A Bioorthogonal Click Chemistry Toolbox for Targeted Synthesis of Branched and Well-Defined Protein–Protein Conjugates

Article abstract of DOI:10.1002/anie.201915079

Bioorthogonal chemistry holds great potential to generate difficult-to-access protein–protein conjugate architectures. Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. Here we present a highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels–Alder cycloaddition with inverse electron demand (DA_inv). With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines. We expect our work to substantially enhance antibody applications such as immunodetection and protein toxin-based targeted cancer therapies.

Full text of DOI:10.1002/anie.201915079

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: A bioorthogonal click chemistry toolbox for targeted synthesis of
branched and well-defined protein-protein conjugates
Authors: Mathis Baalmann, Laura Neises, Sebastian Bitsch, Hendrik
Schneider, Lukas Deweid, Philipp Werther, Nadja Ilkenhans,
Martin Wolfring, Michael J. Ziegler, Jonas Wilhelm, Harald
Kolmar, and Richard Wombacher
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A bioorthogonal click chemistry toolbox for targeted synthesis of
branched and well-defined protein-protein conjugates
Mathis Baalmann,[a]# Laura Neises,[a]# Sebastian Bitsch,[b] Hendrik Schneider,[b] Lukas Deweid,[b] Philipp
Werther,[a] Nadja Ilkenhans,[a] Martin Wolfring,[a] Michael J. Ziegler,[a] Jonas Wilhelm,[a] Harald Kolmar[b]
and Richard Wombacher*[a]
Abstract: Bioorthogonal chemistry holds great potential to generate
difficult-to-access protein-protein conjugate architectures. Current
applications are hampered by challenging protein expression
systems, slow conjugation chemistry, use of undesirable catalysts,
or often do not result in quantitative product formation. Here we
present a highly efficient technology for protein functionalization with
commonly used bioorthogonal motifs for Diels-Alder cycloaddition
with inverse electron demand (DAinv). With the aim of precisely
generating branched protein chimeras, we systematically assessed
the reactivity, stability and side product formation of various
bioorthogonal chemistries directly at the protein level. We
demonstrate the efficiency and versatility of our conjugation platform
using different functional proteins and the therapeutic antibody
trastuzumab. This technology enables fast and routine access to
tailored and hitherto inaccessible protein chimeras useful for a
variety of scientific disciplines. We expect our work to substantially
enhance antibody applications such as immunodetection and protein
toxin-based targeted cancer therapies.
with inverse electron demand (DAinv) between alkenes or
alkynes (dienophiles) and N-heteroaromatic compounds
(dienes) has become the most promising chemoselective bond-
forming chemistry in terms of catalyst-free reaction conditions
and reaction rates.[5] It is thus not surprising that DAinv has
already been adapted for the generation of antibody-drug
conjugates (ADCs),[6] investigated for diagnostic antibody
radiolabeling[7] or related tumor pretargeting[8] approaches.
Advances in the past years gave rise to a variety of co-
translational or posttranslational means to introduce DAinv motifs
site-specifically to proteins to prime them for subsequent site-
specific derivatization.[9] A major challenge still remains in
homogeneously labeling proteins with free choice of modification
site and full independence of the expression system. In contrast
to co-translational approaches, posttranslational labeling
methods entail strict separation of installation of the
bioorthogonal motif from protein translation and the expression
host. This also minimizes exposure time to prevent the reactive
scaffold from losing integrity[10] with the consequence of more
efficient conjugation[11]. Thus, a key consideration toward fast
and quantitative protein labeling is that the installed
bioorthogonal motif is (I) as reactive as possible and (II) entirely
stable until the chemoselective derivatization is performed
(stability-reactivity-tradeoff). With the increasing number of
bioorthogonal DAinv motifs reported in literature,[5, 12] a profound
comparison of the currently used dienophile/diene pairs would
be highly beneficial for experimentalists interested in protein
bioconjugation, also with regard to the production of biologicals,
where well-defined conjugate populations with high purity and
little variability are essential.
Introduction
Bioorthogonal chemistries provide ample opportunities to be
applied in the manufacturing of therapeutic biologics due to their
biocompatible properties. Further they possess power to
generate fusion types that are challenging to access using
common protein expression or conjugation technologies
(Scheme 1).[1] This is particularly the case for proteins that
require special expression systems and thus limits the
production as a fusion protein to a certain expression host, like
e.g. fusion proteins with toxic domains[2] or vaccines with non-
proteinogenic features.[3]
To fully unleash the potential of bioorthogonal chemistry for the
production of advanced biologicals, the method of conjugation
needs to be highly efficient, quantitative and free of undesired
side products to obtain homogeneous products - essential
criteria to meet the high requirements of pharmaceutical
manufacturing.[4]
Among all bioorthogonal reactions, the Diels-Alder cycloaddition
[a]
Dr. M. Baalmann, L. Neises, P. Werther, M. J. Ziegler, N. Ilkenhans,
M. Wolfring, J. Wilhelm, Dr. R. Wombacher
Institute of Pharmacy and Molecular Biotechnology, Heidelberg
University, Im Neuenheimer Feld 364, D-69120 Heidelberg
(Germany)
Scheme 1. Protein fusion and conjugate architectures accessible through
expression (only left), enzymatic ligation strategies or site-specific
bioorthogonal chemistry.
E-mail: wombacher@uni-heidelberg.de
[b]
#
S. Bitsch, H. Schneider, L. Deweid, Prof. Dr. H. Kolmar
Institute for Organic Chemistry and Biochemistry, Technische
Universität Darmstadt, Alarich-Weiss-Straße 4, 64287 Darmstadt
(Germany)
Here we present an extensive comparative study of various
bioorthogonal chemistries for quantitative protein conjugation,
aimed at identifying the ideal reaction partners for the production
of defined and branched protein-protein conjugates by DAinv. We
report the synthesis of a large panel of the most popular
These authors contributed equally
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201915079
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20]
dienophile and diene scaffolds as carboxylic acid derivatives and
tested them as substrates for lipoic acid ligase (LplA). A
methyltetrazine substrate and a stable bicyclononyne (BCN)
substrate was found to be compatible with fast and quantitative
protein derivatization. The combination of both substrates allow
for site-specific and almost quantitative DAinv-based protein-
protein conjugation. We demonstrate the applicability and
efficiency of our conjugation method with the full-length
therapeutic antibody trastuzumab. All protein-protein conjugates
were fully functional as proven in fluorescence, binding, and
stability studies. The speed, robustness and efficiency of the
method guarantees a simple workflow to generate protein-
protein conjugates of choice within hours starting from
recombinant proteins regardless of their expression host. With
the technology presented here, we open up new avenues for
advanced applications in therapeutics or diagnostics.
as well as the 2Z and 4Z isomers of cis-cyclooctenol (CCO*S
and CCOS), and validated them in the HPLC assay. We also
included the equatorial 4E TCO-based substrate first reported by
the Ting group[9b] (denoted as eq.-TCOS) and the norbornene
substrate NorbS previously developed by our group[21] into our
comparison. Complementary to our formerly reported diene-
based methyltetrazinylmethoxycarbonyl (MeTzMeOcS) and
triazine amide (TrzS) substrates,[16] we further synthesized
[22]
methyltetrazinyl alkanoic acids (MeTzAla-e
optimized substrate derivative MeTzAlb.
)
and identified the
Results and Discussion
Synthesis and identification of well-accepted diene and
dienophile substrates for site-specific ligation to peptide
tags
To compare various bioorthogonal motifs for protein modification,
a suitable assay platform is required that allows evaluation of
modification efficiency and product integrity under conditions
close to the desired application.
Building on innovative work by the Ting group,[13] we chose the
engineered lipoic acid protein ligase A LplAW37V from Escherichia
coli that ligates unnatural lipoic acid analogues to the 13 amino
acid recognition motif lipoate acceptor peptide (LAP). LAP can
be attached terminally to as well as internally into recombinant
proteins from any host organism.[13] Several substrates with
bioorthogonal motifs for site-selective tag-based protein
functionalization
including
azides,[14]
norbornenes,[15]
trans-cyclooctenes,[9b] methyltetrazines and triazines[16] have
been described.
We synthesized a panel of potential LplAW37V substrates based
on all commonly used dienes and dieonophiles for DAinv. This
includes the axial 2E regioisomer of trans-cycloctenol
(ax.-TCO*a-d),[10b]
(endo-BCNa-c),[17] mono- and dimethylcyclopropenyl-methanol
(MMCya-d and DMCya-d
[18] derivatized with linear amino acids of
endo-bicyclononynyl-methanol
)
varying length via a carbamate (Scheme 2, Table 1). First, we
used a HPLC-based assay to screen various putative substrate
candidates for Mg2+-ATP-dependent ligation onto LAP within
15 min in phosphate buffer (pH 7.0) at 37 °C by both the
wildtype-LplA and LplAW37V. From each substrate class, an
optimal candidate for maximum ligation efficiency by the
LplAW37V (Table S1) was identified. Carbamates with
5-aminopentanoic acid consistently gave the highest LplAW37V
acceptance which is why we additionally prepared derivatives
with cycloct-2-yn-1-ol (SCOS),[19] the equatorial 2E and axial 4E
regioisomers of trans-cycloctenol (eq.-TCO*S and ax.-TCOS),[10b,
Scheme 2. (A) Two-step chemoenzymatic procedure with LplAW37V and click
chemistry to generate protein-protein conjugates. (B) Substrate scope for
LplAW37V-mediated site-specific protein functionalization and ensuing Diels-
Alder cycloaddition with inverse electron demand (DAinv), strain-promoted
alkyne-azide cycloaddition (SPAAC) or strain-promoted alkyne-nitrone
cycloaddition (SPANC). References; a: Baalmann et al.,[16] b: Liu et al.,[9b] c:
Best et al.[21]
.
This gives a total set of 15 different bioorthogonal motifs as
substrates for LplAW37V, covering the most commonly used diene
and dienophile moieties from literature[12] to conduct DAinv in its
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entire reactivity range (k2 = 10-2 M-1 s-1 – 105 M-1 s-1)[23] (Scheme
2 and Table S1). Notably, the BCN and SCO moieties can
further serve as 1,3-dipolarophiles for strain promoted alkyne-
azide (SPAAC) or alkyne-nitrone (SPANC) cycloaddition,
reactive cis-isomer and TCO* shows elimination after DAinv
-
product formation. Thus, we wanted to evaluate the extent of
these unwanted side-reactions during LplAW37V ligation and DAinv
Although, eq.-TCOS-modified proteins undergo rapid conjugation
with both tetrazine fluorophore probes, we observed this
conjugation to be incomplete limited to a maximum of about
53 % (Figure 1B). This supports the notion that the previously
observed trans-to-cis isomerization tendency in the peptide
assay also takes place during protein labeling (Figure S7).
Comparably, ax.-TCOS-modified EGFP showed higher
isomerization propensity. Interestingly, proteins directly modified
with CCOS were not fully unreactive toward TzBnNH-TAMRA
resulting in non-negligible cycloadduct formation (Figure 1B).
For ax.-TCO*b-modified EGFP treated with TzBnNH-TAMRA, an
unreactive EGFP population substantiates isomerization of
ax.-TCO*, albeit less pronounced as for the related 4E TCO
substrates (Figure 1B). Especially for MeTzBnH-TAMRA-treated
proteins, a protein species with the apparent MW of non-
modified EGFPE172::LAP emerged (Figure 1B), which we attribute
to the elimination of the carbamate function of the cycloadduct.
This was further verified by data from mass spectrometry (Figure
S16). Less elimination was observed when using TzBnNH-
TAMRA, which is in agreement with the previously reported
effect of the tetrazine scaffold for elimination of the TCO*-
tetrazine cycloadduct.[29] High cycloaddition yield, minimal
elimination and low isomerization were achieved solely with the
eq.-TCO*S/TzBnNH-TAMRA combination. However, we were
unable to validate the reported exclusive orthogonality for
MeTzBnNH-substituted probes under the chosen reaction
conditions.[23b] Under the conditions applied, both TCO and
.
expanding the scope of the method to other bioorthogonal
23b, 24]
cycloaddition reactions.[17,
In the HPLC assay, all
substrates resulted in uniform ligation products (Figures S2-S6
and S9-S13), except the TCO-based ones (Table S1 and
Figures S7+S8). Closely examining the retention times and m/z
of the ax./eq.-TCOS ligation products in comparison with CCOS,
trans-to-cis isomerization of the TCO-modified peptides was
apparent (Table S1 and Figure S7B/D). ax./eq.-TCOS stock
solutions showed no sign of cis-isomerization prior to the
LplAW37V ligation reaction, indicating that the assay conditions
lead to the observed trans-to-cis isomerization (Figures S7C).
TCO* substrates (ax.-TCO*b and eq.-TCO*S) with the previously
reported more stable TCO* scaffold[10b] were prone to
isomerization as well, albeit much less pronounced as ax./eq.-
TCOS. For eq.-TCO*S, isomerization was barely detectable
(Table S1 and Figure S8).
Protein labeling, diene/dienophile performance and a tool to
probe protein modification states
With the established substrate platform for peptide modification
with all commonly used dienes and dienophiles, we moved on to
evaluate the best performing LplAW37V substrates for site-specific
modification at the protein level.
During our work with a LAP tag internally inserted in a flexible
)
loop of EGFP (EGFPE172::LAP [25], a change in electrophoretic
mobility in semi-native SDS-PAGE (non-reducing and non-
denaturing conditions) for both, substrate functionalized EGFP
and the corresponding DAinv cycloadducts was observed (Figure
1). Comparable electrophoretic mobility changes due to
lipoylation or octanoylation for E2 and H proteins have been
reported.[26] Amino acid substitutions or posttranslational
modifications can lead to an altered electrophoretic mobility and
TCO* are unsuitable for quantitative conjugation in DAinv reaction.
We then evaluated the reactivity of our novel BCN- and SCO-
LplAW37V substrates. Strained cyclic alkynes do not have an
isomerization-susceptible configuration, are regarded to be
stable and their cycloaddition products are not prone to
elimination.[10b] To our delight, endo-BCNb-functionalized
proteins could be transformed nearly quantitatively to the
cycloadduct with both tetrazine-TAMRA conjugates (Figure 1B
and S17, S19). SCOS-modified EGFP underwent almost full
conversion to the cycloadduct with TzBnNH-TAMRA, but was
only slightly reactive toward MeTzBnNH-TAMRA. This is in
GFP’s intrinsic properties seem to enhance this phenomenon[27]
.
Inspired by the ability to probe the protein modification states
directly at the protein level (Figure 1D), we started to investigate
the ligation yields, DAinv conversion and side-reactions of all
substrates (Figures 1, S14 and S15). Briefly, EGFPE172::LAP was
quantitatively functionalized with each substrate (Figures S14
and S15) and subsequently treated with either MeTzBnNH-
TAMRA or TzBnNH-TAMRA (Figure 1A, 1B, 1C, S14),
respectively, or TCO-Prop-TAMRA and BCN-Pip-TAMRA
(Figure S15). Reactions were quenched with a large excess of
either eq.-TCO-OH or dimethyl tetrazine (MeTzMe), respectively.
Additionally, separate reaction mixtures with LAP-tagged
maltose-binding protein (MBP-LAP) were subjected to mass
determination to further support the gel shifting behavior of
EGFP (Figure S16 and S19).
agreement with
a
previous work[23b]
,
although complete
orthogonality cannot be proven as reported.
Although the strained cyclooctyne substrates already had
demonstrated great potential for quantitative cycloadditions, we
were curious to investigate means to prevent the observed
isomerization of the TCO. Isomerization of TCOs has mainly
been attributed to the influence of thiols via a radical-based
mechanism.[9b] We selected the two radical scavengers Trolox[30]
and ascorbic acid as possible isomerization suppressors in the
ligation mixture with ax.-TCOS without effect (Figure S18A). Next,
we suspected the cysteine residue (Cys85) located in the
binding pocket of the substrate-bound form of the LplAW37V [31] to
be responsible for the observed TCO isomerization. The double
All types of TCOs have remarkably fast kinetics in DAinv that are
particularly useful for intracellular labeling.[28] However, previous
works reported that TCO tends to isomerize to the almost non-
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Figure 1. Individual EGFP modification states during two-step labeling can be traced by semi-native SDS-PAGE. (A) Procedure of two-step EGFP modification
and subsequent analysis. (B) SDS gels assaying the reactivity of different dienophile LplA constructs using EGFPE172::LAP highlights high modification yields
utilizing combinations of endo-BCNb and SCOs with MeTzBnNH-TAMRA and TzBnNH-TAMRA and eq.-TCO*s together with TzBnNH-TAMRA (blue boxes).
CBB = Coomassie Brilliant Blue. M = Marker. †: ~35 kDa. ‡: ~25 kDa. (C) Protein construct used. (D) Scheme illustrating EGFP shifting in the gel during
modification steps. For the exhaustive side-by-side comparison of all DAinv substrates, see Figures S14 and S15. Note: Yields have been corrected for an EGFP
impurity of 6 % that was neither accessible for ligation nor cycloaddition reactions. Crystal structure PDB: 2Y0G[32]
.
mutant LplAW37V/C85A was prepared and displayed ligase activity
for endo-BCNb and eq.-TCOS but did not alleviate or abolish
isomerization of TCO (Figure S18B). This suggests that protein
environments during the ligation reaction are sufficient to trigger
TCO isomerization. Using BCN as a dienophile for DAinv based
post-translational protein modification takes advantage of the
reactivity-stability-tradeoff and outperforms TCO and TCO*.
While maintaining a high reaction rate BCN provides quantitative
conjugation yields. With endo-BCNb, we identified the most
efficient substrate for LplAW37V to prime proteins for efficient
DAinv conjugation. End-, NorbS-, MMCyb- and DMCyb-
functionalized EGFPs were expectedly much less reactive in
DAinv, but their side-by side comparison might be interesting for
some readers (Figure S14). We could also confirm almost
quantitative cycloaddition of tetrazine-modified EGFP for
MeTzMeOcc with TCO and BCN probes and for the novel
MeTzAlb with TCO under the chosen reaction conditions (Figure
S15). Even the triazine substrate TrzS provided reasonable
reactivity with both TCO and BCN probes.
Targeted protein-protein conjugation using DAinv
Ideally, a method for protein-protein conjugation is directly
applicable to the recombinant protein from the expression host
of choice using enzymatic or chemical strategies. Sortase or
split-intein strategies have been used for post-translational
covalent bond formation between two or more native proteins to
33]
a
continuous polypeptide chain.[2,
Especially for larger
multidomain protein fusions, it might be advantageous to
precisely attach proteins at defined internal positions within a
single polypeptide chain. The generation of these internal or
branched protein fusions from individual native proteins purely
by enzymatic means is a challenge. To our knowledge, only the
approaches based on CnaB2 and D4 Ig-like domains
(Spy-/Snoop- × Tag/Catcher) from the Howarth lab provide
enzyme-catalyzed covalent internal protein connectivity in a
building block principle.[34] Amine and thiol reactive chemistry
has enabled internal protein-protein conjugation much long
before the advent of genetic fusions,[35] but is severely limited in
site-specificity and thus not suited for precise construction of
branched protein architectures.[36] Protein-protein conjugation
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Figure 2. Protein-protein conjugation using model proteins MBP-LAP and EGFPQ157 ::LAP. Combination of endo-BCNb with MeTzMeOcs or MeTzAlb was efficiently
used for targeted protein-protein conjugation by DAinv. (A) SDS-PAGE analysis highlights almost quantitative formation of MBP-EGFP conjugate (~ 70 kDa) within
2 h. CBB = Coomassie Brilliant Blue. (B) Schematic view of the model proteins primed for protein-protein conjugation by DAinv. (C) Mass spectrometry analysis of
conjugation reaction. Crystal structure PDBs: 1ANF[37] and 2Y0G
utilizing bioorthogonal reactions has attracted attention over the
last years due to its potential to overcome terminal attachment
restrictions or non-selective reactions. It is surprising that
targeted and conjugation between proteins with internal
connectivity via DAinv has not been investigated yet,given that all
potential tools in genetic code expansion are available for a
couple of years.[38] Apart from conjugations between TCO- and
tetrazine-modified proteins pre-modified via non-site-specific
NHS chemistry,[39] there are only a few examples for targeted
[11b, 40]
internal protein-protein conjugation using DAinv
.
Stimulated by the modularity of the stable LplAW37V substrates
and the quick and near-quantitative ligation reaction, we
envisioned functionalizing proteins with
a methyltetrazine
substrate (MeTzMeOcS or MeTzAlb) and an endo-BCNb
dienophile substrate separately. Subsequently, the primed
proteins can be combined for conjugation by the DAinv
cycloaddition, benefitting from the fast reaction rate of tetrazines
with strained dienophiles and the benign catalyst-free[5]
41]
conditions (Figure 2).[25,
Introduction of LAP in permissive
Figure 3. Reaction kinetics for targeted protein-protein conjugation between
EGFP and mRuby3 monitored by FRET. Left graph shows mRuby3 FRET
acceptor response. Right graph shows determination of second order rate
constant. Error bars in left graph: ±1 standard deviation; in right graph: ±1
highest error estimate. Crystal structure PDBs: 2Y0G and 3U0L[43]
internal positions should provide the basis to construct branched
protein chimeras.[25, 42] We thus chose the conjugation between
the two model proteins MBP-LAP and an EGFP construct with
an internal LAP tag (EGFPQ157::LAP) as a proof of concept. For
1:2 protein ratios at 10 µM and 2-4 h incubation time, the
conjugation gave nearly quantitative yield related to the protein
reaction partner which is not present in excess (fluorescent
protein species at ~ 85 kDa, Figure 2A and Figure S21). Non-
purified reaction mixtures of the protein cycloaddition reactions
were further analyzed by intact protein mass determination
showing full conversion to the cycloaddition product (Figure 2C
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10.1002/anie.201915079
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and S20). Notably, the N- and C-termini of both proteins remain
in their native unmodified form providing additional branching
options for even more complex protein chimeras. To analyze the
kinetics of this protein-protein conjugation method, we incubated
with the LAP motif on the C-termini of each heavy chain
(trastuzumab-hcLAP/lcWT, further referred to as trastuzumab-
(LAP)2). This immunoglobulin targets HER2-postitive cancer
cells and is applied as parental antibody in the FDA approved
drug Herceptin.[44] Very recently, LplAW37V-mediated TCO ligation
was used in combination with transglutaminase for one-pot dual
labeling of trastuzumab.[6a] The data presented in the report
indirectly point toward an incomplete conjugation of TCO
modified trastuzumab-(LAP)2 which is in agreement with our
observation of an impaired stability of TCO for quantitative in
vitro bioconjugation. We reasoned that antibody functionalization
with endo-BCNb might serve as a conjugation hub enabling both
DAinv/SPAAC with small molecules (fluorophores or cytotoxic
drugs) and even whole proteins. Ligation of endo-BCNb to both
LAP tags of intact trastuzumab was monitored by hydrophobic
interaction chromatography (HIC, Figure 4A and S23) and
quantitative conversion of trastuzumab to the doubly BCN-
endo-BCNb-functionalized
EGFPE172::LAP
with
varying
concentrations of methyltetrazine-functionalized mRuby3-LAP
and monitored the Förster resonance energy transfer (FRET)
acceptor fluorescence of mRuby3 over time. Under pseudo-first
order kinetics, remarkably high second order rate constants of
~ 70 M-1s-1 and ~ 50 M-1s-1 for the endo-BCNb/MeTzMeOcS and
endo-BCNb/MeTzAlb
combinations,
respectively,
were
determined at 37 °C in PBS (Figure 3 and S22). In line with our
expectations, FRET partners both equipped with endo-BCNb did
not show any FRET response (Figure 3).
Functional protein-antibody conjugates
Next, we wanted to demonstrate the power of our conjugation
procedure for the generation of therapeutic protein chimeras.
We genetically equipped the monoclonal antibody trastuzumab
functionalized
species
within
3 min
was
observed,
demonstrating rapid and clean antibody functionalization.[45]
Figure 4. Protein-antibody conjugation using DAinv with endo-BCNb and MeTzMeOcs/MeTzAlb yields a functional protein conjugate with the full-length antibody
trastuzumab. (A) Ligation of endo-BCNb to the doubly LAP-tagged trastuzumab, BAR = BCN-to-antibody ratio. See Figure S23 for expanded ligation time. (B)
SDS-PAGE analysis shows quantitative conjugation EGFP to trastuzumab. hc = heavy chain, lc = light chain. (C) Scheme of trastuzumab-(LAP•EGFP)2 conjugate
architecture. (D) Confocal microscopy with the trastuzumab-(LAP•EGFP)2 conjugate shows specific binding to SK-BR-3 and no binding to CHO-K1 cells. Scale
bar = 20 µm. Conjugation control: trastuzumab-(LAP)2 and EGFPQ157::LAP both functionalized with endo-BCNb. (E) Flow cytometry analysis shows concentration-
dependent binding of trastuzumab-(LAP•EGFP)2 and trastuzumab-(LAP•TAMRA)2 conjugates to SK-BR-3 cells. fl. = fluorescence.
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SK-BR-3 cells and CHO-K1 cells was performed (Figure 5).
Potent, subnanomolar (IC50 = 0.31 nM) cell-killing properties
were shown for trastuzumab-(LAP•MMAE)2 on HER2-postive
cells, while the construct was found innocuous to HER2-
negative cells. As expected, trastuzumab-(LAP)2 lacking a toxin
warhead was found non-toxic for both HER2-postive and
negative cells. The introduced peptide may have antigenic
potential upon in vivo administration as for several other
antibody tags that are currently used for orthogonal modification
of antibodies. Consequently, careful evaluation of the
antigenicity of the LAP sequence that may not only depend on
the peptide sequence, but also on other factors such as the
mode of administration or the antibody concentration will be
necessary in frame of potential medical applications.
With fast, targeted and efficient posttranslational protein
conjugation to antibodies being a hitherto unmet challenge, we
sought to directly assess this concept with internally tetrazine-
functionalized EGFP. Protein conjugation to trastuzumab
smoothly went to near completion at 40 µM of tetrazine-modified
EGFPQ157::LAP and 10 µM of doubly BCN-functionalized
trastuzumab-(LAP)2 at 37 °C for 4 h in PBS (Figure 4B).
Encouraged by these promising results, the biological activity of
doubly EGFP-modified trastuzumab (trastuzumab-(LAP•EGFP)2,
Figure 4C) was assessed via immunofluorescence staining of
living mammalian cells using confocal microscopy (Figure 4D
and S24A). As expected, the fluorescent signal of EGFP-
conjugated trastuzumab accumulated at the outer cell
membrane of the HER2-positive breast cancer cell line SK-BR-3,
but not at the HER2-negative control cell line CHO-K1. No
fluorescent signal was detected for the negative control
conjugation (Figure 4D), supporting the lack of mutual cross
reactivity between BCN-functionalized proteins (Figure 2A and
4B). Incubation of both cell lines with
a 1:1 mixture of
trastuzumab-(LAP•EGFP)2 and trastuzumab-(LAP•TAMRA)2
resulted in colocalizing fluorescent signals of only on the HER2-
positive cell line (Figure S24B).
To examine how the LAP tag and subsequent conjugation with a
probe, influence biological properties of the antibody,
subsequent binding studies were performed. A comparative
binding study of trastuzumab-(LAP•EGFP)2, trastuzumab-
(LAP•TAMRA)2 and trastuzumab-(LAP)2 on HER2-positive cells
revealed potent binding of all constructs (Figure 4E and S25). To
further substantiate these findings, we determined the binding
constants on cells applying flow cytometry in vitro (Figure S26).
Trastuzumab with the C-terminal LAP motif revealed single-digit
nanomolar binding properties (KD = 5.6 nM) equal to unmodified
wildtype trastuzumab (KD = 6.3 nM).[46] Notably, trastuzumab
equipped with EGFP exhibited equipotential binding
(KD= 7.9 nM). Additionally, the further modified trastuzumab-
(LAP•TAMRA)2 construct revealed only a negligible loss of
binding potential (KD = 12.9 nM).
Figure 5. Generation of the potent antibody-drug conjugate trastuzumab-
(LAP•MMAE)2 by endo-BCNb ligation followed by SPAAC with N3-PEG3-vc-
PABC-MMAE. Top: Conjugate architecture. vc = valine-citrulline, PABC =
para-aminobenzyl carbamate, MMAE = monomethyl auristatin E. Dotted lines
indicate cleavable bonds for drug release. Scissors indicate cleavage site for
lysosomal proteases. Bottom: Cell proliferation assays with SK-BR-3 and
CHO-K1 cells treated with trastuzumab-(LAP)2 and trastuzumab-
(LAP•MMAE)2 (DAR2). Error bars in graphs: ±1 standard deviation.
Encouraged by these results, we further investigated the impact
of the LAP motif itself and the conjugation to EGFP on the
stability of the antibody. Thermal shift assays revealed equal
melting points for the trastuzumab-(LAP)2 compared to the
unmodified wildtype antibody. Additionally, no loss in stability
was found for trastuzumab-(LAP•EGFP)2, as three melting
points equal to the Fc- and the Fab-part of the unmodified
trastuzumab and to solitaire EGFP were obtained (Figure S27
and Table S28).
Conclusion
In conclusion, we have developed a highly efficient method to
synthesize well defined, homogeneous protein(-protein)
conjugates utilizing enzyme-mediated protein modification and
bioorthogonal DAinv chemistry. Various diene and dienophile
substrates were analyzed for ligation to proteins using LplAW3V
as well as for their ability to yield homogeneous conjugates in
subsequent DAinv. For this we utilized the unique gel shifting
behavior of EGFP to evaluate stability and reactivity of
bioorthogonal motifs at the protein level. We identified novel
tetrazine- and BCN substrates to yield quantitative
homogeneous protein-protein conjugates without any detectable
by-products or toxic remnants, within a single working day
starting from purified protein. By efficiently labeling the
Antibody-drug conjugates (ADCs) by SPAAC click
chemistry
To investigate, whether our strategy of site-specific protein
conjugation allows for the efficient generation of antibodies
equipped with highly potent cytotoxins, we generated an ADC in
a two-step procedure. Trastuzumab-(LAP)2 was modified with
endo-BCNb by LplAW37V-catalysis followed by conjugation of
highly toxic monomethyl auristatin
E (MMAE) by SPAAC
applying N3-PEG3-vc-PABC-MMAE, resulting in trastuzumab-
(LAP•MMAE)2. Next, we assessed the in vitro potency of the
trastuzumab-(LAP•MMAE)2. For this, a cell proliferation assay on
This article is protected by copyright. All rights reserved.

10.1002/anie.201915079
Angewandte Chemie International Edition
RESEARCH ARTICLE
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Acknowledgements
R.W. and H.K. acknowledge funding from DFG / SPP1623.
M. B. gratefully acknowledges funding from the Landes-
graduiertenförderung Baden-Württemberg. M.J.Z. thanks the
Carl-Zeiss-Stiftung for a fellowship. We thank H. Rudy, T.
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Keywords: click chemistry • protein modifications • antibody-
drug conjugate • bioorthogonal chemistry • protein-protein
conjugates • protein ligation
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RESEARCH ARTICLE
Mathis Baalmann#, Laura Neises,#
Sebastian Bitsch, Hendrik Schneider,
Lukas Deweid, Philipp Werther, Nadja
Ilkenhans, Martin Wolfring, Michael J.
Ziegler, Jonas Wilhelm, Harald Kolmar
and Richard Wombacher*
Page No. – Page No.
Text for Table of Contents
A bioorthogonal click chemistry
toolbox for targeted synthesis of
branched and well-defined protein-
protein conjugates
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