Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates

Abstract

Spontaneous isopeptide bond formation, a stabilizing posttranslational modification that can be found in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here we adapted this technology to establish a novel modular antibody labeling approach which is based on isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy allows the attachment of a reporting cargo of interest to an antibody scaffold by fusing it chemically to KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody with SpyTag. This strategy was successfully used to engineer site-specific antibody-drug conjugates (ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of antibody conjugates based on peptide-tags that have minimal effects on protein structure and function, thus expanding the toolbox of site-specific antibody conjugation.

Full text

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Scientific RepoRts | 6:39291 | DOI: 10.1038/srep39291
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Spontaneous Isopeptide Bond
Formation as a Powerful Tool for
Engineering Site-Specic Antibody-
Drug Conjugates
Vanessa Siegmund1,2, Birgit Piater2, Bijan Zakeri3, Thomas Eichhorn2, Frank Fischer2,
Carl Deutsch2, Stefan Becker2, Lars Toleikis2, Björn Hock2, Ulrich A. K. Betz2 & Harald Kolmar1
Spontaneous isopeptide bond formation, a stabilizing posttranslational modication that can be found
in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide
ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here
we adapted this technology to establish a novel modular antibody labeling approach which is based on
isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy
allows the attachment of a reporting cargo of interest to an antibody scaold by fusing it chemically to
KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody
with SpyTag. This strategy was successfully used to engineer site-specic antibody-drug conjugates
(ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of
antibody conjugates based on peptide-tags that have minimal eects on protein structure and function,
thus expanding the toolbox of site-specic antibody conjugation.
e conjugation of small molecule drugs to antibodies represents a promising strategy for the development of
cancer therapeutics. By harnessing the capacity of antibodies to home in on specic targets and combining that
with the cytotoxic capability of small molecule drugs, antibody-drug conjugates (ADCs) can be generated to
deliver lethal payloads to cancer cells with precision, while minimizing the o-target eects of cytotoxic drugs to
increase the therapeutic index1.
First generation ADCs employing statistic conjugation of cytotoxic payloads via reduced cysteines or lysines
led to heterogenous populations with limited therapeutic index suering from a low ecacy and inconsistent
in vivo performance2,3. Initial attempts to generate homogenous ADCs with a dened stoichiometry relied on the
mutation of selected interchain cysteines to serines and conjugation of the cytotoxic payload to the remaining
accessible cysteines originating from reduction4. Since then, several elegant methods have been developed to
conjugate drugs to antibodies in a site-specic manner5,6. Chemical methods include the site-specic chemical
conjugation through engineered cysteines7 or selenocysteines8,9, cysteine containing tag with peruoroaromatic
reagents10 and conjugation to reduced intermolecular disuldes by re-bridging dibromomalemides11, bis-sulfone
reagents12, and dibromopyridazinediones13. In addition, several enzymatic and chemoenzymatic conjugation
approaches have been reported including the use of engineered galactosyl- and sialyltransferases14, formyl
glycine generating enzyme (FGE)15, phosphopantetheinyl transferases (PPTases)16, sortase A17, and microbial
transglutaminase18–20, an enzyme forming an isopeptide bond between a glutamine side-chain and an
amine-donor substrate.
Here we sought to explore a new method for engineering ADCs. Spontaneously forming intramolecular iso-
peptide bonds—peptide bonds that form outside of the protein main chain—were rst discovered a decade ago
and were found to provide remarkable stability to outer-membrane proteins of Gram-positive bacteria21. Using
these protein scaolds, Zakeri et al. engineered a series of genetically programmable peptide-protein partners that
are able to spontaneously reconstitute via covalent and irreversible isopeptide bond formation22,23. One of these
1Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt, 64287 Darmstadt, Germany.
2Merck KGaA, Frankfurter Straße 250, 64293 Darmstadt, Germany. 3EMD Serono Research & Development Institute,
Inc., 45A Middlesex Turnpike, Billerica, MA 01821, USA. Correspondence and requests for materials should be
addressed to H.K. (email: kolmar@biochemie-TUD.de)
Received: 19 August 2016
Accepted: 21 November 2016
Published: 16 December 2016
OPEN

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peptide-protein partners was obtained by engineering and splitting the C-terminal beta-strand of the CnaB2
domain from the bronectin adhesion protein FbaB of Streptococcus pyogenes that is able to reconstitute with the
protein by forming an intramolecular isopeptide bond between an aspartate and a lysine residue catalyzed by an
opposed glutamate22. Further engineering of the CnaB2 domain and splitting it into three parts generated the
synthetic enzyme SpyLigase that is able to direct the formation of an isopeptide bond between the two peptides
SpyTag and KTag (Fig.1A)24. Since the tags can be genetically fused to various proteins, these protein super-
glues have emerged as useful tools to covalently and specically assemble linear and branched protein structures,
thereby enabling the generation of new protein architectures via modular assembly25.
Results
Expression of peptide-tagged IgG1 antibody Fc domains, SpyLigase and synthesis of labeled
peptides. To test whether the SpyLigase-catalyzed peptide ligation approach could also be applied for the
covalent attachment of small reporting molecules such as uorescent dyes, biotin or cytotoxins to antibody scaf-
folds, we fused SpyTag (13aa) and KTag (10aa) genetically to the C-terminus of an Fc domain from an IgG1 anti-
body via a short GS-linker. In order to facilitate analysis of conjugates, an N297A mutation was introduced into
the IgG1-Fc gene by site-directed mutagenesis to remove the natural glycosylation site. e peptide-tagged anti-
body fragments were transiently expressed in HEK293F cells and subsequently puried by protein A anity chro-
matography. SpyTag and KTag-peptides with adjacent N-terminal GSG-spacer were synthesized on solid support
by semi-automated SPPS using a Rink amide (RAM) resin yielding peptide carboxamides aer cleavage. KTag was
further extended with a GY-dipeptide at the carboxy terminus as described24. 5/6-Carboxytetramethylrhodamine
(TAMRA), a uorophore commonly used for the preparation of protein conjugates, and biotin were coupled to
the resin-bound peptide amino terminus via standard amide coupling chemistry using 2-(1H-benzotriazol-1-
yl)-1,1,3,3-tetramethyluronium hexauorophosphate (HBTU) and N,N-Diisopropylethylamine (DIPEA). Aer
Figure 1. Site-specic conjugation of Spy-tagged IgG1-Fc with 5/6-carboxytetramethylrhodamine
(TAMRA)-KTag by SpyLigase-mediated isopeptide bond formation. (A) Cartoon illustrating the splitting
strategy to form SpyLigase from the CnaB2 domain. e two peptide tags, KTag (orange) with the reactive
lysine and SpyTag (blue) with the reactive aspartic acid, can be ligated by the remaining protein domain
(SpyLigase, green) by isopeptide bond formation. Active-site residues involved in the reaction are indicated
(PDB 2X5P). (B) SDS-PAGE, Coomassie staining (top), and in-gel uorescence (bottom) of the reduced Fc-
uorophore conjugates. Reactions were conducted with increasing concentration of SpyLigase (1, 3, and 10 mol
eq. over Fc) and 10-fold excess of TAMRA-KTag. Control reactions (Ctrl) were performed by using 10 mol eq. of
SpyLigase EQ. (C) Scheme of SpyLigase-mediated IgG1-Fc-SpyTag labeling using TAMRA-KTag.

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cleavage from the resin, labeled peptides were puried by semi-preparative RP-HPLC and analyzed by ESI-MS.
SpyLigase and its inactive mutant SpyLigase EQ were obtained in yields of 20 mg per liter by expression in
Escherichia coli and purication by anity chromatography followed by size-exclusion chromatography (SEC)24.
e purity of the proteins was > 95% as determined by denaturing SDS-PAGE gel electrophoresis (Fig.1B, upper
panel, and S1, lane 2–4).
Conjugation of labeled peptides to antibody Fc domains by SpyLigase-mediated isopeptide
bond formation. We next performed conjugation reactions of labeled-peptides to the peptide-tagged Fc
domains at pH 7.0 in the presence of the protein stabilizer trimethylamine N-oxide (TMAO) for 18–24 h at 4 °C.
Each reaction contained peptide-tagged IgG1-Fc, labeled peptide counterpart (20 eq.), and SpyLigase (1–10 eq.).
All possible combinations of SpyTag and KTag with the respective TAMRA or biotin labeled counterpart were
assayed. Covalent ligation reactions were analyzed by boiling the samples for 5 min in SDS-loading buer and
subsequent SDS-PAGE analysis. Reaction products were visualized by Coomassie staining, uorescence read-
out or western blotting (not shown). For all combinations the formation of a new product, stable to boiling in
SDS, with a slightly slower migration behavior consistent with isopeptide bond formation between SpyTag and
KTag was observed (Fig.1B, upper panel, and S1, lane 5–7). is product was not observed when SpyLigase was
replaced by its inactive Glu77 to Gln mutant (SpyLigase EQ). is mutant was not able to catalyze the sponta-
neous isopeptide bond formation due to the lack of the required glutamate residue (Fig.1B, upper panel, and
S1, lane 8). ese results suggested that SpyLigase covalently links KTag and SpyTag in a site-specic manner.
e conjugation eciencies for Fc-SpyTag with TAMRA-KTag were estimated to be 40–60%. Here, reactions
with three equivalents of SpyLigase seemed to be most ecient (Fig.1B, upper panel, lane 6). Conjugation of the
Fc-KTag with TAMRA-SpyTag was observed to be less ecient, which may be caused by a shorter GS-linker that
was used to fuse KTag to the Fc (FigureS1 lane 5–7).
SpyLigase-mediated conjugation of labeled peptides to Spy-tagged antibodies. Having
proven our site-specic labeling approach on antibody domains, we extended the application for the prepara-
tion of dened antibody conjugates of the anti-EGFR monoclonal antibody cetuximab (Erbitux® ). e cetux-
imab fusion proteins with SpyTag at the C-terminus of the heavy chains, the light chains, and at both chains
were obtained by expression in HEK293F cells and puried by protein A anity chromatography. e yields
were comparable to those obtained for the unmodied antibody (40–60 mg per liter cell culture). SEC analy-
sis revealed no signicant aggregation behavior of the peptide-tagged antibodies (FigureS2). Antibodies were
then conjugated to TAMRA-KTag using SpyLigase under previously determined reaction conditions. Reaction
products were analyzed by in-gel uorescence and Coomassie staining following SDS-PAGE gel electrophoresis.
Fluorescent bands corresponding to the conjugated antibody’s heavy and light chains and a mobility shi of the
conjugated light chain conrmed that SpyLigase-promoted site-specic labeling can also be applied on full-length
IgG1-antibodies (FigureS3, lane 2–4). ese results indicated a chemically highly specic conjugation reaction
due to the required assembling of SpyTag/KTag and SpyLigase to form an active complex mediating isopeptide
bond formation.
Synthesis of cytotoxin-peptide payloads and conjugation to Spy-tagged antibodies. Next,
we wanted to investigate whether SpyLigase-catalyzed conjugation could also be applied for the coupling
of drug molecules to antibodies to generate site-specic ADCs. For this purpose, we designed and synthe-
sized a set of Monomethyl auristatin E (MMAE)-peptide payloads diering in the nature of the used linker
and the chemistry used for peptide-coupling (Fig.2, compound 5–7). MMAE was designed with either a
non-cleavable (nc) maleimide-thiol linker (compound 5), without linker (compound 6), or with a cleava-
ble valine-citrulline-p-aminobenzylcarbamate (vc-PABC) linker7,26,27 (compound 7). In general, all payloads
contained polyethylene glycol (PEG) spacer-units to increase the overall toxin solubility and to increase the
toxin-peptide distance. To persue two dierent chemical strategies, toxins were either provided with an azide
functionality for copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) or with an carboxyl group for amide
coupling. For peptide-toxin coupling in solution by CuAAC, resin-bound KTag was rst coupled to propargy-
lacetic acid to obtain an N-terminal alkyne moiety before it was cleaved from the resin and puried. e carboxy
functionalized MMAE was coupled directly to the N-terminus of the resin-bound peptide, requiring an acidic
cleavage step of the entire MMAE-payload. As a proof of principle, cetuximab fused to SpyTag at the heavy chains
was used for conjugation. ese reactions were carried out in phosphate-citrate buer pH 7.0 in the presence of
TMAO for 18–24 h at 4 °C. Each reaction contained Spy-tagged antibody, 15 eq. of the toxic payload, and 3 eq.
of SpyLigase. Reactions were analyzed by hydrophobic interaction chromatography (HIC) and drug-to-antibody
ratios (DAR) were determined. Using payload 5, the highest observable DAR was only 0.57 (FigureS4). With the
payloads from peptide-coupling via click-chemistry, formation of about 80% of the desired species with two drug
molecules was obtained, resulting in a DAR of 1.76 for payload 5 and a DAR of 1.66 for 7. Control reactions with
either SpyLigase EQ, without SpyLigase, and without payload did not result in the formation of a new product.
is was analyzed by HIC, conrming also the specicity of the reaction (FigureS5).
MS analysis of Spy-tagged ADCs. Next, we conrmed the identity of the peptide-tagged ADCs and the
specicity of the conjugation reaction by mass spectrometry. Intact mass analysis of ADCs by MALDI-MS indi-
cated the attachment of two payload molecules per antibody by a mass shi of 5252 Da (calc. 5349 Da) for pay-
load 6 and 6202 Da (calc. 6159 Da) for payload 7 (FigureS7) compared to unmodied Spy-tagged cetuximab.
is observation was veried by mass analysis of reduced ADCs showing a mass shi of 2735 Da (calc. 2675 Da)
and 3116 Da (calc. 3080 Da) for payload 6 and 7, respectively (FigureS8). In order to conrm the identity of the
ADCs, a more accurate mass determination was obtained by LC-ESI-MS showing a mass shi of 2677 Da for

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compound 6 (calc. 2675 Da) (FigureS9). Tryptic digestion of the ADCs resulted in a peptide fragment com-
posed of amino acids derived from both SpyTag and KTag which are connected to each other via an isopeptide
bond between Asp and Lys residues (FigureS13). is fragment was successfully identied in both ADC samples
but not in the unconjugated antibody control. Further identication of the peptide was conducted by sequenc-
ing using Tandem-MS (FiguresS14–S16). Unmodied SpyTag fragment, however, was only observed in minor
amounts conrming the eciency of our approach (FigureS17). No unspecic conjugation of the antibody light
chain was observed (FigureS18).
In vitro cytotoxicity of Spy-tagged ADCs. To determine the cell killing activities of the Spy-tagged
cetuximab-based ADCs in vitro, ligation reactions were scaled up and ADCs were puried by protein A chro-
matography in order to remove excess payload and SpyLigase. In vitro cytotoxicity was determined using two
dierent breast cancer cell lines, MDA-MB-468 and MCF-7, the former one expressing high levels of EGFR (epi-
dermal grow factor receptor, EGFR+ ), the latter one without EGFR expression (EGFR-)28. ADCs with payloads
that were synthesized by CuAAC with either non-cleavable linker (compound 6) or with cleavable linker (com-
pound 7) were compared to each other to investigate an eect of the dierent linkers on ecacy and potency.
e ADCs were tested in a serial dilution with the highest concentration of 50 nM for three days. As expected,
cetuximab-based ADCs effect on MDA-MB-468 cells in a dose dependent manner (Fig.3, upper panels).
For the ADC with a non-cleavable linker the half-maximal cytotoxic eect was in the subnanomolar range
(IC50 = 0.2 nM) (Fig.3A, ■ ). In contrast the ADC with a cleavable linker showed an even higher in vitro potency
(IC50 = 0.1 nM) (Fig.3B, □ ) which was probably due to the cathepsin B-mediated cleavage of the linker in the lys-
osome releasing the free toxin upon cellular uptake. No eect on cell viability was observed with the same ADCs
on the EGFR-negative cell line MCF-7 (Figure3, lower panels). To conrm the receptor-mediated internalization
and cell killing of the anti-EGFR ADCs, anti HER2 (human epidermal grow factor receptor 2) Spy-tagged tras-
tuzumab ADCs were prepared with the same payloads under identical reaction conditions and tested as isotype
controls. e free payload was also used as control. MDA-MB-468 cells do not express HER2 whereas MCF-7
cells exhibit a low expression of HER228. e cell viability of both cell lines was not aected by the isotype control
ADCs (Fig.3, ○ and ● ), demonstrating that the anti-EGFR ADCs generated by using the catalytic activity of
Figure 2. Overview of dierent cargoes of interest coupled to the N-terminus of KTag or SpyTag used in this
study: (1) TAMRA-KTag, (2) Biotin-KTag, (3) TAMRA-SpyTag, (4) Biotin-SpyTag, (5) MMAE-nc-KTag (amide
bond), (6) MMAE-nc-KTag (click chemistry), (7) MMAE-vc-KTag (click chemistry). Payloads 1–4 were
subjected to SpyLigase-mediated IgG1-Fc conjugation whereas payloads 1 and 5–7 were used for conjugation to
the antibody cetuximab as SpyTag fusion.

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SpyLigase selectively kill EGFR-overexpressing cells in a receptor-dependent manner. A cell killing eect of free
payload 7 was observed at the highest concentration of 50 nM (Fig.3B, ▲).
Conclusion
In conclusion, we have developed a modular bioconjugation approach based on isopeptide bond formation
between two peptide tags catalyzed by SpyLigase. is approach was used as a tool for the site-specic conju-
gation of small reporting molecules like uorescent TAMRA and biotin to antibodies and for the generation of
homogenous ADCs. ADCs prepared by fusing SpyTag to the C-terminus of the anti-EGFR monoclonal antibody
cetuximab and attaching a cytotoxic compound to the chemically synthesized KTag were characterized regarding
their biophysical and cytotoxic properties in vitro. e obtained ADCs performed specically and were observed
to exhibit subnanomolar IC50 values. We assume that this technology can also be used for engineering ADCs
with a higher payload density since it has been described that the used peptide tags were also reactive when
placed at internal sites of a protein24. e feasibility of such a peptide-tagged ADC approach has recently been
proven by the introduction of small substrate sequences for enzyme-promoted conjugation at dierent constant
region loops positions of trastuzumab for ecient site-specic ADC preparation16. e possibility to place the
peptide-tags at multiple sites within the antibody scaold would clearly provide an advantage compared to cova-
lent peptide labeling via sortases that exclusively allow for C-terminal payload conjugation17,29–31. In addition to
microbial transglutaminase our approach oers the opportunity for site-specic and ecient antibody conjuga-
tion based on a stable isopeptide bond.
Methods
Expression and purication of SpyLigase. e plasmid coding for SpyLigase (pDEST14-SpyLigase)
was purchased from Addgene (Addgene ID 51722). e plasmid coding for the inactive form of SpyLigase
(pDEST14-SpyLigase EQ) was generated by Quick Change Side-Directed Mutagenesis by introducing an E77Q
mutation according to the literature22,24. SpyLigase expression in E. coli BL21 DE3 pLysS (Stratagene) and puri-
cation via immobilized metal ion anity chromatography (IMAC) was conducted according to the literature22,24.
Aer dialyzing proteins in a 1,000-fold excess of PBS overnight at 4 °C, size-exclusion chromatography (SEC) was
Figure 3. Cytotoxicity induced by Spy-tagged cetuximab ADCs using EGFR overexpressing MDA-MB-468
and EGFR-negative MCF-7 breast cancer cell lines. (A) In vitro cell killing of ADCs with non-cleavable
(nc) MMAE payload 6 (■ ). (B) In vitro cell killing of ADCs with cleavable (vc) MMAE payload 7 (□ ).
Incubation times were 3 days. Trastuzumab ADCs were used as isotype controls (○ and ● ). Compound 6
(▼) and compound 7 (▲ ) were also assayed as controls. IC50 values were calculated as mean values from two
independent experiments.

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performed using a HiPrep (16/60) Sephacryl S-200 HR column (GE Healthcare) and PBS. Proteins were concen-
trated by centrifugation dialysis using Amicon Ultra-15 (Merck Millipore, NMWL 3000 Da) and protein concen-
trations determined at 280 nm using a nano spectrophotometer (MW = 11474.3 g/mol, ε280 = 15930 M−1m−1).
A protein yield of about 20 mg per liter culture could be obtained. Proteins were supplemented with 10% (v/v)
glycerol, aliquots frozen in liquid nitrogen, and stored at − 80 °C.
Expression and purification of IgG1 Fc and antibodies fused to peptide tags. IgG1 Fc
(pEXPR-IgG1 Fc) was fused to either SpyTag (AHIVMVDAYKPTK) or KTag (ATHIKFSKRD) with additional
GY-dipeptide at the C-terminus by using standard molecular cloning techniques. Native IgG1 glycosylation at
position N297 was eliminated by introducing a N297A mutation by Quick Change Side-Directed Mutagenesis.
Plasmids coding for chimeric cetuximab (C225, Erbitux® ) and trastuzumab (Herceptin® ) were kindly provided
by Merck KGaA (Darmstadt). Cetuximab and trastuzumab variants containing SpyTag at the C-terminus of either
the heavy chains, the light chains, or at both chains were prepared by standard molecular cloning techniques. A
(GSG)2-linker was introduced between the C-terminus of the antibody and SpyTag. IgG1 Fc and full-length anti-
bodies were transiently expressed from HEK293F cells using the Expi293 Expression System (Life Technologies).
Supernatants containing secreted proteins were conditioned and applied to spin columns with PROSEP-A Media
(Montage, Merck Millipore). Columns were washed with 1.5 M Glycine/NaOH, 3 M NaCl, pH 9.0 and proteins
eluted with 0.2 M Glycine/HCl pH 2.5 into 1 M Tris/HCl pH 9.0. Eluted proteins were dialyzed in 1 × DPBS (Life
Technologies) using Amicon Ultra-15 (Merck Millipore, NMWL 10000 Da) and stored at 4 °C.
Conjugation of peptide-tagged IgG1 Fc. Antibody fragments (IgG1 Fc) fused to either SpyTag or KTag
at the C-terminus were mixed at 5 μ M with 50 μ M of TAMRA-KTag or TAMRA-SpyTag, respectively, and incu-
bated in the presence of 5–50 μ M of SpyLigase (1–10 mol eq.) for 24 h in 40 mM Na2HPO4, 20 mM citric acid
buer, pH 5.0, with addition of 1.5 M trimethylamine N-oxide (Sigma-Aldrich) to give a nal pH of 7.0 (PCT
buer) at 4 °C. Control reactions using 50 μ M of an inactive SpyLigase (EQ, E77Q) were performed simulta-
neously. Reactions were stopped by the addition of SDS loading buer and samples heated at 95 °C for 5 min.
SDS-PAGE was performed on 15% polyacrylamide gels at 40 mA for approximately 45 min. Gels were analyzed by
uorescence readout using a Versa Doc Imaging System 5000 (Bio-Rad) and aerwards stained with Coomassie
Blue.
SpyLigase-mediated antibody conjugation. Cetuximab or trastuzumab fused to SpyTag at the
C-terminus of the heavy chain, light chain, or at both chains was mixed at 6 μ M with 120 μ M of TAMRA-KTag
and incubated in the presence of 18 μ M of SpyLigase for 24 h in 40 mM Na2HPO4, 20 mM citric acid buer, pH
5.0, with addition of 1.5 M trimethylamine N-oxide (Sigma-Aldrich) to give a nal pH of 7.0 at 4 °C (PCT buer).
Antibodies conjugated to TAMRA-KTag were analyzed by SDS-PAGE on 15% polyacrylamide gels and uores-
cently labeled heavy and/or light chains visualized by uorescence readout using a Versa Doc Imaging System
5000 (Bio-Rad). For toxin conjugations, cetuximab or trastuzumab fused to SpyTag at the C-terminus of the heavy
chain was incubated at 6 μ M with 90 μ M of the respective MMAE-KTag payload and incubated in the presence
of 18 μ M of SpyLigase under the above described reaction conditions. Antibody drug conjugates were evaluated
by hydrophobic interaction chromatography (HIC) on a TSKgel Butyl-NPR column (Tosoh Bioscience, 4.6 mm x
3.5 cm, 2.5 μ m) using an Agilent Innity 1260 HPLC. e HIC method was applied using a mobile phase of 1.5 M
(NH4)2SO2, 25 mM Tris-HCl pH 7.5 (Buer A) and 25 mM Tris-HCl pH 7.5 (Buer B). ADCs (45 μ g) in 0.75 M
(NH4)2SO2 were loaded and eluted with a gradient consisting of 2.5 min 0% Buer B followed by a linear gradient
into 100% Buer B over 35 min with a ow rate of 0.9 ml/min. For preparative ADC preparations, reactions were
scaled up and ADCs puried by protein A magnetic beads (Promega) according to the manufacturers protocol.
Excess payload and SpyLigase were removed by rigorous washing with PBS before eluting ADCs with 100 mM
citrate buer pH 3.0 into 1 M Tris-HCl pH 9.0 neutralization buer. ADCs were buered into PBS by using PD
MiniTrap G-25 colums and concentrated by centrifugation dialysis (100 K Amicon Ultra 0.5 Centrifugal Filters).
In vitro cytotoxicity of peptide-tagged ADCs. In vitro cytotoxicity of peptide-tagged anti EGFR ADCs
was determined by using MDA-MB-468 and MCF-7 breast cancer cells. MDA-MB-468 cells have a high EGFR
expression level, but no HER2 expression28. MCF-7 cells show no EGFR expression, but a weak HER2 expression28.
Cells were cultivated in appropriate culture media according to the manufacturer protocol. Cells were detached by
trypsination, diluted and allowed to settle down in 96-well plates at a seeding density 12.5 × 104 cells/ml (0.08 ml/
well) for 24 h at 37 °C (5% CO2). ADCs and free payloads were diluted in appropriate culture media and added to
the cells in various concentrations (ranging from 50 nM to 7.6 pM) following a three-day incubation at 37 °C (5%
CO2). All concentrations were assayed in duplicates. Cell proliferation was determined by adding CellTiter-Glo
(Promega) and luminescence readout (Synergy 5, BioTek).
General for peptide chemistry. All chemicals and solvents for peptide synthesis, analysis, and isolation
were purchased from Iris Biotech, Agilent Technologies, Sigma-Aldrich, or Roth and used without any further
purication. MMAE-linker payloads were obtained from Syngene.
Peptide solid-phase synthesis. Peptides were synthesized at a 0.1 mmol scale on an AmphiSpheres
40 RAM resin (Agilent, 0.37 mmol/g) by microwave-assisted Fmoc-SPPS using a Liberty BlueTM Microwave
Peptide Synthesizer. Activation of the respective carboxyfunctional amino acid (0.2 M) was performed by 1 M
Oxyma/0.5 M N,N′ -Diisopropylcarbodiimide (DIC). Deprotection of the aminoterminal Fmoc-group was
achieved using 20% piperidine in DMF in the presence of 0.1 M Oxyma. During the synthesis cycles all amino
acids were heated to 90 °C (histidine to 50 °C). Peptides were cleaved from the resin for analytical purposes by a
standard cleavage cocktail of 94% TFA, 2% triethylsilane, 2% anisole, 2% H2O. Methionine containing peptide 2

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Scientific RepoRts | 6:39291 | DOI: 10.1038/srep39291
was cleaved in the presence of dithiotreitol (DTT) to prevent oxidation. Aer 1 h of cleavage, peptides were pre-
cipitated in cold diethylether, washed with diethylether and dried in vacuo prior to LC-MS analysis.
RP-HPLC. Peptides were analyzed by chromatography using an analytical RP-HPLC from Agilent Technolgies
(920-LC or 1260 Innity) using either a Phenomenex Hypersil 5 u BDS C18 LC column (150 × 4.6 mm, 5 μ m,
130 Å) or a Phenomenex Luna 5 u C18 (2) column (150 × 4.6 mm, 5 μ m, 100 Å). Peptides were isolated by
semi-preparative RP-HPLC (Varian) using a Phenomenex Luna 5 u C18 LC column (250 × 12.2 mm, 5 μ m ,
100 Å). Eluent A (water) and eluent B (90% aq. MeCN) each contained 0.1% triuoroacetic acid (TFA).
ESI-MS analysis of peptides. ESI mass spectra were measured on a Shimadzu LCMS-2020 equipped with
a Phenomenex Synergi 4 u Hydro-RP LC column (250 × 4.6 mm, 4 μ m, 80 Å) by using an eluent system consisting
of eluent A (0.1% aq. formic acid, LC-MS grade) and eluent B (100% acetonitrile containing 0.1% formic acid,
LC-MS grade).
MALDI-TOF-MS analysis of intact masses. 20 μ g of protein was used for non-reduced and reduced
(30 min at 60 °C with 5 mM Dithiothreitol) sample preparation. Prior MALDI analysis on an Ultraex III (Bruker,
Bremen), samples were desalted and concentrated with C4 ZipTips (Millipore, Cork) corresponding to the
manufactures guide. e prepared samples were directly mixed in equal volumes with saturated alpha-Cyano-
4-hydroxy-cinnamic acid matrix (Bruker, Bremen) on a MTP AnchorChip 384 TF plate (Bruker, Bremen). e
Ultraex III was used in a linear positive mode and data acquisition was performed in a mass range between m/z
10000 to 180000. Per sample, 4000 laser shot were accumulated and nally smoothed and baseline subtracted.
CapLC-ESI-MS analysis of intact masses. For intact mass analysis of heavy and light chain, 20 μ g of each
sample was mixed with 1% mercaptoethanol nal concentration and reduced for 30 min. at 37 °C. Reduced sam-
ples were loaded directly onto a CapLC-MS system (1100 series, Agilent Technologies) coupled to a Synapt G1
HDMS (Waters, Milford, MA) operated in MS positive ion mode. 0.1% triuoroacetic acid in water was used as
solvent A and 0.1% triuoroacetic acid in 70% n-propanol as solvent B. e samples were separated via a Zorbax
SB300 C8 column (3.5 μ m, 150 × 0.3 mm, Agilent Technologies), tempered at 75 °C. e gradient started with 3%
B and a ow rate of 10 μ L/min followed by a linear gradient from 2 to 39 min from 10% B to 60% B. Protein signals
were recorded with a DA-detector at 214 and 280 nm. MS spectra were acquired from m/z 500 to 3000. Finally,
intact protein mass was calculated applying the MaxEnt1 deconvolution algorithm.
NanoLC-ESI-MSe analysis of tryptic peptides. Investigation of the payload conjugation site was per-
formed by peptide mapping analysis using a slightly modied RapiGest SF Surfactant care and use protocol
(Waters, Milford, MA). Briey, 10 μ g of each sample was mixed with RapiGest 0.1% nal concentration and
reduced for 30 min at 60 °C with 5 mM Dithiothreitol. Alkylation was performed with 15 mM iodoacetamide
for 30 min at RT. Finally 1.5 μ L of a 1 μ g/μ l trypsin solution were added and incubated overnight at 37 °C. Aer
digestion, the sample was acidied by addition of 0.5% triuoroacetic acid and analyzed by Nano-LC-MSe using
a nanoAcquity UPLC coupled to a Synapt G1 HDMS (Waters, Milford, MA) operated in MSe mode. 0.1% for-
mic acid in water was used as solvent A and 0.1% formic acid in acetonitrile as solvent B. Tryptic peptides were
injected and trapped for 3 min on a Symmetry C18 pre-column (5 μ m, 180 μ m × 20 mm, Waters, Milford, MA)
with a ow rate of 10 μ l/min. Separation was performed using an UPLC 1.7 μ m BEH130 column (C18, 75 μ
m × 100 mm, Waters) with a ow rate of 450 nl/min, starting with 2% B from 0 to 2 min followed by a linear
gradient from 8–35% B for 67 min. e used MSe mode combines an alternating MS and MSe (full mass range
fragmentation) function each second. Spectra were acquired from m/z 50 to 1600 and extracted ion chromato-
grams were analyzed manually.
Synthesis of peptide conjugates 1–4. Resin-bound peptides were functionalized with
5/6-carboxytetramethylrhodamine (TAMRA, mixed isomers) or biotin at the free amino terminus by
double-coupling using a preactivated carboxyl functionality. For activation, 2 eq. of TAMRA or biotin were incu-
bated with 1.95 eq. of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexauorophosphate (HBTU) in the
presence of 4 eq. of N,N-Diisopropylethylamine (DIPEA) in DMF for 10 min shaking. Aerwards, the mixture
was added to the resin-bound peptides (preswelled in 1:1 DMF/DCM) and coupling allowed to proceed for 2 h,
respectively. Peptides were cleaved from the resin by using a standard cleavage cocktail of 94% TFA, 2% triethyl-
silane, 2% anisole, 2% H2O. Methionine containing SpyTag-peptides were cleaved in the presence of dithiotreitol
(DTT) to prevent oxidation. Aer 2 h of cleavage, peptides were precipitated in cold diethylether, washed twice
with diethylether, dried in vacuo, and aerwards puried by semi-preparative RP-HPLC.
Synthesis of KTag with N-terminal alkyne functionality. K Ta g -peptide was equipped with an
N-terminal alkyne functionality by coupling with propargylacetic acid (4-pentynoic acid) on resin. For this
purpose, 4 eq. of the building block was preactivated for 10 min with 3.95 eq. of 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexauorophosphate (HBTU) and 8 eq. of N,N-Diisopropylethylamine (DIPEA)
in DMF and aerwards added to the resin-bound peptide (preswelled in 1:1 DMF/DCM). Coupling was per-
formed for 1.5 h twice and the resin washed subsequently with DMF, DCM, and diethylether for drying. e
alkyne-functionalized peptide was cleaved from the resin using a standard cleavage cocktail of 94% TFA, 2%
triethylsilane, 2% anisole, 2% H2O. Aer 2 h of cleavage, the peptide was precipitated in cold diethylether, washed
twice with diethylether and dried in vacuo. e product was analyzed by LC-MS and puried by semi-preparative
RP-HPLC.

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Synthesis of peptide-toxin conjugate 5. Resin-bound KTag-peptide was incubated with 1.2 eq. MMAE
bearing a carboxy group in the presence of 1.14 eq. 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hex-
auorophosphate (HBTU) and 2.4 eq. of N,N-Diisopropylethylamine (DIPEA) in DMF for 5 h at room tempera-
ture. Aerwards, the resin was washed twice with DMF, DCM, and diethylether. e toxin-functionalized peptide
was cleaved from the resin using a cleavage cocktail of 85% TFA, 5% triethylsilane, 5% anisole, and 5% H2O for
1 h at room temperature. e peptide-toxin conjugate was precipitated in cold diethylether, washed twice with
diethylether and dried in vacuo. Compound 5 was isolated by RP-HPLC.
Synthesis of peptide-toxin conjugates 6 and 7. KTag was coupled to monomethyl auristatin E
(MMAE) via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). For this purpose, the peptide bearing an
amino terminal alkyne functionality (1 mg/ml) was reacted with 1.5 eq. MMAE holding an azide functionality in
the presence of 4 eq. of Cu(II)-TBTA complex, 4 eq. of N,N-Diisopropylethylamine (DIPEA), and 4 eq. of sodium
ascorbate for 5 h at room temperature in water. e water was degassed by ushing it with argon prior to usage.
TBTA was dissolved at a concentration of 10 mM in 1:4 DMSO/tBuOH and the complex formed by addition
of Cu(II)SO4 5xH2O. e toxin was pre-dissolved in 50% MeCN. e reaction was monitored by HPLC-MS.
Peptide-toxin conjugates 6 and 7 were isolated by semi-preparative RP-HPLC.
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Scientific RepoRts | 6:39291 | DOI: 10.1038/srep39291
Acknowledgements
is project has been funded by Merck KGaA Darmstadt in context of the Merck Biopharma Innovation Cup. e
authors thank the members of the team Chemo- and Bioengineering Pedro Matos, Sara Cleto, Darryl Gibbings-
Isaac, Reswita Dery G from the Innovation Cup 2013 and Siegfried Neumann for giving inspiration for this
project. It was also inspired by Deutsche Forschungsgemeinscha SPP1623. e authors thank Dirk Müller-
Pompalla (Merck KGaA) for SEC-analysis of peptide-tagged antibodies.
Author Contributions
V.S., B.P., F.F., and H.K. conceived and designed the experiments, V.S. and T.E. conducted the experiments, C.D.
designed and supplied the cytotoxic compounds used for peptide coupling, V.S., B.P., T.E., F.F., S.B., L.T., B.H.,
U.A.K.B., and H.K. analyzed the results, V.S., B.P., B.Z., and H.K. wrote the manuscript, and all authors reviewed
the manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: B.Z. receives royalties from Oxford University on spontaneous isopeptide bond
formation technology and declares competing nancial interest.
How to cite this article: Siegmund, V. et al. Spontaneous Isopeptide Bond Formation as a Powerful Tool for
Engineering Site-Specic Antibody-Drug Conjugates. Sci. Rep. 6, 39291; doi: 10.1038/srep39291 (2016).
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