Multifunctional, histidine-tagged polymers: Antibody conjugation and signal amplification

Article abstract of DOI:10.1039/d0cc04591h

A polymer scaffold, with multiple reactive centres, was synthesised by RAFT polymerisation and conjugated to the antibody herceptin. A hexahistidine RAFT agent enabled the rapid and simple purification of polymer-protein conjugates, while the tetrazine conjugation strategy allows myriad cargos to be attached and amplified.

Full text of DOI:10.1039/d0cc04591h

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Multifunctional, Histidine-tagged Polymers: Antibody Conjugation
and Signal Amplification†
Received 00th January 20xx,
Accepted 00th January 20xx
Yichuan Zhang,a,b‡ Alessia Gambardella, a,‡ Muhammed Üçüncü, Jin Geng, a,b Jessica
Clavadetscher,a Mark Bradley,a,* and Annamaria Lilienkampf a,*
DOI: 10.1039/x0xx00000x
A
polymer scaffold, with multiple reactive centres, was uses pre-synthesised polymers for bioconjugation but suffers
synthesised by RAFT polymerisation and conjugated to the from complications, such as how to separate conjugated
antibody Herceptin. A hexahistidine RAFT agent enabled the rapid proteins from non-conjugated materials.11
and simple purification of polymer–protein conjugates, while the Here, we report a rapid amplification approach, based on
tetrazine conjugation strategy allows myriad cargos to be attached poly(acrylamide)–antibody conjugates bearing norbornene
and amplified.
reactive centres for both “switch-on” of quenched tetrazine-
linked fluorophores and cargo amplification in the form of
metal ions (Fig. 1). The polymers were synthesised through
reversible addition-fragmentation chain transfer (RAFT)
The ability to amplify reporter signals and cargos is important
in a number of areas ranging from fluorescent imaging to
therapeutic radiotherapy and is of particular importance with
low abundance targets. Several signal enhancement methods
have been developed with numerous techniques associated
with the polymerase chain reaction and variants.1 Non-nucleic
acid based amplification methods include horseradish
peroxidase-driven signal amplification of tyramide-conjugates2
or luminol,3 and alkaline phosphatase driven amplification.4
Although these enzyme-based amplification methods work
well, they typically require the use of secondary antibodies and
bespoke substrates, with colorimetric or fluorescent outputs
often able to diffuse away from the site of generation. Non-
enzymatic fluorescence amplification techniques include e.g.
the recruitment of antibody-GFP fusion proteins to a target
protein tagged with multiple copies of the antibody epitope,5
Avidin-conjugated viral capsids loaded with dyes for the
detection of biotinylated targets,6 and biological self-assembly
of biotin and streptavidin surface engineered quantum dots.7
Synthetic polymers are an attractive approach to biomolecule
labelling and fluorescence or mass-tag signal amplification.8
Conjugation of polymers to proteins can be categorised as
“grafting-from” and “grafting-to” approaches,9 the former
starting with the formation of a “macro-activator” (i.e., linkage
of an initiator or a chain transfer agent to a protein), followed
by polymerisation; however, this approach is limited by the
need to avoid protein denaturation and the restricted choice
of suitable functional monomers.10 The “grafting-to” approach
polymerisation, using the synthesised chain transfer agent
2
that contained the ubiquitous hexahistidine tag (His-Tag),
designed to allow rapid polymer–antibody conjugate
purification by metal affinity chromatography. Upon treatment
with a quenched BODIPY–tetrazine conjugate, rapid “switch-
on” and amplification in fluorescence were observed.
Five polymers with different molecular weights and densities
of the norbornene reactive centres were synthesised (P1–P5)
to explore how composition affected conjugation, antibody
binding affinity, and the level of cargo amplification (Fig. 2a).
N,N-dimethylacrylamide (DMA) was used due to its low
cytotoxicity and the high water solubility of its polymers,12
with the norbornene-based acrylamide
1 (Scheme S1)
providing the site for tetrazine ligation. The polymers were
efficiently synthesised by RAFT polymerisation with the
desired molecular weights (7–30 kDa) with polydispersities
between 1.2–1.8 (Table S1, Fig. S1) using a His-Tag-RAFT agent
2
(synthesised on solid-phase13 in high yield/purity, Scheme
S2), which allowed the widely used method of metal ion
affinity chromatography to be applied to polymer-conjugate
purification.
To achieve simultaneous “switch-on” and amplification of
fluorescence by the polymer–protein conjugates, two
fluorogenic BODIPY–tetrazines were synthesised (Fig. 2b).
Tetrazine-quenched fluorophores, that are activated via an
inverse electron-demand Diels–Alder reaction (INVDA), have
found use in fluorescent imaging,14–18 allowing direct target
visualisation without extensive washing steps. Tz1 was
synthesised as reported previously,14 whereas the published
route to Tz215 could not be reproduced and instead was
synthesised from 3-formylbenzonitrile, via the tetrazine
aldehyde S9 16
which was treated with 2,4-dimethylpyrrole,
,
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Fig. 1 Signal amplification using a polymer–protein conjugate. The antibody Herceptin was conjugated to a polymer scaffold that carried multiple norbornenes (for
signal amplification via tetrazine ligation) and was synthesised using a hexahistidine tagged-RAFT agent. Subsequent treatment with a tetrazine-quenched
fluorophore
or
a
metal-bearing
tetrazine,
“switches-on”
and/or
amplifies
the
signal.
followed by oxidation with DDQ and complexation with analysis of the labelling efficiency of the polymer–antibody
BF3·OEt2 to give the BODIPY–tetrazine Tz2 in high yield conjugates on HER2 receptor positive breast cancer cells (SK-
(Scheme S3).17 The metal chelating tetrazine Tz3 was BR-3) with BODIPY-P1-Her
, BODIPY-P2-HER and BODIPY-P3-
synthesised by coupling the DOTA–NHS ester to an amino- Her and BODIPY-Her. Incubation of SK-BR-3 cells with small
functionalised tetrazine,19 followed by Cu(II) loading (Scheme molecule labelled BODIPY-Her (Scheme S5) resulted in rapid
S4).
labelling of the cells with fluorescence saturation of cells
The reactivity of the norbornene-decorated polymer P1 (12 observed after 2 h. Similar labelling and fluorescence
μM) with the tetrazines Tz1 and Tz2 (1 μM) was investigated saturation of cells was also observed by polymer conjugated
by monitoring the time-dependent increase in fluorescence, antibodies BODIPY-P2-Her and BODIPY-P3-Her, whereas
triggering fluorescence increases of 21 and 9-fold respectively, BODIPY-P1-Her showed slower labelling with fluorescence
in PBS after 30 min (Fig. S3 and S4). However, stability studies saturation reached after 4 h incubation (Fig. 3b). MCF-7 cells,
showed that although Tz1 and Tz2 were robust in PBS and which do not express the HER2 receptor, showed no significant
serum-free media, Tz2 underwent undesired tetrazine changes in fluorescence intensity when incubated with the
hydrolysis in serum, while in contrast, Tz1 showed good polymer–Herceptin conjugates (Fig. S10). None of the
stability and was thus used in preference for all investigations polymer–antibody conjugates (P1-Her to P5-Her), BODIPY-Her
(Fig. S5).
or Tz1 showed cellular toxicity at 10 nM, 10 nM and 1 µM,
The antibody Herceptin facilitates tyrosine kinase receptor respectively (Fig. S11).
HER2 degradation and is used clinically to treat aggressive The fluorescence “turn-on” and signal amplification with the
breast cancer,20 and was used here to demonstrate both cell polymer–antibody conjugates and tetrazine Tz1 was
labelling21 and cargo amplification. Polymers conjugated to demonstrated in live SK-BR-3 cells by flow cytometry and
Herceptin (P1-Her–P5-Her) were purified in 45–88% yields microscopy (Fig. 3c–d). Cells treated with BODIPY-Her (4 h, 10
utilising the His-Tag (Fig. 2a, Table S2), with SDS PAGE showing nM) showed a moderate increase in fluorescence intensity;
successful conjugation (Fig. S9). When P1-Her was treated with however, treatment with the conjugates P1-Her to P5-Her (4
tetrazine Tz1 (Fig. 2a), intense fluorescent labelling of the h, 10 nM), followed by the quenched fluorophore Tz1 (30 min,
antibody resulted, with selectivity between the polymer- 1 µM) resulted in remarkable increases in cellular fluorescence
tagged antibody and the unmodified variant observed (Fig. 3a, (83-fold increase for P3-Her and P5-Her, compared to BODIPY-
lane 2 vs 4). In addition, SDS-PAGE analysis indicated that the Her). The fluorescence intensity could be tuned by either
majority of polymer conjugation took place on the heavy changing the molecular weight of the polymers (45, 10 and 83-
chains of the antibody. The influence of the polymers on the fold increases for P1-Her, P2-Her and P3-Her, respectively) or
antibody’s binding affinity was investigated by flow cytometry the density of the reactive centres (6 and 83-fold increase for
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P4-Her and P5-Her, bearing 2.8 and 18 norbornes per polymer treated with P1-Her and P3-Her, which had different numbers
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DOI: 10.1039/D0CC04591H
chain, respectively). Confocal microscopy images of the cells
Fig. 2 (a) Synthesis of the multifunctional polymers P1
were synthesised as random copolymers (see ESI Table S1 for full characterisation). Polymer synthesis was followed by their antibody conjugation (10 eq. polymer per
antibody added) and metal-affinity chromatography purification of the polymer–antibody conjugates (P1-Her P5-Her) and subsequent INVDA mediated dequenching
to give the fluorescent conjugates. The NHS-conjugation strategy produces heterogenous polymer–antibody population, with multiple conjugation sites possible;
however, only one modification site on the antibody is shown for clarity. (b) The tetrazines explored for signal amplification.
–P5 and the ratios of 1, 2
and DMA found in the synthesised polymers (based on 1H NMR analysis). All polymers
–
Fig. 3 (a) SDS-PAGE showing successful conjugation and fluorescence “switch-on”. Lane 1 Herceptin; Lane 2 Herceptin (2.5 μM) incubated with Tz1 (25 μM); Lane 3
P1-Her and Lane 4 P1-Her (2.5 μM) incubated with Tz1 (25 μM) (reaction mixtures were purified using centrifugal size exclusion filters prior to analysis). (b) Polymer–
Herceptin conjugate labelling of SK-BR-3 cells (quantified by flow cytometry with fluorescence intensity normalised to fluorescence saturated cells, n = 3) compared to
the traditional small molecule labelled Herceptin BODIPY-Her (Scheme S5). (c) Fluorescence intensity of HER2 receptor positive SK-BR-3 cells treated first with the
polymer–Herceptin conjugates (10 nM) followed by Tz1 (1 μM) (normalised to cells treated with P3-Her followed by Tz1). The increase in fluorescence intensity
between BODIPY-Her and BODIPY-labelled P1–P5-Her was 45, 10, 83, 6 and 83-fold, respectively. Insert: the flow cytometry histograms of SK-BR-3 cells treated with
BODIPY labelled Herceptin BODIPY-Her or Tz1, or the polymer–antibody conjugate P1-Her followed by Tz1 (green). Data were analysed using one-way ANOVA with
Dunnett post-test (ns, not significant, *** P < 0.001 , **** P < 0.0001). (d) Confocal fluorescence microscopy images of SK-BR-3 cells treated with BODIPY-labelled
Herceptin BODIPY-Her (10 nM), and polymer–antibody conjugates P1-Her and P3-Her (10 nM) followed by Tz1 (1 μM). The cell nucleus was stained with Hoechst
33342 (blue, λex/em = 353/483 nm) and the plasma membrane stained with CellMaskTM Deep Red (red, λex/em = 649/666 nm). “Switched-on“ BODIPY from Tz1 is shown
in green (λex/em = 488/512 nm). Scale bar = 10 μm. The norbornene content and monomer ratios for P1–P5 (based on 1H NMR analysis) are given in Fig. 2.
of reactive centres (8 vs 21), followed by Tz1 showed much treated cells, although with similar cellular localisations, i.e.,
brighter fluorescence (λex/em = 488/512 nm) than BODIPY-Her cytoplasma and membrane (Fig. 3d and Fig. S12).
This journal is © The Royal Society of Chemistry 20xx
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This amplification method could also be translated to
amplifying the loading density of radioisotopes.22 As a proof of
concept, P5-Her was pre-labelled with Tz3 (Fig. 2a and S11,
Scheme S6) bearing Cu(II) on a DOTA ligand (copper was used
as a model as 64Cu is widely used in nuclear medicine23). The
amplified metal loading on SK-BR-3 cells was confirmed by
inductively coupled plasma-mass spectrometry (ICP-MS) where
the Cu(II) loaded polymer–antibody conjugate Cu-P5-Her
showed a 6-fold higher copper loading (1.6 pg/cell) in
comparison to the conventionally radiolabelled antibody Cu-
Her (Fig. S14, Scheme S7).
In summary, a linear poly(acrylamide) scaffold bearing
norbornenes and a His-Tag was synthesised, conjugated to a
clinically used antibody Herceptin, and used for fluorescence
and metal amplification. Compared to the standard small
molecule fluorophore labelled antibody, the polymer–antibody
conjugates gave up to an 83-fold increase in fluorescence
without affecting antibody binding. This approach of receptor
targeted polymer conjugates enables simultaneous “switch-
on” and amplification of fluorescent signal with possible
contributions to fluorescence-guided surgery and other
fluorescence-based applications, especially those where
signals are inherently weak due to low numbers of receptors.
The approach was applied to the amplification of metal loading
with possible uses in nuclear medicine, but the designed
polymer scaffold would also enable the “multiplication” of
other molecules (e.g. drugs). The new His-Tag-RAFT agent
provides a powerful new approach to access various functional
polymers with an endogenous purification handle to enable
the rapid and simple purification of both “graft-to” or “graft-
from” polymer–conjugates.
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