Light-Activated Protein Conjugation and 89Zr-Radiolabelling with Water-Soluble Desferrioxamine Derivatives

Article abstract of DOI:10.1002/chem.202001755

Protein-conjugates are vital tools in biomedical research, drug discovery and imaging science. For example, functionalised monoclonal antibodies (mAbs) coupled to the desferrioxamine B (DFO) chelate and radiolabelled with ⁸⁹Zr⁴⁺ ions

Full text of DOI:10.1002/chem.202001755

Accepted Article
Accepted Article
Title: Light-activated protein-conjugation and 89Zr-radiolabelling with
water-soluble desferrioxamine derivatives
Authors: Amaury Guillou, Daniel Earley, and Jason P. Holland
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To be cited as: Chem. Eur. J. 10.1002/chem.202001755
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01/2020

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Light-activated protein-conjugation and 89Zr-radiolabelling with
water-soluble desferrioxamine derivatives
Amaury Guillou,[a] Daniel F. Earley[a] and Jason P. Holland*[a]
Abstract: Protein-conjugates are vital tools in biomedical research,
drug discovery and imaging science. For example, functionalised
monoclonal antibodies (mAbs) coupled to the desferrioxamine B
(DFO) chelate and radiolabelled with 89Zr are used as
radiopharmaceuticals for diagnostic positron emission tomography
(PET). In this context, protein functionalisation requires the formation
of a covalent bond which must be achieved without compromising the
biological properties of the mAb. Photochemistry offers new synthetic
routes toward protein-conjugates like 89Zr-mAbs but to harness the
potential of light-induced conjugation reactions new photoactivatable
reagents are required. Here, we introduce two photoactivatable DFO-
derivatives functionalised with an aryl azide (ArN3) for use in light-
activated conjugation and radiosynthesis of 89Zr-mAbs. Incorporation
of a tris-polyethylene glycol (PEG)3 linker between DFO and the ArN3
group furnished water-soluble chelates that were used in the one-pot,
photoradiosynthesis of different 89Zr-radiolabelled protein-conjugates
with radiochemical yields up to 72.9±1.9%. Notably, the DFO-PEG3
chelates can be readily synthesised in accordance with Good
Laboratory Practice (GLP), which will facilitate clinical trials with
photoradiolabelled 89Zr-mAbs.
and radiolabelling process or by a novel one-pot approach.[6–11]
Simultaneous 89Zr-radiolabelling and light-induced conjugation
using a photoactivatable DFO-ArN3 derivative produced viable
PET radiotracers (Scheme 1).[7,11] Additionally, the photochemical
conjugation process is compatible with standard formulation
buffers, which allows direct synthesis of radiolabelled-mAbs from
approved drugs like HerceptinTM. Experimentally, the efficiency
and reproducibility of the photochemistry depends on several
factors including the reaction geometry, light intensity, emission
wavelength, and the stability and solubility of the reagents in basic
conditions.
Scheme 1. Illustration of the general concept of the one-pot, light-activated
photoradiolabelling to produce 89Zr-mAbs.
There have been many reports of alternative, octadentate
chelates for selective radiolabelling of proteins with 89Zr4+
ions.[12,13] In the clinic, DFO remains the chelate of choice for PET
imaging with 89Zr-mAbs. DFO is normally conjugated to mAbs via
reactive groups such as activated esters or benzyl-
isothiocyanates that are introduced to the terminal primary amine
of the chelate. A limitation is that functionalisation of the primary
amine of DFO often compromises the water-solubility, and can
lead to problems controlling the conjugation chemistry.
Reagents that undergo photochemical activation are striking
alternatives to the well-established thermochemically activated
routes toward protein functionalisation.[1,2] The absorption of light
by compounds containing benzophenone, diazirine or ArN3
groups generates highly reactive diradical, carbene or nitrene
intermediates, which under optimised conditions can form
covalent bonds to protein. In a biologically compatible medium,
these reactive intermediates exhibit lifetimes in the nanosecond
(ns) to microsecond (µs) range. Such extreme reactivity is
Two approaches to circumvent solubility issues have been
reported. Richardson-Sanchez et al. hijacked the biosynthetic
machinery of Streptomyces pilosus to install ether groups into the
backbone of the hexadentate DFO ligand, bioisosterically
replacing the –(CH2)5– unit with a –(CH2)2O(CH2)2– moiety to give
the water-soluble DFO-O3 ligand (Figure 1).[14] Similar methods
were also used to introduce redox-active disulfide bonds.[15] In
parallel, Briand et al. used solid-phase synthesis to produce the
water-soluble, octadentate chelate oxoDFO* which incorporated
four ether groups in the ligand backbone.[16] Computational
studies revealed that the ether functionalities in DFO-O3 and
oxoDFO* could potentially increase the thermodynamic stability
of 89Zr4+ complexes by reducing ligand strain.[17] Although
biosynthetic chemistry and solid-phase techniques are advanced
routes to new chelates, and solubility characteristics were
improved, these chemistries present challenges for making GLP-
compliant materials for use in clinical translation. Here, we report
a simple approach to the synthesis of water-soluble DFO
derivatives coupled to a photoactivatable ArN3 group via a
polyethylene glycol (PEG)3 linker.
inaccessible
from
traditional
thermochemically-mediated
processes and mechanistically implies that if successful
bimolecular protein-conjugation is to be achieved, the coupling
step must kinetically outcompete other non-productive
background reactions including hydrolysis, protonation or
quenching by oxygen.[3–5]
We recently demonstrated that metal ion binding chelates
functionalised with a photoactivatable ArN3 group can be used to
prepare radiolabelled antibodies by either a two-step conjugation
[a]
Dr A. Guillou, Dr D. F. Earley, Prof. Dr J. P. Holland*
University of Zurich, Department of Chemistry, Winterthurerstrasse
190, Zurich, CH-8057, Switzerland
E-mail: jason.holland@chem.uzh.ch
Tel: +41.44.63.53.990
Homepage: http://www.hollandlab.org
Supporting information for this article is given via a link at the end of
the document.
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Figure 2. Radioactive chromatography showing: (A) radio-ITLC
chromatograms of
[
89Zr]ZrDFO-PEG3-ArN3 before (blue) and after (red)
Figure 1. Chemical structures of DFO and four water-soluble DFO-derivatives.
Compounds 4 and 9 are reported in this work.
photolysis at 395 nm. The elution profile of [89Zr][Zr(DTPA)]– (black) is shown as
a control. (B) HPLC chromatograms shown the elution profile of compound 4
(black trace), the nonradioactive natZr-complex (green), and the [89Zr]ZrDFO-
PEG3-ArN3 before (blue) and after (red) photolysis at 395 nm.
The functionalised DFO-PEG3-ArN3 (4; Scheme 2) and DFO-
PEG3-EtArN3 (9; supporting Scheme S1) metal binding chelates
were synthesised by using standard chemical transformations
with overall yields of 36% and 14%, respectively. Full
1
experimental details and characterisation data, including H and
13C{1H} NMR spectroscopy and high-resolution electrospray
ionisation mass spectrometry for compounds 1 to 9, are provided
in the supporting information (Figures S1–S37). The synthesis of
compound 4 was achieved on a gram scale with simple isolation
by reversed-phase C18 flash chromatography.
Scheme 3. Simultaneous (one-pot) protein conjugation and 89Zr-radiolabelling
of proteins using light-induced activation of DFO-PEG3-ArN3 (4). Successful
protein radiolabelling was achieved with HSA (MW = 69.08 kDa), onartuzumab
(99.16 kDa) and trastuzumab (MW ~150 kDa).
Scheme 2. Multi-step synthesis of the water-soluble, photoactivatable chelate,
DFO-PEG3-ArN3 (4).
Aliquots of the photoactivatable compounds 4 or 9 were mixed
with the separate protein solutions in transparent glass vials, and
then [89Zr][Zr(C2O4)4]4– was added to give final reaction volume of
150 µL. Reaction mixtures were stirred gently and irradiated (395
nm) for 15 min. at room temperature to ensure complete
photoactivation. Aliquots of the crude reaction mixtures were
retained for analysis and fractions were purified by preparative
size-exclusion gel filtration (PD-10) methods. Crude and purified
samples were analysed by radio-ITLC, analytical PD-10 size-
exclusion chromatography (SEC), and automated HPLC coupled
to a SEC gel-filtration column (Figure 3 and supporting Figures
S38–S41).
89Zr-radiolabelling reactions were performed at room
temperature by adding aliquots of [89Zr][Zr(C2O4)4]4– (89Zr-oxalate)
to samples of compounds 4 or 9 dissolved in water and adjusting
to pH8.0–8.5 with Na2CO3 (the optimum range for our
photoradiosynthesis).[6,7] 89Zr-radiolabelling was characterised by
using radio-instant thin layer chromatography (radio-ITLC) and
radio-HPLC methods (Figure 2). Quantitative 89Zr-radiolabelling
yields were obtained in <5 min. The chemical identity and
radiochemical purity (RCP) of the products were confirmed by
comparison of the HPLC elution profiles to that of the
corresponding non-radioactive natZr-complexes (Figure 2B; blue
and green traces; Figure S37 for nat/89Zr-9+ data). Irradiation of the
[89Zr]ZrDFO-PEG3-ArN3 (nat/89Zr-4+) samples with a powerful light-
emitting diode (LED) with peak emission at 395 nm confirmed the
photochemical activity of the complex (Figure 2B, red trace).
Complete photoactivation of nat/89Zr-4+ was observed in <15 min.
After irradiation, isolated and decay-corrected (d.c.)
radiochemical yields (RCYs) for the [89Zr]ZrDFO-PEG3-azepin-
protein reactions with compound 4 were 72.9±1.9% for HSA,
64.5±6.7% for MetMAbTM, and 58.3±3.4% for HerceptinTM (n = 3
independent measurements for each reaction; with final protein
concentrations in the reaction mixtures of 175 µM, 141 µM, 77 µM,
respectively). For comparison, reactions to form [89Zr]ZrDFO-
PEG3-azepin-HSA by irradiation at a shorter wavelength (365 nm)
gave an average RCY of 70.7±2.3% (n = 3). For each reaction the
radiochemical purity (RCP) of the isolated products was >90%
(determined by HPLC). Protein aggregation was <5%, and when
compared to the analysis of the protein stock solutions, did not
increase after irradiation. The equivalent 89Zr-radiolabelling HSA
Next, we evaluated the efficiency of compounds 4 and 9 in the
photoradiosynthesis of a series of 89Zr-radiolabelled proteins.
Formulated protein substrates included human serum albumin
(HSA), the engineered antibody onartuzumab (formulated as
MetMAbTM), and the humanised IgG1 immunoglobulin
trastuzumab (formulated as HerceptinTM). Simultaneous (one-pot)
photoradiolabelling reactions were performed in accordance with
Scheme 3.[7,11]
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onartuzumab per animal while the same
activity dose but an increased protein
mass (823–834 µg of onartuzumab per
animal) were administered to the control
(blocking) group to modulate the tumour
uptake by saturating the available c-MET
receptors. Quantitative volume-of-interest
(VOI) analysis from the PET images
revealed high accumulation of 89Zr-
radioactivity in the tumours of the normal
group reaching 14.3±1.7 %ID cm-3 (n = 5)
by 72 h post-administration (supporting
Figure S45). In comparison, radiotracer
uptake in tumours of the blocking group
showed a statistically significant decrease
of over 40% and reached only
8.5±0.9 %ID cm-3 (n = 5) after 72 h
(Student’s t-test P-value < 0.001). These
data confirmed that [89Zr]ZrDFO-PEG3-
Figure 3. Radioactive chromatography showing: (A) radio-ITLC chromatograms of purified (blue)
[
89Zr]ZrDFO-PEG3-azepin-onartuzumab (MetMAbTM). (B) Analytical SEC (PD-10) elution profiles showing
the crude (black) and purified (blue) samples of [89Zr]ZrDFO-PEG3-azepin-onartuzumab. (C) Radioactive
and electronic absorption HPLC chromatograms acquired using SEC gel-filtration showing analysis of the
crude (black) and purified (blue) samples. Note (*) corresponds to a protein aggregate fraction; (D)
corresponds to the photolysed [89Zr]ZrDFO-PEG3-ArN3 byproducts.
azepin-onartuzumab
immunoreactive and displayed specific binding to the c-MET
remained
with compound 9 to form [89Zr]ZrDFO-PEG3-Et-azepin-HSA was
also performed and gave an isolated d.c. RCY of 59.6±3.6% (n =
3). Although both compounds 4 and 9 are water-soluble, the
dissolution of compound 9 in water was improved by adding <1%
DMSO/H2O. The addition of DMSO does not appear to interfere
with the photoactivation or protein-conjugation steps. In contrast
to our previous work with DFO-ArN3, the facile reactions and high
reproducibility of these experiments likely stems from the
enhanced water-solubility of compounds 4 and 9.[7]
receptor in vivo.
The slightly improved solubility profile of compound
4
compared with compound 9 (which contains a more lipophilic
ethylene group) led us to pursue further biological studies using
[89Zr]ZrDFO-PEG3-azepin-onartuzumab.
Chelate
challenge
Figure 4. Coronal and axial PET images taken through the centre of the
tumours showing the spatial distribution of
onartuzumab over time after intravenous administration in mice bearing MKN-
45 tumours. T = tumour, H = heart, L = liver, K = kidney.
experiments demonstrated that [89Zr]ZrDFO-PEG3-azepin-
onartuzumab remained stable with respect to changes in RCP
during incubation with excess diethylenetriamine pentaacetate
(DTPA; >50 mM, pH7, 37 oC, 94 h; supporting Figure S42). After
incubation for 4 days, <16% of the radioactivity was found to
transchelate to DTPA to give [89Zr][Zr(DTPA)]– which is consistent
with the excellent stability of the ZrDFO complex under
physiological conditions.[18] Saturation binding experiments were
also performed by incubating [89Zr]ZrDFO-PEG3-azepin-
onartuzumab with human gastric carcinoma MKN-45 cells
(supporting Figure S43). These cellular studies revealed that
[89Zr]ZrDFO-PEG3-azepin-onartuzumab was immunoreactive and
displayed specific binding to the human hepatocyte growth-factor
receptor (c-MET).[19]
[
89Zr]ZrDFO-PEG3-azepin-
As additional controls, PET images were also recorded at
multiple time points (0 – 16 h post-administration) in mice that
received [89Zr]ZrDFO-PEG3-ArN3 (Figure S46; synthesised in the
dark) or a photolysed sample of [89Zr]ZrDFO-PEG3-azepin that
was irradiated at 395 nm for 15 min. at room temperature in the
absence of protein (Figure S47). VOI analysis (supporting Figure
S48 and Table S1) revealed that [89Zr]ZrDFO-PEG3-ArN3 or the
photolysed
byproducts
have
dramatically
different
pharmacokinetic profiles when compared with [89Zr]ZrDFO-PEG3-
azepin-onartuzumab. [89Zr]ZrDFO-PEG3-ArN3 and photolysed
[89Zr]ZrDFO-PEG3-azepin were cleared rapidly from the
circulation as demonstrated by the absence of 89Zr-activity in the
heart/blood pool. Both samples showed unusually high uptake in
the gall bladder with rapid elimination of 89Zr-activity observed via
both intestinal and renal pathways. By 2 h post-administration,
89Zr-activity in the bladder and kidneys was almost entirely
cleared whereas activity in the abdominal region migrated from
the small to the large intestine. These PET data indicate that
purification of our photoradiolabelled proteins by preparative size-
exclusion small-molecule effectively removed all small-molecule
contaminants since no uptake in the gall bladder or intestines was
Encouraged by the experimental results in vitro, we pursued
small-animal PET imaging and biodistribution experiments in
female athymic nude mice bearing subcutaneous MKN-45 human
xenografts on the right shoulder (Figure 4; maximum intensity
project [MIP] images are given in supporting Figure S44). The
photoradiosynthesis of [89Zr]ZrDFO-PEG3-azepin-onartuzumab
was scaled up and the isolated product was formulated in sterile
PBS (pH7.4). Two groups of animals (n = 5 mice/group) received
between 0.46–0.60 MBq of 89Zr radioactivity. The first group
received a normal dose containing a total mass of 59–72 µg of
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observed in the PET images recorded with [89Zr]ZrDFO-PEG3-
azepin-onartuzumab.
Full experimental details and characterisation data are provided
in the Supporting Information.
Biodistribution studies were performed at 72 h on all animals
after the final imaging time point (Figure 5; supporting Figures
S49–S51 and Table S2). Ex vivo analysis corroborated the PET
data and confirmed the specific tumour uptake and low retention
of [89Zr]ZrDFO-PEG3-azepin-onartuzumab in background tissues.
Acknowledgements
JPH thanks the Swiss National Science Foundation (SNSF
Professorship PP00P2_163683 and PP00P2_190093), the Swiss
Cancer League (Krebsliga Schweiz; KLS-4257-08-2017), and the
University of Zurich (UZH) for financial support. This project has
received funding from the European Union’s Horizon 2020
research and innovation programme / from the European
Research Council under the Grant Agreement No 676904, ERC-
StG-2015, NanoSCAN. We thank all members of the
Radiochemistry and Imaging Science group at UZH.
Keywords: Radiochemistry • photochemistry • positron emission
Figure 5. Bar chart showing ex vivo biodistribution data (%ID g-1) for the uptake
of [89Zr]ZrDFO-PEG3-azepin-onartuzumab in the (normal group, white; blocking
group, blue) in mice bearing MKN-45 tumours.
tomography • antibody conjugates • zirconium-89
[1]
[2]
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2195.
Our experimental studies confirmed that photoradiosynthesis
of 89Zr-radiolabelled proteins with water-soluble DFO-derivatives
produces viable PET radiotracers. However, proteins contain
different nucleophiles that could attack the ketenimine
intermediate produced by photoactivation of ArN3 groups. To
investigate the reaction coordinate for the light-induced activation
of ArN3 and to probe the potential reactivity of different
nucleophiles, we used density functional theory (DFT; supporting
Scheme S2, Table S3 and Figures S52–S55) calculations.
intramolecular rearrangement and bimolecular reactions with
AcO– (modelling Glu / Asp residues) nucleophiles. Computational
results indicated that nucleophilic attack by MeNH2 (modelling Lys
residues) at the ketenimine formed by photoactivation of
compound 4 has a free energy barrier of +51.3 kJ mol-1. In
contrast, a significantly higher barrier of +62.8 kJ mol-1 was
obtained with AcO– as the incoming nucleophile (modelling Glu /
Asp residues). Differences in the kinetic barriers (labelled as TS4
in Table S3) suggest that lysine residues are more likely to react
than Glu/Asp carboxylate groups. Notably, extensive DFT studies
were unable to find appropriate transition states for the attack of
H2O, HO–, MeOH, MeO–, MeSH, MeS– nucleophiles at the
ketenimine intermediates indicating that reactions with cysteine,
threonine or serine residues are unlikely to occur by this
mechanism. Further experiments are required to confirm the
proposed chemoselectivity of light-induced protein-conjugation
using photoactivatable ArN3 compounds.
J. P. Holland, M. Gut, S. Klingler, R. Fay, A. Guillou, Chem. – A Eur.
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In conclusion, accessing water-soluble DFO-derivatives
bearing discrete (PEG)3 linkers is simple and facilitates the use of
photochemistry in the synthesis of 89Zr-mAbs. Further studies are
underway to evaluate the impact of the PEG unit on the metabolic
stability of protein-conjugates in vivo. Compound 4 has been
produced in accordance with GLP regulations which will expedite
the synthesis of clinical-grade 89Zr-mAbs by photoradiosynthesis.
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Experimental Section
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Entry for the Table of Contents
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Amaury Guillou, Daniel F. Earley and
Jason P. Holland*
Page No. – Page No.
Light-activated protein-conjugation
and 89Zr-radiolabelling with water-
soluble desferrioxamine derivatives
Photoradiosynthesis of 89Zr-antibodies: Water-soluble, photoactivatable chelates
derived from desferrioxamine B (DFO) have been synthesised and allow rapid, one-
pot, protein-conjugation and 89Zr-radiolabelling to produce antibody-based
radiotracers for use in positron emission tomography (immuno-PET).
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