5 min, 1500g) using sodium
phosphate buffer (50 mM, pH 10.0) to dilute the sample. The reaction
mixture was further shaken at 37 °C for 22 h. After this time, small
molecules were removed from the reaction mixture by loading the
sample onto a Zeba Spin desalting column previously equilibrated with
sodium phosphate buffer (50 mM, pH 8.0). The sample was eluted via
centrifugation (2 min, 1500g). Samples for LC−MS analysis were
prepared by aliquoting 5 μL of the reaction mixture followed by
dilution with 5 μL of sodium phosphate buffer (50 mM, pH 8.0). Ten
μL of this diluted sample was injected on the LC−MS. Complete
conversion to a single product with a mass corresponding to the
formation of two Dha in the light-chain was observed after 24 h
(calculated mass light-chain bearing two Dha, 23373, observed mass,
23376). The heavy-chain remained intact after Dha formation.
Antigen Binding Properties of Thiomab-Dha. Biotinylation of
antibodies. Nonmodified Thiomab and Thiomab-Dha were conjugated
to a biotin linker (Biotin-(PEG)4-N-hydroxysuccinimide, Thermo-
fisher Scientific) in order to carry out Biolayer Interferometry (BLI)
experiments using Streptavidin (SA) Biosensors. A solution of EZ-Link
NHS-(PEG)4-Biotin (20 μL, 200 mM in PBS, pH 7.4) was added to
the corresponding antibody (20 μL, 20 mM in PBS, pH 7.4) and was
shacked at room temperature for 30 min. The crude reaction mixture
was buffer exchanged (3×) with PBS pH 7.4 to remove the excess of
biotin-linker, obtaining a biotin-to-antibody ratio of ∼1 to 2
(determined using the Pierce Biotin Quantitation Kit, ThermoFisher
Scientific). Biolayer interferometry. Binding assays were performed on
involved in 17 disulfide bridges, some of which are solvent
exposed, it can successfully be modified using the reported
postexpression chemical conversion of Cys to Dha followed by
aza-Michael ligation. Following modification, the Recombumin-
NHBn34 retained reversible pH dependent receptor binding.
We also confirmed that modified C2Am is stable under
reducing conditions and in human plasma, while retaining the
protein’s native biological function. Importantly, the scope of
the method could be expanded to build an ADC with a precise
DAR of 2 through direct site-selective conjugation of the
piperidine motif present in the anticancer drug crizotinib. Its
antibody-mediated intracellular delivery resulted in a 10-fold
improvement in cancer cell killing activity.
Taken together, we have explored the potential of amine
additions to Dha as a successful, disulfide compatible protein
modification reaction for site-selective introduction of secon-
dary and tertiary amine linked modifications into proteins. This
is demonstrated by the application of this reaction to three
structurally distinct proteins, making it a ligation of potential
importance for the construction of homogeneously labeled
proteins for imaging (C2Am and Annexin-V) and therapeutic
(Recombumin and Trastuzumab) applications. Considering the
simple setup of this bioconjugation method and the use of
easily derivatized amine reagents, we anticipate that this
method will become a much-used tool for the chemical site-
selective modification of proteins with diagnostic and
therapeutic relevance.
EXPERIMENTAL SECTION
■
Dha Formation on Proteins. To an aliquot of purified, reduced
protein, a freshly prepared solution of α,α′-dibromo-adipyl(bis)amide
10 in DMF was added and the resulting mixture was vortexed for 30 s
and then was shaken at room temperature and/or warmed to 37 °C.
The reaction progress was monitored by LC−MS with samples taken
after defined time points by aliquoting 2.5 μL of the reaction mixture
and diluting it with 8 μL of 50 mM sodium phosphate buffer at pH 8.0.
Ten μL of this diluted sample was injected on the LC−MS. Full
conversion to the expected Dha product was observed after several
hours, with the time to completion depending on the protein. Small
molecules were removed from the reaction mixture by loading the
sample onto a Zeba Spin Desalting Column previously equilibrated
with 50 mM sodium phosphate buffer at pH 8.0. The sample was
eluted via centrifugation (2 min, 1500g). The protein solution was
then flash frozen with liquid nitrogen and stored at −20 °C.
Aza-Michael Ligation on Proteins. An aliquot of Dha-modified
protein in 50 mM sodium phosphate buffer at pH 8.0 was thawed. A
nitrogen nucleophile was added at room temperature (reaction
concentration ranging from 5−138 mM) and the resulting mixture
vortexed for 30 s. Of note, the final pH of the reaction mixture may
vary between 8 to 9 depending on the amine nucleophile used. The
reaction progress was monitored by LC−MS with time points taken at
defined time points ranging from 15 min to 48 h. Time points were
taken by aliquoting 5 μL of the reaction mixture and diluting it with 6
μL of 50 mM sodium phosphate buffer at pH 8.0. Ten μL of this
diluted sample was injected into the LC−MS.
Surface Plasmon Resonance Analysis of Recombumin-
Cys34 and Recombumin-NHBn34. SPR experiments were
performed using a Biacore 3000 instrument (GE Healthcare). Flow
cells of CM5 sensor chips were coupled with soluble human FcRn
(1505 RU) using amine coupling chemistry as described in the
protocol provided by the manufacturer (GE Healthcare). The coupling
was performed by injecting 10 μg/mL of the protein in 10 mM sodium
acetate pH 4.5 (GE healthcare). Phosphate buffer (25 mM Na-acetate,
25 mM NaH2PO4, 150 mM NaCl, 0.01% T-20, pH 5.5) was used as
running buffer and dilution buffer. Regeneration of the surfaces were
performed using injections of HBS-EP buffer (0.01 M HEPES, 0.15 M
NaCl, 3 mM EDTA, 0.005% surfactant P20) at pH 7.4 (GE
́
an Octet Red Instrument (forteBIO). Ligand immobilization, binding
reactions, regeneration and neutralizations were conducted in wells of
black polypropylene 96-well microplates. Thiomab and Thiomab-Dha
were immobilized on Streptavidin (SA) Biosensors in PBS pH 7.4 with
0.1% BSA and 0.02% tween at 30 °C. Binding analysis were carried out
at 25 °C, 1,000 r.p.m. in PBS pH 7.4 with 0.1% BSA and 0.02% tween.
Association time was 600 s, followed by 2,200 s of dissociation, using
different concentrations (200, 66.6, 22.2, 7.4, and 2.47 nM) of ErbB2/
Her2 Recombinant Protein Antigen to obtain the association curve.
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Glycine pH 2.0 was used as a regeneration buffer. Data were analyzed
using Data Analysis (forteBIO), with Savitzky-Golay filtering. Binding
́
Gonzalo Jimenez-Oses: 0000-0003-0105-4337
Gonca̧ lo J. L. Bernardes: 0000-0001-6594-8917
́
́
was fitted to a 2:1 Heterogeneous ligand model, steady state analysis
was performed to obtain the binding kinetics constants (KD).
Aza-Michael Addition of Crizotinib 30 to Thiomab-Dha. A 40
μL aliquot of Thiomab-Dha (10 μM, 399 pmol) in 50 mM sodium
phosphate buffer at pH 8.0 was thawed. Crizotinib 30 (0.54 μL of a
222 mM solution in DMF) was added at 37 °C and the resulting
mixture vortexed for 30 s. The reaction progress was monitored by
LC−MS. Small molecules were removed from the reaction mixture by
loading the sample onto a Zeba Spin Desalting Column previously
equilibrated with sodium phosphate buffer (50 mM, pH 8.0). The
sample was eluted via centrifugation (2 min, 1500g). When the
reaction was scaled up for in vitro studies, this procedure was repeated
3 times to optimize the efficiency of the method. These columns are
described to have at least 95% retention (removal) of salts and other
small molecules (<1000 MW). Samples for LC−MS analysis were
prepared by aliquoting 5 μL of the reaction mixture and diluting it with
5 μL of sodium phosphate buffer (50 mM, pH 8.0). Ten μL of this
diluted sample was injected on the LC−MS. Complete conversion to
Thiomab-30 was observed after 24 h (calculated mass light-chain,
23822, observed mass, 23826). The heavy-chain remained intact after
aza-Michael addition of 30 to Thiomab-Dha.
Cell Viability Assay. 10 000 cells/well were seeded in 96 well-
plates and were treated with crizotinib, Thiomab or Thiomab-30 24 h
after seeding, to allow the cells to stabilize. The cells were incubated
with several concentrations of crizotinib (0.5, 1, 2.5, 5, 10, 15, 25, 50,
75, 100 μM), Thiomab (0.5, 1, 2.5, 5, 8 μM) and Thiomab-30 (0.5, 1,
2.5, 5, 8 μM) for 24 h. After this incubation period, the culture
medium was removed and the cells were incubated with CellTiter-Blue
(Promega) for 90 min at 37 °C. Cell viability was evaluated by
measuring the Emission Intensity in RFUs, relative fluorescent units,
with an Infinite M200 plate reader. IC50s were calculated using
GraphPad Prism5 software.
Binding Affinity Determined by Flow Cytometry Analysis.
The binding affinity of the antibody Thiomab-30 was determined by
Flow Cytometry analysis. For this purpose, SKBR3 cells (with high
expression of Her2 receptor) and Hek293T cells (with low expression
of Her2 receptor) were plated in 96 well plates (100 000 cells per well)
and incubated with 10 μL of 1 μM Thiomab-30 at room temperature.
After 1 h of incubation, cells were washed with medium and were
incubated with 50 μL/well of Goat anti-Human IgG (H+L) cross-
adsorbed secondary antibody (10 μg/mL, Alexa Fluor 647, Thermo
Scientific), for 1 h. After this incubation period, cells were washed by
adding 100 μL of 10% FBS in PBS pH 7.4 and centrifuged for 5 min at
400g.The supernatant was then removed, the cells were resuspended
in 400 μL of 10% FBS in PBS pH 7.4 and transferred to flow
cytometry tubes. Acquisition was performed using a BD LSR Fortessa
set up with a 640 nm laser and a 670/14 nm band-pass filter
(combination used for APC detection). Data analysis was performed
with FlowJo (version 6.3.4, FlowJo) software. Data represents mean
s.d. of 3 biological replicates and only single-cell events are shown.
Author Contributions
#A.M.F., M.J.M., O.B., F.C., and A.G. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the Gates-Cambridge Trust Foundation (scholarship
to A.M.F.), Xunta de Galicia (Postdoctoral Fellowship to
M.J.M.), Campus Iberus and MINECO (“Programa de ayudas
para la movilidad del personal docente e investigador” and
CTQ2015-67727-R both to F.C.), the European Commission
(Marie Curie CIG to G.J.L.B., Marie Curie IEF to O.B., Marie-
Skłodowska-Curie IF to T.R. and the Marie Curie ITN project
ProteinConjugates to G.J.L.B. and P.A.), MINECO (CTQ2015-
70524-R and RYC2013-14706 grants to G.J.O.), FCT Portugal
(FCT Investigator to G.J.L.B. and Ph.D. studentship to A.G.),
and the EPSRC for financial support. BIFI (Memento cluster)
and UR (Beronia cluster) are acknowledged for computer
support, Dr. Mike Deery and Ms. Julie Howard for help with
mass spectrometry analysis. We also thank Dr. Justin Chalker
for useful discussions and Genentech, Inc. for providing
purified 4D5 LC-V205C THIOMAB. G.J.L.B. is a Royal
Society University Research Fellow and holds an ERC Starting
Grant (TagIt).
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.7b10702.
■
S
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AUTHOR INFORMATION
Corresponding Author
*gb453@cam.ac.uk, gbernardes@medicina.ulisboa.pt
ORCID
Omar Boutureira: 0000-0002-0768-8309
Francisco Corzana: 0000-0001-5597-8127
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DOI: 10.1021/jacs.7b10702
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