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A Review of Approaches to 18F Radiolabelling Affinity Peptide & Proteins
O. Morris1,2, M. Fairclough1,2, J. Grigg3, C. Prenant 1,2 & A. McMahon1,2
1. Wolfson Molecular Imaging Centre, The University of Manchester, UK2. CRUK/EPSRC Imaging Centre in Cambridge & Manchester, The University of Manchester, UK
3. GE Healthcare, Amersham Place, Little Chalfont, Bucks, UK
Abstract
Affinity peptide and protein- (APP) based radiotracers are an increasingly popular class of
radiotracer in positron emission tomography (PET), which was once dominated by the use of
small molecule radiotracers. Radiolabelled monoclonal antibodies (mAbs) are important
examples of APPs yet a preference for smaller APPs, which exhibit fast pharmacokinetics
and permit rapid PET aided diagnosis, has become apparent. 18F exhibits favourable physical
characteristics for APP radiolabelling and has been described as an ideal PET radionuclide.
Notwithstanding, 18F radiolabelling of APP is challenging and this is echoed in the literature
where a number of diverse approaches have been adopted. This review seeks to assess and
compare the approaches taken to 18F APP radiolabelling with the intention of highlighting
trends within this expanding field. Generic themes have emerged in the literature, namely the
use of mild radiolabelling conditions, a preference of site-specific methodologies with an
impetus for short, automated procedures which produce high-yielding [18F]APPs.
Introduction
The field of PET was dominated by the use of radiolabelled small molecules. Over recent
decades, however, radiolabelled affinity peptides & proteins (APPs) have become an
important class of radiotracer including radiolabelled somatostatin analogues and octreotides
[1]. The field of 18F APP radiochemistry is diverse and dynamic yet, despite the extensive
literature reporting 18F APP radiolabelling methods, very few have progressed to clinical-
trials or gained widespread use. A review by Stephanopoulos et al. [2] iterates the sentiments
in this review regarding the usefulness of APPs as molecular vehicles in PET and contains a
valuable flow-diagram that highlights the decision process for choosing an appropriate APP
radiolabelling strategy. The current work seeks to build on this topic and evaluate the various
approaches to APP radiolabelling with 18F whilst highlighting trends in the radiochemistry.
Separated into non-site-specific and site-specific methods, the review will comment on the
cost/benefit analysis of the approaches.
Affinity peptides & proteins in PET
The high selectivity and affinity of biological protein-protein interactions initially motivated
their exploitation in biomedical fields. Key developments in the knowledge of APP antigen-
binding, speed of generation using solid-phase and recombinant techniques, as well as
progressions in affinity selection techniques have strengthened their utility in a number of
areas [1]. APP-based pharmaceuticals represent a rapidly growing market in pharmaceutical
industry, despite some challenging absorption, distribution, metabolism and excretion
(ADME) properties of APPs, such as low cell membrane permeability, short biological half-
life and limited stability [3]. The advantages of their use including exquisitely high target
binding affinity and specificity alongside low toxicity and immunogenicity have
overwhelmed the challenges associated with their use [3].
Naturally, their utility generated attention in the field of PET, where APPs could be
radiolabelled and their high target selectivity and affinity could be exploited in vivo. The
protein-nature of many disease targets and their natural binding partners, including tumour
cell surface receptors and hallmarks of dementia such as tau and amyloid plaque, also
reinforces the use of radiolabelled APPs in the disease diagnoses and highlights their utility in
both peripheral and neurological pathologies [1].
Monoclonal antibodies (mAbs) (~150 kDa) are an important subclass of immunoglobulin; the
extensive use of mAbs is evident in the number of therapeutic antibodies that are available
and used in a clinical setting, with many more in the latter stages of clinical development [4].
The popularity of mAbs is credited to their naturally-evolved target selectivity and specificity
[4]; their ubiquity in other areas of medicine led to mAbs filtering across into the field of
nuclear medicine and literature as early as the 1950s can be found, documenting the use of
antibodies in oncology radiolabelled with iodine-131, a therapeutic radionuclide [5]. Koehler
et al. in 1975 [6] reported an in vitro method for the generation of high volumes of target-
specific antibodies and described the importance of this method in both medical and
industrial fields. The method is recognised as a key development in the use of radiolabelled
mAbs and more recent achievements in recombinant techniques, permitting humanisation and
chimerisation, have strengthened their clinical utility [7]. Encouraging results using
radiolabelled mAbs in clinical oncology trials have been reported, where their use has
permitted diagnosis, patient stratification and identification of the most appropriate treatment
[8].
The rate of uptake from the blood to the biological target is approximately inversely
proportional to the molecular mass of the APP, consequently target accumulation of mAbs is
relatively slow [9]. For this reason, longer-lived radioisotopes such as 89Zr and 64Cu have been
conventionally used to radiolabel mAbs [10, 11].
The slow biodistribution of radiolabelled mAbs can lead to poor target tissue to background
contrast unless the mAb is given sufficient time to accumulate at its biological target [12]. This
is time and labour intensive and the use of longer lived radioisotopes contribute to a high
patient radiation dose. Alternatively, pre-targeting approaches to mAb radiolabelling permit
the use of shorter-lived radioisotopes, however these methods are still relatively new [13].
The slow biodistribution and requirement for longer-lived radioisotopes are considered to be
significant limitations to the use of radiolabelled mAbs. In addition to this, large proteins
have complex tertiary structures with multi-domain features, formed by glycosylation
patterns and disulphide bridges, resulting in thermal and chemical vulnerability that tightly
constrains the radiolabelling method [14, 15]. The issue of fragility is underlined in the literature,
where the use of physiological temperatures and aqueous buffers for radiolabelling
procedures is consistently described [16-18]. These limitations have complicated the translation
of radiolabelled mAbs to routine clinical practice and, as a result, a change in the landscape
of APP-based radiotracers is evolving. This corresponds to a preference for smaller APPs that
exhibit rapid pharmacokinetics and enhanced robustness, whilst retaining high target affinity
and selectivity [4, 19]. Importantly, the rapid pharmacokinetics of small APPs permits the use of
short lived radioisotopes. The diagnostic value of small APPs has not yet been validated; yet
their exploitation in diagnostic PET has the potential to be a powerful tool helping to guide
patient stratification and identify those who would benefit from mAb-based therapy.
Radionuclide Nuclear equation Max. Positron energy (MeV)
Branching ratio
Half-life
Short lived
68Ga 70Zn(p,n)68Ga
Germanium-68 generator:68Ge/68Ga
1.8 0.81 68.3 mins
18F 18O(p,n)18F 0.6 0.97 109.80 mins
44Sc Titanium-44 generator:44Ti/44Sc
0.6 0.94 3.9 hrs
Long lived
64Cu 64Ni(p,n)64Cu 0.6 0.18 12.7 hrs89Zr 89Y(p,n)89Zr 0.8 0.23 3.30 days
124I 124Te(p,n)124I 1.5 0.23 4.2 daysTable 1) Commonly used radionuclides in APP radiolabelling [20, 21]
Table 1 documents the radioisotopes that have been utilised in APP radiolabelling; the Table
has been separated into short and long lived radionuclides. The majority of the radionuclides
listed are most commonly produced using a cyclotron however 68Ga is normally generator-
produced, which is considered a significant advantage. The wide range of radionuclides
available is highly advantageous in order to match the diverse biological half-lives of
different APPs.
Somatostatin analogues were amongst the first small APPs to be radiolabelled and approved
for clinical use in oncology, in the 1980s [1]. Early APP-based radiotracers used in PET,
including somatostatin analogues, typically made use of radioisotopes such as 68Ga [1]. Use of
18F radiolabelled APP in clinical trials would not be documented for another 20 years and this
has been ascribed to challenges associated with the radiochemistry [1].
Given the prevalence of mAbs, unsurprisingly, the utility of antibody fragments, such as Fab
(antigen-binding fragment, Mr ~50 kDa) and scFv (single-chain variable fragment, Mr ~25
kDa), as APP-based radiotracers have been investigated. However the target affinity of the
parent antibodies was rarely retained and issues of poor solubility and aggregation were
apparent [7]. The loss of affinity observed in mAb fragments was also observed in a anti-
HER2 Fab fragment, derived from trastuzumab, possibly related to the contribution of mAb
bivalency to antigen affinity [22].
Nanobodies (Mr ~15 kDa) are fragments of camelid heavy chain only antibodies and are
alternative immunoglobulin-based APPs. They exhibit high target affinity and selectivity
together with rapid biodistribution, whilst avoiding the issues of poor solubility and
aggregation observed in non-camelid derived antibody fragments [23]. Since the nanobody is
camelid-derived, it is necessary to humanise the APP for use in therapeutic applications [24].
Vaneycken et al.[25] described the use of an anti-HER2 (human epidermal growth factor
receptor 2) nanobody; the ease of nanobody production as well as improved affinity selection
techniques permitted efficient deduction of lead nanobodies that were able to identify HER2
expressing lesions and also regions of metastases [25]. This is indicative of the work that has
underlined the potential of nanobodies and they have thus been labelled the ‘magic bullet’ of
molecular imaging and are predicted to have an important future role in the field [23].
Advances in APP engineering have facilitated the arrival of non-immunoglobulin based APP
[4]. Designed using combinatorial protein libraries, non-immunoglobulin based APPs have
been engineered to possess characteristics, such as enhanced solubility or stability that
challenge some of the limitations of immunoglobulin-based APPs [4, 14]. The affibody
molecule is a promising example of a non-immunoglobulin APP and originated from
staphylococcal protein A (SPA). The affibody is derived from the B-domain: one of the five
domains that make up SPA. Its potential as a radiotracer was recognised based on its high
stability and aqueous solubility. Key mutations were subsequently made that afforded
enhanced stability of the newly named Z-domain; it is upon this protein-scaffold backbone
that more than 60 target-specific affibodies have been constructed [14]. Literature cited in this
review, describing the use of affibodies in oncology, unanimously reported successful and
rapid tumour targeting alongside fast clearance from non-target tissue [26-28]. Indeed, Kramer-
Marek et al. [27] reported tumour uptake as early as 20 minutes post-injection, thus stressing
the potential of the APP in rapid PET-guided diagnosis. It should, however, be noted that
tumour accumulation at tumour points exhibits lower image contrast due to accumulation of
the radiotracer in excretory organs.
Great variety in the characteristics of APPs used in PET exists: from large immunoglobulin-
based mAbs to short-polypeptide chains such as anti-αvβ3 RGD motif-containing APPs used
as angiogenesis radiotracers. Further tailoring of APP characteristics is reported and
conjugation of biomolecules, such as poly-histidine and albumin, [3, 28] or inert polymers, such
polyethylene-glycol (PEG), used to manipulate the in vivo behaviour of the APP. Kalia and
Raines [29] reported the enhanced water-solubility, reduced immunogenicity and enhanced
biological half-life of PEGylated APPs.
The characteristics of smaller APPs described above are profitable attributes in PET; a key
advantage is their compatibility with short lived radioisotopes, in particular 18F which has
been described as an ideal PET radionuclide [1, 30]. However, as previously discussed, APP
radiolabelling with 18F can be challenging and this was outlined in a review article by Richter
et al. [1] where the incompatibility of direct fluorination and lack of automation of complex
multi-step methods was said to have impeded the translation of 18F radiolabelled APP to
clinical practice [1]. The review stresses the importance of process automation in aiding
conformance with GMP but at the same time highlighted the difficulties associated with
automation of multi-step approaches required for APP radiolabelling with 18F [1]. The
importance of process automation is reflected in the successes of 68Ga radiolabelled APPs,
where all-in-one, self-shielded, fully-automated radiosynthesisers are used to prepare the
radiotracers, stimulating an exponential growth in their use [31, 32]. Gallium-68 radiochemistry
lends itself more favourably to process automation due to one-step chelation methods, unlike
the current multi-step approaches to 18F radiolabelling. However, the advantageous physical
characteristics of 18F (Table 1) make the automation of 18F radiolabelling approaches, a
profitable endeavour [20, 33].
[18F]Fluoride
The biological half-life of a small or intermediately sized APP is complemented by a t1/2
=109.8 min, 18F radiolabel (≤ 60 kDa).
The introduction of fluorine into a molecule, at the outset, initiated much debate given the
limited natural abundance of fluorine in biological molecules [30]. It was argued that its
addition may alter the physical characteristics of any tracer in view of the electronegative
character of fluoride. It has, however, been found that its electronic properties can lead to
improved pharmacological characteristics and the strength of the C-F bond (112 kcal/mol), in
comparison to the C-H bond (98 kcal/mol), enhances its metabolic stability [30]. The small
size of fluorine (van der Waals radius 1.47 Å) bears similarity to hydrogen (1.2 Å) and
electronic nature is comparable to a hydroxyl group [30]. This, therefore, provides scope for
their replacement in a whole host of molecules, where fluoride can be described as a
bioisostere [34].
The highly favourable decay characteristics of 18F permit more complex radiochemistry and
longer imaging studies [1, 30]. This is of particular importance when radiolabelling APPs,
which frequently necessitates multi-step radiosynthetic strategies as direct fluorination of the
APP is often not possible due to the necessary harsher radiolabelling conditions and multiple
fluorination sites of the APP. The half-life also enables transportation to different clinical
locations; this is important as it permits off-site radiotracer synthesis.
Non-site-specific radiolabelling
Prosthetic groups are small molecules that conjugate to the APP under mild conditions, they
are 18F-fluorinated before conjugation to the APP thereby avoiding issues arising from the
harsher reaction conditions often required for direct fluorinations [35, 36].
Non-site-specific radiolabelling is commonly achieved using carbonyl-containing 18F labelled
prosthetic groups which can target the N-terminal or ε-amine belonging to lysine residues and
thus making use of the inherent nucleophilic sites of the APP. These nucleophilic sites have
been identified in Figure 1, which provides an example APP and where the N-terminus and
lysine ε-amines have been marked with a dashed ring.
Table 2 provides a summary of commonly used 18F prosthetic groups, the Table summarises
key features of prosthetic groups radiosynthesis allowing comparisons to be drawn. The
authors refer readers to a review by Schirrmacher et al. [37] for a more detailed review of small
prosthetic groups used in 18F radiochemistry.
Reductive alkylation, using amine-reactive prosthetic groups, is a commonly employed
strategy. It involves the formation of an imine (or Schiff base) which is subsequently reduced
with an appropriate reducing agent. [18F]fluorobenzaldehyde (FBA) is a popular example of
amine-reactive 18F prosthetic groups used in reductive alkylation [38]. More recently
[18F]fluoroacetaldehyde ([18F]FA) and [18F]fluoropyridinecarboxaldehyde ([18F]FPCA) were
reported, as water-soluble prosthetic group alternatives [39-42].
Figure 2 gives the mechanism of reductive alkylation in both acidic and basic conditions.
Reductive alkylation proceeds in both pH ranges but acidic conditions appear to be superior
Figure 1) Example of an APP where N-terminal or lysine ε-amines have been identified with dashed rings
[38, 39, 41]. The instability of the imine and production of H2O or hydroxyl group, which is
thermodynamically disfavoured in aqueous media, is overcome upon addition of an
appropriate reducing agent. NaCNBH3 is often used as a reducing agent on account of its mild
reducing properties, stability in water and compatibility with the pH range required for imine
formation [43].
Non-site-specific radiolabelling: [18F]Fluorobenzaldehyde ([18F]FBA)
[18F]FBA is a popular 18F prosthetic group and is generally synthesised by direct fluorination
of the commercially-available 4-formyl-N-N-N-trimethylammonium-triflate, this can be seen
in Figure 3.
The presence of an ortho/para (o/p) electron withdrawing group and powerful leaving group
permits nucleophilic aromatic substitution of the precursor, which without these elements
A
B
Figure 2) Reaction mechanism showing reductive alkylation in A) acidic and B) basic media
would not be susceptible to nucleophilic attack. Many of the arene-based 18F prosthetic
groups make use of a trimethylammonium precursor owing to the solubility of ammonium
salts, ease of separation of the 18F-labelled prosthetic group from the ammonium salt
precursor and stability of the trimethylamine leaving group. [18F]Fluoromethane has been
reported as a possible side-product, however its generation is disfavoured when substituents
with large Hammett constants are present on the arene in o/p positions [44].
Speranza et al. [45] report the automation of [18F]FBA radiosynthesis using a GE TRACERlab
FX-FN. Automated radiosynthesis of [18F]FBA was completed in 45 minutes and
radiochemical yields (RCY) of 90 % decay-corrected (d.c.) were reported [45]. The method
made use of SPE purification, rather than HPLC, for a number of reasons including ease of
automation, time-saving aspects and reproducibility [45]. Importantly the group report
comparable [18F]FBA recovery between the two methods and [18F]FBA radiochemical purity
(RCP) measurements of > 99 % were described [45]. The group insist on the importance of
automation in order to provide a reliable tool to produce large quantities of [18F]FBA to
radiolabel APPs for use in routine clinic [45].
Apana et al. [38] report the radiolabelling of an APP via reductive alkylation with [18F]FBA.
The APP, anginex, is a 33-residue peptide with anti-angiogenic properties and has been
reported to sensitise tumours to radiotherapy and inhibit tumour growth; [18F]anginex was
Figure 3) Reaction scheme to show [18F]FBA radiosynthesis
evaluated as an anti-angiogenic radiotracer in pre-clinical models [38]. [18F]Anginex yields of
76% were reported alongside molar activity measurements of 14.4 GBq/μmol [38].
[18F]Anginex was evaluated in tumour bearing mice and, although high liver uptake was
observed, tumour uptake of [18F]anginex was 16 % higher than the maximal liver uptake and
accounted for 30 % of the total body distribution. The authors concluded that [18F]anginex
had retained target selectivity, achieved high target to background ratios and is thus a
potentially useful antiangiogenic diagnostic radiotracer [38]. The yield and molar activity of
[18F]anginex were significantly improved by removal of polytetrafluoroethylene (PTFE)
which is a material used in tubing and as a surface coating for various components of
radiochemistry synthesisers. PFTE derived stable fluoride has a carrier effect and its removal
yielded ~25 % improvement in [18F]anginex yields [38, 46]. PTFE removal from the
radiosynthesis of [18F]FBA proved to be a profitable modification; the average mass of carrier
fluoride was calculated to be between 400-800 nmol, which was reduced to 25 nmol after its
elimination [38].
Non-site-specific radiolabelling: [18F]Fluoroacetaldehyde ([18F]FA)
[18F]FA is a small, water-soluble prosthetic group reported by Prenant et al. [42] and used to
radiolabel a recombinant human interleukin-1 receptor antagonist (rhIL1RA, 17.5 kDa) via
reductive alkylation [41]. A remotely-controlled semi-automated configuration was used for
[18F]FA radiosynthesis and subsequent rhIL1RA radiolabelling; the method achieved [18F]FA
yields of 34 ± 3 % (d.c., n = 3) and overall [18F]rhIL1RA RCY of 11.4 ± 4 % (d.c., n = 17) in
100 minutes [41, 42]. The non-site-specificity of reductive alkylation is a drawback of this
radiolabelling approach [41]. Nonetheless, ex-vivo binding assays revealed retention of
[18F]rhIL1RA binding specificity and it was thought that the small size of [18F]FA was, in
part, responsible [41].
Full process automation of [18F]FA radiosynthesis and subsequent APP radiolabelling was
later reported, where rhIL1RA was radiolabelled via reductive alkylation, in order to make
direct comparisons between the semi- and fully- automated procedures [39]. The automation
permitted larger starting quantities of [18F]fluoride which contributed to larger quantities of
[18F]FA, despite some observed loss in [18F]FA yields (~25 % loss) [39]. Overall [18F]rhIL1RA
yields of 5 ± 3 % (d.c., n = 5) and apparent molar activity measurements of 13.5 GBq/μmol
were reported. [18F]rhIL1RA apparent molar activity measurements reported using the fully-
automated radiosynthesis [39] are significantly higher than measurements described by Prenant
et al. [41] (0.9 – 5.0 GBq/μmol) despite the inability of both methods to separate the product
from unlabelled rhIL1RA. This was largely attributed to the ability to use higher starting
levels (15-fold higher) of radioactivity [39, 41, 42].
Non-site-specific radiolabelling: [18F]Fluoropyridinecarboxaldehyde ([18F]FPCA)
The ease with which pyridine can be fluorinated at both the C2 and C4 positions, attributable
to the presence of the nitrogen ‘electron-sink’, has led to an increasing number of pyridine-
based prosthetic groups [40, 47, 48]. One such example is [18F]FPCA, a water-soluble 18F
prosthetic group developed by Morris et al. [40]. Its radiosynthesis has been fully-automated
using a customised GE Tracerlab FX-FN with [18F]FPCA RCY of 28 ± 2 % (d.c., n = 10)
reported [40]. Morris et al. [40] describe the replaced purification of [18F]FPCA with SPE for
time-saving reasons and to simplify automation and improve reproducibility [40], as was
reported by Speranza et al. [45] for the purification [18F]FBA. The automation of [18F]FPCA
was thereby extended to include the subsequent radiolabelling of an APP.
Non-site-specific radiolabelling: N-Succinimidyl-4-[18F]fluorobenzoate ([18F]SFB)
[18F]SFB, similarly to [18F]FPCA, radiolabels APPs through conjugation with the inherent
nucleophilic sites of the APP. Unlike the other amine-reactive prosthetic groups described,
[18F]SFB has the advantage that it does not require a reducing agent. Alkylation of APPs with
[18F]SFB is commonly carried out between pH 8 and 9 to deprotonate APP amine groups [49].
Automation of [18F]SFB radiosynthesis has been described by Zijlstra et al. [49]; in brief:
[18F]SFB was produced by first synthesising ethyl 4-[18F]fluorobenzoate through fluorination
of a trimethylammonium-precursor [49]. 4-[18F]Fluorobenzoic acid was then produced by
base hydrolysis of the fluorinated ester [49]. In the final step, a succinimide-containing synthon
(O-[N-succinimidyl]-N,N,N,N-tetramethyluronium tetrafluoroborate) (TSTU) was incubated
with 4-[18F]fluorobenzoic acid to yield [18F]SFB in yields of ≤ 35 % (d.c.) and RCP of 98 %
[49].
High RCP values were reported despite having to reuse SPE cartridges owing to limitations
of the automated radiochemistry module [49]. [18F]SFB was used to radiolabel the apoptosis
radiotracer, annexin-V (30 kDa), and RCY of ≤ 20 % (d.c.) were reported [49]. In vitro
experiments using apoptotic cells verified the selectivity of [18F]annexin-V and cell uptake of
the radiotracer was dose dependent in experiments where varying concentrations of
unradiolabelled annexin-V were used, thereby verifying retention of the biological activity of
[18F]annexin-V [49]. Conjugation of [18F]SFB to an APP, such as annexin-V, can be seen in
Figure 4.
Figure 4) Reaction scheme to show APP radiolabelling with [18F]SFB
Non-site-specific radiolabelling: Evaluation of non-site-specific methods
Thus far, this review has described the use of 18F prosthetic groups to radiolabel APPs via a
non-site-specific pathway. Apana et al. [38] sought to establish the preferential site of non-site-
specific radiolabelling and their results indicated that the singly-substituted labelled APP,
also the major product, was N-terminally labelled suggesting this as the preferred site and the
likely site of [18F]FBA radiolabelling [38]. This was a useful undertaking in order to understand
the impact of labelling on the interaction of the APP with its binding site.
Table 2 summarises the radiosyntheses of the 18F-prosthetic groups discussed above. The
literature shows widespread use of [18F]FBA and [18F]SFB: they are well-established methods
and can achieve attractive RCY (≤ 90% d.c.) [45]. The commercial availability of the
[18F]FBA precursor augments its popularity and the additional advantage of not having to use
a reducing agents adds to the appeal of [18F]SFB. The popularity of the [18F]FBA and
[18F]SFB prosthetic reagents is reflected in the commercial availability of bespoke
molecularly imprinted polymer (MIP) SPE for their purification following nucleophilic
aromatic substitution of ammonium-containing precursors [40, 50].
Process automation has been reported for all the above prosthetic groups with radiosyntheses
not exceeding 45 minutes. This conforms to the generally held view that radiotracer
synthesis, purification, formulation and analysis should not exceed two half-lives of the PET
radionuclide [1]. The automated radiosynthesis of all prosthetic groups uses SPE purification
[40, 45], with the exception of [18F]fluoroacetaldehyde which uniquely uses distillation
purification [39, 41, 42]. There are, however, significant challenges and considerations to process
automation. Losses in yield are reported [39], as are considerations relating to the component
materials of imposed by automated radiosynthesisers: limitations in the number of
compatible valves and ports that are available can place restrictions on process automation
[49]. Additionally, the use of radiochemistry synthesisers can increase the exposure of 18F to
stable fluoride owing to the multiple reactors and tubing used to transfer a radiolabelling
reaction mixture in a multi-step radiosynthesis [38, 46].
The ability to use larger starting levels of 18F and the consistency and reliability that process
automation can bring means that its challenges are outweighed by its advantages. This
importance of process automation is reflected in the literature, where there is a trend towards
automation of prosthetic group synthesis.
Alongside the encouraging movement towards automated radiosynthesis is the advent of
water-soluble 18F prosthetic groups, such as [18F]FA and [18F]FPCA. The water-solubility of
these prosthetic groups allows milder radiolabelling conditions; radiolabelling of APP with
[18F]FBA, for example, is often carried out in an organic solvent, such as methanol [38].
Non-site-specific radiolabelling: A summary
Radiosynthesis simplification, process automation and compatibility of a radiolabelling
strategy with mild reaction conditions are key characteristics that have emerged regarding
methods of non-site-specific radiolabelling with 18F. The developments are indicative of the
measures being taken to align with the generic features that are required of a versatile
platform for 18F radiolabelling and would facilitate the translation of [18F]APPs to GMP.
The ability to radiolabel the native APP using its naturally-occurring nucleophilic sites,
without pre-modification, is the foremost advantage of this approach [38, 41, 49]. Despite this,
there has been significant interest in the development of site-specific APP radiolabelling
methods. The appeal of these methods is not only characterised by site-selectivity but also the
faster rates of reaction that often result [19, 29, 51].
18F prosthetic
group
RCY d.c.(approx.
%)
Purification No. of steps
Automation Radiosynthesis time (min)
APP radiolabelling
temp (°C)
Comments Refs
SFB 35 SPEMIP SPE
3 Yes 45 (estimated from data in the paper)
30 1. No reducing agent2. Availability of bespoke MIP SPE cartridge
[49, 50]
FBA 90 C18 SPEMIP SPE
1 Yes 45 60 1. High RCY2. Availability of bespoke MIP SPE cartridge3. Commercial availability of precursor
[38, 45]
FA 26 Distillation 2 Yes 45 40 1. No SPE or HPLC purification2. Water-soluble3. Full process automation3. Commercial availability of precursor
[39, 41, 42]
FPCA 28 MIP SPE 1 Yes 45 50 1. Water-soluble2. Full process automation3. Facile synthesis of precursor
[40, 52]
Table 2) Summary of selected carbonyl-containing 18F prosthetic groups used in non-site-specific APP radiolabelling
18F site-specific radiolabelling
Site-specific APP radiolabelling addresses the concerns of possible radiolabel conjugation
at or near the antigen binding site [53]. By virtue of their chemistry, these methods can offer
faster rates of reaction and thus are more high yielding approaches to APP radiolabelling,
given that synthesis is time-limited.
By way of comparison, Table 3 documents the rates that have been reported in the literature
for non-site-specific and site-specific methods of bioconjugation. Consequently site-specific
approaches have become preferable means by which to radiolabel APPs and safeguard
biological activity [1, 19].
Approach Method Rate (M-1s-1) Refs
Non-site-specific Reductive alkylation
(imine formation)
k1 = 10-1 [54]
Site-specific Oxime & Hydrazone
Huisgen 1,3-dipolar cycloaddition
Inverse electron demand Diels-Alder
k = 10-3 – 1.0
k = 2.3
k = 104 - 105
[51]
[55]
[56]
Table 3: A comparison of rates between non-site-specific and site-specific bioconjugation methods
Rate constants for site-specific methods have been included, however overall rate constants
of reductive alkylation are not readily available in the literature, for this reason, k1 values of
imine formation have been included [54].
Site-specific approaches to APP radiolabelling include 18F acceptor chemistries and click-
chemistry methods; the methods and their functional groups have been summarised in Table
4.
Site specific labelling method
Functional group on APP 18F prosthetic group Radiolabelled APP Refs
([18F]Al)2+
[18F]AlCl3
[57-59]
Isotopic exchange
[58, 60,
61]
Oxime bond formation
[15, 40,
52, 62]
Hydrazone formation
[51, 55]
Thiol-Michael addition
[53]
CuAAC [63, 64]
SPAAC [65]
IEDDA [66]
Table 4) Summary of site-specific methods and the functional groups required for the chemistry
Site-specific radiolabelling: 18F acceptor chemistry
Richter et al.[1] described 18F acceptor synthons as attractive alternatives to the use of
prosthetic groups. Two types of 18F acceptor chemistry used to radiolabel APPs will be
discussed: ([18F]AlF)2+ complexation and isotopic exchange. The authors refer readers to a
recent review of recent 18F radiochemistry advances, which includes 18F acceptor approaches
[58] and also a review of ([18F]AlF)2+ coordination chemistries by Kumar et al. [59].
Table 5 summarises the key features of site-specific 18F APP radiolabelling approaches.
Site-specific radiolabelling: ([18F]AlF)2+
([18F]AlF)2+ has been used to radiolabel a number of APPs in a one-pot, high yielding
reaction characterised by Al chelation followed by the formation of a strong Al-F bond, first
reported by McBride et al. [58, 67, 68].
Da Pieve et al.[57] used the ([18F]AlF)2+ method to radiolabel a HER3-specific affibody with a
view to assessing its usefulness as radiotracer in oncology; the method mirrored conditions
reported by Glaser et al. [69]. The affibody was derivatised with 1,4,7-triazacyclononane-1,4,7-
triacetic acid (NOTA) and incubated with AlCl3. Final incubation of the complex with 18F
gave rise to yields of 38.8 ± 5.8 % non-decay-corrected (n.d.c) and molar activity
measurements of 6.0 – 11.9 GBq/μmol were reported [57]. A reaction by-product was
observed, presumed to be an APP degradation product as a result of the radiolabelling
conditions [57, 69], as a result Da Pieve et al. [57] report the requirement for both RP-HPLC and
SPE purification to ensure a final RCP of > 98 %. In vitro analysis using HER3 expressing
cells confirmed retention of [18F]Al-F-NOTA-affibody target specificity and affinity,
indicated by preservation of the affibody’s low nanomolar binding constant [57]. These results
were echoed by the in vivo analysis, using HER3 tumour-bearing mice, where successful
tumour targeting and fast blood clearance of Al[18F]NOTA-affibody was observed [57].
A recent publication from the group describes full process automation of the approach,
having been encouraged by the results of the method and the recognised requirement for
automated processes in GMP production [70]. High temperatures and a water-miscible solvent
were, again, used in the fully-automated procedure which resulted in efficient radiolabelling
of two model APPs which required single SPE-purification of the final radiolabelled product
[70]. A clear advantage of the approach is the simplicity of the single-vial method and ability to
use commercially available aqueous [18F]fluoride/H2O. Application of the fully-automated
approach to a NOTA-derivatised octreotide, chosen for its commercial availability, achieved
RCY of >45 % and RCP measurements of >98 % within 26 minutes [70].
In a similar vein, McBride et al. [67] report the development of single-vial lyophilized kits as a
method to simply and rapidly label APPs with ([18F]AlF)2+ and although not automated, the
group demonstrate the feasibility of translating the simplified single-vial method into a
clinical setting, owing to the ease with which freeze-dried APPs can be radiolabelled and
purified using SPE [67].
The potential of the ([18F]AlF)2+ radiolabelling method is clear and encouraging achievements
in process automation will surely stimulate the appeal of the approach and an additional
advantage to the technique is the ability to exploit the myriad NOTA-derivatised APPs that
are commercially available and used as precursors for 68Ga radiolabelling. However, the
electronic and/or steric influences of a chelate and metal ion complex on the in vivo
performance of an APP should be considered. Strand et al.[26] report the difference in tumour
accumulation of chelate-derivatised affibodies, which was in agreement with previous work
reported by the group that demonstrated a significant influence of the radiolabelling method
on the in vivo behaviour of affibodies. It is also important to consider the toxicity of Al and
that exposure limits to aluminium, ranging from 5-15 mg/m3, have been established in Europe
and the US [71].
Site-specific radiolabelling: Isotopic exchange
Silicon-based isotopic exchange agents are attractive alternatives to 18F radiolabelled
prosthetic groups owing to the higher rate of fluorination at silicon relative to that at carbon
and greater stability of the Si-F bond, compared to that of C-F (142.3 and 116.4 kcal/mol
respectively) [72, 73]
Rosenthal et al. [72] report the development of a silicon-fluoride acceptor (SiFA),
[18F]fluorotrimethylsilane ([18F]FTMS), by nucleophilic substitution of a
chlorotrimethylsilane with 18F, achieving RCY of 80 % (d.c.). However, the in vivo instability
of the Si-F limited its applicability but encouraged developmental of isotopic exchange
agents with bulky alkoxy substituents which exhibit enhanced in vivo stability, whilst
retaining high RCY [60, 72]. The stabilising effect of bulky alkoxy substituents is in agreement
with the work of Brinker et al. [74] where steric factors were shown to have the greatest impact
on the hydrolytic stability of organosilanes.
Schirrmacher et al. [60] reported the development of an isotopic exchange agent, [18F]fluorodi-
tert-butylphenylfluorosilane, described as a highly efficient and hydrolytically stable SiFA.
The group reported a RCY of 80-95 % and molar activity between 194-230 GBq/μmol; the
competitive RCY and molar activity measurements were ascribed to the rapid rates of isotope
exchange [60].
The group identified the requirement for a simplified and, if possible, single-vial method to
yield [18F]APPs to act as an attractive alternative to multi-step radiosyntheses [60]. In order to
achieve this, they bonded the SiFA to a benzaldehyde-derivative for site-specific conjugation
with an aminooxy-functionalised APP; RCY of 60 ± 5 % were attained alongside RCP
measurements of > 98 % [60]. The group describe the APP radiolabelling conditions as the
mildest documented to date. This is true of the pH and room temperature conditions used, yet
the use of 100 % acetonitrile as the reaction medium could denature some APPs. The
approach was also applied to non-azeotropically dried [18F]fluoride; comparable yields to
those attained using azeotropically-dried 18F were only attained at higher temperature (95
°C), yet highlights the translation of the method to a single-vial approach.
The method was further developed by Niedermoser et al. [75] where somatostatin analogues
bearing a SiFA motif were radiolabelled with dried 18F achieving RCY of 50 ± 6 % (d.c.)
within 25 minutes. The radiolabelled APPs was SPE purified and did not require HPLC
purification which contributed significantly to the short overall radiosynthesis time.
Encouragingly, the [18F]APPs exhibited higher tumour accumulation and, although, signal to
noise ratios were lower than those achieved using a 68Ga analogue the single-vial approach
was described as being advantageous for routine clinical use [75].
Site-specific radiolabelling: Click-chemistry
Click-chemistry is characterised by high-yielding and chemoselective reactions that are
compatible with benign solvents and do not generate offensive by-products; see to the
comprehensive list of criteria provided by Sharpless et al. [76]. Click-chemistry reactions were
originally divided into two types of reactions: ring-opening and cycloaddition reactions [76],
however a number of other reactions are now included in the definition, which will be
discussed.
The hallmarks of click-chemistry reactions make clear its appeal in radiochemistry and in
APP radiolabelling terms allows the site-specific conjugation of a radiolabelled synthon,
containing a reactive group, to a complementary reactive group on the APP.
A number of click-chemistry approaches have been reported for APP radiolabelling with 18F;
including oxime bond and hydrazone formation, the products of the reaction between amine-
reactive prosthetic groups and aminooxy- and hydrazide- functional groups respectively [15, 51,
62]. The efficiency of hydrazone and oxime bond formation can be explained by the alpha
effect, where an adjacent lone-pair enhances the nucleophilicity of the amine group by raising
the energy of the highest occupied molecular orbital (HOMO) and stabilisation of the
transition state [77].
Another click-chemistry approach is the Michael-addition of thiol-containing APPs with
maleimide containing prosthetic groups [53]; thiol-Michael additions are the only click-
chemistry approaches for APP radiolabelling using a naturally-occurring residue: thiol-
containing cysteine.
An additional, and very important, category of click-chemistry approaches are cycloadditions
in which new σ-bonds are formed at the expense of π-bonds to form a cyclic compound [78];
examples include copper catalysed and strain-promoted azide-alkyne cycloadditions and
inverse electron demand Diels-Alder cycloadditions [13, 64, 65].
Site-specific radiolabelling: Oxime bond formation
Aminooxyacetyl groups, or aminooxy (Aoa), are important functionalities in site-specific
conjugation to APPs and have been used in a number of fields, including the synthesis of
complex proteins from peptide fragments [62]. As such, oxime bond formation for the purpose
of APP radiolabelling has become an increasingly popular approach; moreover the oxime
bond has been described as hydrolytically stable thereby highlighting its suitability for in vivo
studies [51, 55]. Figure 6 shows the conjugation of an Aoa-functionalised APP with a carbonyl-
bearing prosthetic group, such as [18F]FBA.
Poethko et al. [62] sought to verify the site-specificity of the conjugation and for this [18F]FBA
was incubated with a range of amino-acids both in the presence and absence of aminooxy-
acetic acid. Results indicated that, in the presence of aminooxy-acetic acid, oxime bond
formation accounted for 93 % of the radiolabelled product; [18F]FBA showed some reactivity
with cysteine but, nevertheless, site-specificity was high [62]. [18F]FBA was subsequently used
to radiolabel Aoa-derivatised model APPs: an RGD peptide and a somatostatin analogue,
with overall RCY of ≤ 40 [62].
The highest yields of radiolabelled APPs (60-80 %), were reported at a pH of 2-3 [62]. The
radiolabelled APPs were evaluated in vivo to confirm hydrolytic stability of the oxime bond
but results also showed good tumour-to-background ratios [62].
Namavari et al. [15] adopted the same approach to radiolabel an Aoa-derivatised dimeric
HER2 affibody with [18F]FBA to assess its use as a radiotracer for HER2 detection: HER2 is
another member of the human epidermal growth factor family and is also associated with a
more aggressive tumour phenotype and poorer patient prognosis [22]. [18F]HER2 RCY of 27 ±
3 % (d.c.) were reported [15]; the lower yields of Aoa-functionalised APP, compared to those
described by Poethko et al. [62], were ascribed to the use of a higher pH (pH 4). Namavari et
al. [15] had wanted to assess the applicability of the method to APPs with more complex
proteins that might be susceptible to the use of low pH, especially in combination with
elevated temperatures, such as 60 °C [15]. The group report the success with which the APP
was radiolabelled via oxime bond formation and the high in vivo stability of the oxime bond
[15].
Low pH conditions are widely used in conjunction with oxime-bond formation [40, 62], this
reflects results reported by Dirksen et al. [79] where slow kinetics of formation are observed at
neutral pH. Further work by Dirksen et al. [79] demonstrated the utility of aniline to catalyse
oxime-bond formation. Aniline catalysis was used in the work by Glaser et al. [69], who
radiolabelled an Aoa-functionalised APP with yields of 13 ± 3 % (n.d.c, n=5) and where
aniline catalysed oxime-formation even at neutral pH. Figure 5 shows the mechanism of
aniline catalysis on oxime bond formation.
Full process automation of APP radiolabelling via oxime bond formation has recently been
reported [40] with a 4-fold reduction in overall yields of radiolabelled APP ascribed to the
challenges of translating the radiosynthesis from a semi-manual to an automated approach [40,
52]. This was recompensed by the enhanced consistency, reliability and process robustness
afforded by the automated radiosynthesisers, alongside the ability to use larger starting levels
of 18F [40].
Site-specific radiolabelling: Hydrazone formation
Hydrazone formation is an additional chemoselective approach to site-specific APP
radiolabelling [51]. Hydrazide-derivatised APPs can be radiolabelled using well-established
amine-reaction 18F prosthetic groups. Hydrazone formation has, arguably, had limited utility
in APP radiolabelling and this can be ascribed to the limited stability of the hydrazone
product. Oxime bonds possess greater hydrolytic stability compared to hydrazones [51]; this is
supported by the work of Kalia et al. [55] which documents the hydrolytic half-lives of an
oxime (25 d) and a corresponding hydrazone (2 h).
The rates of hydrazone and oxime bond formation have been compared [51]; Keq of oxime
formation were reported to be two orders of magnitude larger than those for hydrazone
formation despite a 20-fold smaller k1, attributable to the instability of the hydrazone product
Figure 5) Reaction scheme to show the catalysis of oxime bond formation with aniline
[51]. The work provides evidence for the limited utility of hydrazone formation in APP
radiolabelling; despite encouraging rates of hydrazone formation, the stability of the
radiolabelled product would translate to low radiolabelled APP yields and does not align with
some of the generic features of 18F APP radiolabelling, where fast rates of reaction coupled
with high yields as well as in vivo stability are important in order to motivate the translation
of 18F radiolabelled APP into the clinic. Figure 6 shows the conjugation of an hydrazide-
functionalised APP with a carbonyl-bearing prosthetic group, such as [18F]FBA
Site-specific radiolabelling: Thiol-Michael addition
N-[2-(4-18fluorobenzamido)ethyl]maleimide ([18F]FBEM) is a maleimide-containing 18F
prosthetic group, which site-specifically radiolabels thiol-containing APPs. The relative ease
with which thiol-groups can be incorporated in APPs, in comparison to Aoa-derivatives,
drove the developments in the area [15]. As seen in Figure 7, [18F]FBEM can be synthesised
through addition of N-(2-aminoethyl)maleimide to [18F]SFB in the presence of a non-
nucleophilic base, typically N,N-diisopropylethylamine (DIPEA).
B
A
Figure 6) Reaction scheme to show oxime bond (A) and hydrazone formation (B)
Conjugation of [18F]FBEM to an N-terminal cysteine proceeds via a Michael addition, the
mechanism of which can be seen in Figure 8. Radiolabelling mixtures containing thiol-
bearing APPs require a reducing agent (such as tris(2-carboxyethyl)phosphine (TCEP) ) to
prevent disulphide bridge formation between two cysteine residues.
Cai et al. [53] reported using [18F]FBEM to site-specifically radiolabel a thiolated RGD peptide
with yields of 80 % (n.d.c.) and molar activity measurements of 100-150 GBq/μmol, yet the
overall RCY was impacted by the low [18F]FBEM RCY of 5 ± 2 % (n.d.c.). In vitro assays in
cells verified the binding specificity of [18F]RGD and were able to inhibit binding of the avβ3
antagonist echistatin. In vivo assessment showed specific accumulation of the radiotracer
alongside good metabolic stability [53].
Cai et al. [53] described the method as a high-yielding approach to radiolabel thiol-containing
APPs with 18F with high molar activity and the mild reaction conditions highlight the
versatility of the technique [53]. The relevance of this approach for routine production was
unclear, due to the time-consuming multistep radiosynthesis (150 ± 20 min) and low yields,
Figure 7) Reaction scheme to show [18F]FBEM radiosynthesis from [18F]SFB
until the radiosynthesis was later automated by Kiesewetter et al. [80]. The automated approach
reported increased RCY of 17 ± 6 % and reduced synthesis time of 115 minutes (n=21).
Site-specific radiolabelling: CuI catalysed azide alkyne cycloaddition (CuAAC)
CuAAC is a prevalent click-chemistry technique, characterised by the catalysis of azide and
alkyne conjugation by copper (CuI) and the formation of a stable triazole [81]. The chemistry
of CuAAC, a type of Huisgen 1,3-dipolar cycloaddition, originally necessitated the use of
high reaction temperatures or pressures. Important methodical improvements reported by
Rostovtsev et al. [81] involved the use of a copper catalysts which eliminated the requirement
for such harsh reaction conditions [81]. CuI is often generated in situ by reduction of Cu(II)SO4,
often with sodium ascorbate. Understanding of the mechanism of copper catalysis has
*
Figure 8) Reaction scheme to show [18F]FBEM Michael addition to N-terminal cysteine bearing APP
evolved, from originally hypothesising the formation of copper(I)acetylide, via coordination
to the alkyne, to, more recently, the supposition that two copper atoms are involved. This is
discussed in more detail in a review by Pretze et al. [82].
Glaser et al. [64] compared the CuAAC approach with oxime bond formation to radiolabel an
RGD peptide. For the investigation, the azide-containing prosthetic group
[18F]fluoroethylazide ([18F]FEA) was conjugated to an alkyne-derivatised APP, in the
presence of a copper catalyst [76]. [18F]RGD RCY of 47 ± 8 %, (d.c., n=3) were reported;
comparable to those attained using oxime bond formation (40 ± 12 %, d.c., n=5). Optimised
CuAAC radiolabelling was reported in slightly basic conditions; the use of pH 8, however,
led to the formation of a side-product, which was thought to be a copper catalyst adduct.
Generation of the by-product was minimised by reducing the amounts of Cu(II)SO4 and
ascorbic acid [64], an adaptation that would also be of benefit to APPs that are vulnerable to
reduction and susceptible to structural rearrangements to coordinate CuII [56].
Further optimisation of CuAAC approaches would promote its use in APP radiolabelling; of
particular importance is the necessity to ensure the formulated radiotracer is purified from
copper. Pretze et al. [83] reported the formation of copper complexes with amino acid residues:
serine, histidine and arginine and the difficulties in separating these copper species from the
radiolabelled APP. Additional concerns surround the use of Cu(I) stabilising ligands, such as
tris(benzyltriazolylmethyl)amine (TBTA), which are also cytotoxic and must be purified
from the formulated radiotracer.
Site-specific radiolabelling: Bioorthogonality & pre-targeting
More recently, click-chemistry criteria have matured to include a preference for
bioorthogonal methods, which are highly site-selective and compatible with physiological
conditions and inert to biological reactions [56]. Strain promoted azide-alkyne cycloaddition
(SPAAC) and inverse electron demand Diels-Alder (IEDDA) are click-chemistry techniques
that have seen significant attention for their bioorthogonality [56].
Bioorthogonality has enabled the development of a pre-targeting approach: a two-step
methodology involving the labelling of a prosthetic group followed by reaction with an APP
functionalised with a click reagent, which can selectively react in vivo. Firstly, the APP is
administered, and allowed to accumulate at its biological target in accordance with its
relatively slow pharmacokinetics, followed by administration of the more rapid, radiolabelled
prosthetic group. Pre-targeting has been applied in clinical practice, most particularly in
therapeutic oncology applications for localised radiotherapy or chemotherapy rather than
imaging [84].
Site-specific radiolabelling: Strain promoted cycloaddition
A highly strained alkyne, characterised by constrained bond angles flanking the alkyne
moiety (< 180°, linearity of sp-hybridisation), can be used to drive the cycloaddition and
lower the activation barrier and thus eliminate the requirement for a catalyst. A number of
highly strained alkyne reagents are commercially available; those bearing electron
withdrawing groups have reportedly faster kinetics due to alkyne polarisation - although a
trade-off exists between alkyne stability and reactivity [85].
Site-specific radiolabelling: Strain promoted azide-alkyne cycloaddition (SPAAC)
Like CuAAC, strain-promoted azide-alkyne cycloadditions (SPAAC) involve the formation
of a triazole product.
Kim et al. [65] radiolabelled an RGD peptide using the SPAAC approach in order to evaluate
its potential as a PET radiotracer in oncology. The APP was functionalised with a
dibenzocyclooctyne (DBCO) moiety for strain-promoted click-labelling with an azide-
bearing 18F prosthetic group [65]; the radiosynthesis of the prosthetic group had previously
been reported on and achieved a RCY of 63 % (d.c.) [86].
The bioorthogonality of SPAAC click-chemistry enabled use of ambient temperature and
neutral pH; quantitative conjugation of the azide-bearing prosthetic group was reported and
[18F]RGD RCY of 92 % (d.c.) were attained [65]. In vivo analyses of [18F]RGD, in tumour-
bearing mice, showed good tumour accumulation and tumour to background ratios and thus
highlighted the preservation of the APP’s biological activity [65].
Figure 9 shows the reaction pathway for SPAAC click-radiolabelling with
[18F]fluoroethylazide, a commonly used azide-bearing prosthetic group, and a DBCO
functionalised APP. Owing to the robustness and adaptability of the approach, both 18F azides
and DBCO prosthetic groups are reported for use in APP radiolabelling via SPAAC [56].
Site-specific radiolabelling: Inverse electron demand Diels-Alder cycloadditions (IEDDA)
The [4+2] Diels-Alder cycloaddition is the most common pericyclic reaction and can be
reversible or irreversible depending on the diene and dienophile substituents. Figure 10
shows the differences between reversible and irreversible Diels-Alder reactions and, as can
be seen, the inverse electron demand Diels-Alder (IEDDA) occurs between an electron
deficient diene and a dienophile [87].
Figure 9) Reaction scheme to SPAAC between DBCO-functionalised APP and radiolabelled azide
The absence of charged intermediates permits a solely aqueous reaction medium and
proceeds well, via a single-step, on heating due the stability of the transition state which has
six delocalised π-electrons.
Tetrazine and transcyclooctyne (TCO) have been reported as useful IEDDA synthons as a
result of their fast reaction rates [66, 82].
Selvaraj et al.[66] adopted the approach to successfully radiolabel a tetrazine-derivatised RGD
with a fluorinated TCO synthon. The mechanism of IEDDA between tetrazine and TCO can
be seen in Figure 11; the endo product is formed (the kinetic product). [ 18F]TCO RCY were
not provided, but [18F]RGD yields of 90 % (n.d.c.) were achieved within 5 minutes and at
room temperature.
Keliher et al. [88] adopted the IEDDA approach to radiolabel an exendin derivative, targeting
glucagon-liked peptide 1 (GLP-1). For this, a radiolabelled TCO synthon was produced,
achieving RCY of 46 ± 12 % (d.c.), and conjugated to a tetrazine-modified exendin
derivative which yielded d.c. RCY of 47 ± 17 % [88]. Previous attempts to modify exendin
analogues had been reported to have a significant impact on its pharmacokinetics;
importantly, the selectivity of the exendin derivative was retained when radiolabelled using
the IEDDA approach [88].
Figure 10) Irreversible inverse electron demand Diels-Alder and B) reversible normal electron demand Diels-Alder
The mild reaction conditions and compatibility of IEDDA to a range of solvents including
cell media and buffer highlight the bioorthogonality of the approach [66]. These favourable
characteristics promoted an investigation into the application of IEDDA in pre-targeting
methods [12].
Meyer et al. [13] notably used the IEDDA reaction between a tetrazine and TCO to radiolabel a
mAb with 18F using a pre-targeting approach [13]. For the investigation, an [18F]Al-F-NOTA-
radiolabelled tetrazine synthon was synthesised achieving RCY of 60 ± 5 % (d.c.) [13]. The
mAb, which targeted the tumour-associated antigen CA19.9, was derivatised with TCO and
administered to mice bearing CA19.9-expressing tumours before administration of the
radiolabelled tetrazine. In vivo evaluation of the pre-targeting method confirmed that tumour
delineation was possible after 1 hour post administration of Al[18F]NOTA-tetrazine and good
tumour to background ratios were reported between 2 and 4 hours [13]. The in vivo results are
encouraging, yet a real merit of the technique is the ability to radiolabel this influential class
of APP with 18F, an ideal PET radionuclide [1, 30].
Figure 11) Reaction scheme to show IEDDA cycloaddition between tetrazine and TCO
Site-specific radiolabelling: Evaluation of methods
A review by Meyer et al. [56] describes the importance of site-specific APP radiolabelling and
the superiority of the approaches over non-site-specific methods by providing a ‘homogenous
product’ with better in vivo behaviour [56]. A number of site-specific approaches to APP
radiolabelling with 18F have been adopted, Tables 4 and 5 provide a summary of these
methods.
Encouraging features of fluoride acceptor chemistries have emphasised their appeal and
highlighted their potential as attractive alternatives to click-chemistry based approaches [1].
Full process automation of an ([18F]AlF)2+ radiolabelling method [70] and the development of
single-vial methods using lyophilized APPs [67] are key developments in the approach that
enhance the ease with which the methods can be translated to GMP. The ability to use
commercially available aqueous [18F]fluoride and removal of azeotropic drying steps
simplifies the overall procedure and increases the ease at which the methods can be
implemented in a clinical setting. The versatility of the fluoride acceptor approaches may,
however, be limited to APPs where the use of organic solvents and/or high temperatures does
not lead to loss of function [15].
Oxime bond formation and thiol-Michael additions are additional approaches to APP
radiolabelling that have yielded good results. The flexibility of the methods to a variety of
media, including aqueous buffers, and numerous APPs, including those with more complex
structures, highlights the versatility of the approaches [15, 27, 53]. Alongside this are the
developments towards automated radiosyntheses that have improved the consistency and
reliability of multi-step methods [40] and, in the case of [18F]FBEM, resulted in higher yields
[80]. The compatibility of the methods with mild labelling conditions also underlines their
appeal and although neutral pH had been reported to significantly impact yields attained
using oxime bond formation, the use of an aniline catalyst circumvents this issue [69, 79].
Approaches to APP radiolabelling via cycloaddition are very encouraging; the faster rates of
reaction that are afforded using cycloaddition chemistries have given rise to a number of
advantages. Glaser et al. [64] report near complete consumption of crude radiolabelled
synthon using CuAAC, thereby underlining the robustness and high-yielding nature of the
method and highlights the feasibility of a single-vial method of APP radiolabelling.
Comparable RCY using oxime bond formation required a 3-fold increase in reaction time [64].
The ability to use mild reaction conditions opened the door to bioorthogonality and the
applicability of an APP radiolabelling method to physiological conditions and in vivo
labelling. SPAAC and IEDDA are very promising methods, their use of mild reaction
conditions highlights their potential in pre-targeting approaches, which promises to be a very
powerful technique. However, the size and hydrophobic nature of the
dibenzocyclooctatriazole SPAAC and bicyclic IEDDA products and their impact on the
pharmacokinetics of the APP should be considered [56]. An additional disadvantage of SPAAC
is isomerisation in the final radiolabelled APP product, which raises issues of product
consistency and homogeneity.
The use of pre-targeting methods overthrows the convention of matching a PET radioisotope
to the pharmacokinetics of an APP and enhances the versatility of the approach, removing the
need for longer-lived radioisotopes, where radioactive burden to non-target tissues is a
considerable limitation of the technique. Meyer et al. [13] report effective dose measurements
of an 18F pre-targeting approach that were 60-fold lower than directly labelled mAb using
89Zr. Yet the translation of a pre-targeting method to clinical practice may be complicated by
the inconvenience of the technique, where a lag time of 24-72 hours between administration
of the APP and radiolabelled prosthetic group is not uncommon [13, 56], relative to a method
that involves a single injection and rapid scanning protocol which would be more suitable.
Site-specific radiolabelling: A summary
Alongside process automation and radiosynthesis simplification, a trend towards click-
chemistry methods of APP radiolabelling is apparent in the literature with some momentum
towards bioorthogonal approaches. The notion of a generic radiolabelling platform by which
APPs can be radiolabelled with 18F appeared unfeasible due to the striking diversity of APPs
yet the encouraging developments in these bioorthogonal methods, most particularly pre-
targeting methods, and the flexibility and versatility that these methods can afford relieve
some of the challenges of APP radiolabelling with 18F.
Labelling method
Reaction conditions
Overall yields (%)
Comments Refs
Click-chemistry Oxime pH 3-4,50-60°C
≤ 40 (d.c.u.) 1. Good stability of oxime-product2. Aniline catalyst enhances rate, even at neutral pH3. Full process automation
[40, 52,
62, 79]
Hydrazone - - 1. Instability of hydrazone product has limited the appeal of the approach, despite rapid rates of hydrazone formation- k1 of hydrazone and oxime (aniline catalysed), respectively: 170 ± 10 and 8.2
± 1.0 M-1
[51]
Thiol-Michael
pH 7-7.5, room temp
~4 (n.d.c, calculated)
1. Physiological reaction conditions2. Low overall yield as a result of low [18F]FBEM RCY3. Requirement for reducing agent for disulphide bridging (TCEP.HCl)4. Process automation of [18F]FBEM synthesis
[53, 80]
CuAAC slightly basic,80°C
≤ 55 (d.c.) 1. Issues of Cu(I) toxicity2. Higher temperatures often used3. Slightly basic pH enhanced yield
[64]
SPAAC Neutral, room temp
~58 (d.c., calculated)
1. Bioorthogonalitya. No Cu(I) toxicityb. Mild reaction conditions
[65]
IEDDA Neutral, room temp
~60 (d.c.) 1. Bioorthogonalitya. No Cu(I) toxicityb. Mild reaction conditions
2. Pre-targeting application
[13]
Fluoride acceptor chemistry
([18F]AlF)2+ pH 4,100°C
≤ 45 (n.d.c.) 1. Water miscible solvent enhances rate2. Single-vial/fully-automated methods3. No azeotropic distillation4. Electronic/steric impact of macrocyclic on APP
[57-59,
67, 68,
70]
Isotopic exchange
pH 9, room temp
≤ 65 (d.c.u.) 1. Mild APP radiolabelling conditions2. No azeotropic distillation3. Competition with boron exchange (BF3) side reactions
[58, 60]
Table 5) Summary of site-specific methods using 18F (non-decay corrected (n.d.c), decay-corrected (d.c.), decay-correction status unknown (d.c.u))
18F radiolabelling methods: final remarks
A number of approaches have been adopted to radiolabel small and intermediately sized
APPs with 18F. Non-site-specific methods, generally achieved using amine targeting
prosthetic groups, offer a rapid approach to radiolabel native APPs allowing the potential of
the radiolabelled APP to be rapidly validated. The importance of this is reiterated in a
publication by Wester et al. [89], where it was argued that the ability to make a ‘go’ or ‘no go’
decision as early as possible is vital in order to help focus efforts towards the most promising
radiotracers. Non-site-specific radiolabelling methods can, arguably, be used to identify these
strong APP candidates, yet it should be noted that different radiolabelling methods can alter a
radiotracer’s pharmacokinetics, metabolism and excretion [26, 28, 90].
Site-specific methods of APP radiolabelling have become the preferred approach for APP
radiolabelling [19]. Site-specific methods help safeguard the biological properties of the APP
antigen-binding site, provide consistency in the final radiolabel product and also permit
exploitation of the faster reaction rates [15, 19]. However, site-specific approaches often require
APP-functionalisation and whilst functionalisation of smaller APPs is often possible during
solid phase peptide synthesis, functionalisation of larger, more complex, multi-domain APPs
often requires a different approach. Theile et al. [91] report an enzyme-mediated method by
which APPs can be functionalised. The enzyme, sortase, is expressed by gram-positive
bacteria and involved in cell-wall synthesis. An APP and a derivatised small peptide, bearing
the sortase recognition motif, are conjugated by the enzyme [91]. It is clear that APP-
functionalisation has time and financial implications, most particularly using multi-step,
enzyme-mediated derivatisation methods. Such investments may well be worthwhile, in order
to exploit the advantage of site-specific methods, if the potential of an APP as a radiotracer
has already been established.
It may, therefore, be concluded that non-site-specific radiolabelling approaches serve as rapid
tool to first, determine the feasibility of APP radiolabelling and second, the usefulness of the
labelled APP as a PET radiotracer before adapting the approach to permit site-specific APP
radiolabelling.
Although mAbs are likely to remain the prevalent APP PET tracers, there are a number of
very promising 18F radiolabelled APPs. The potential of [18F]APPs hasn’t yet met its full
potential due to the inherent challenges of fluoride radiolabelling and this is plain from the
dearth of [18F]APPs in routine clinical use [1]. In response to the recognised need to harness
the unmet potential of [18F]APPs in the clinic, momentum in the field has unfolded and a
diverse range of approaches have been reported. Despite the diversity of the field, a number
of generic features of 18F APP radiolabelling methods have manifested, including process
automation and site-specificity of methods that are high-yielding and mild. The propensity
for mild APP radiolabelling conditions has given rise to an appetite for bioorthogonality. The
coming together of these generic features has afforded the arrival of very promising APP
radiolabelling methods that are high-yielding, site-specific and bioorthogonal. Whilst these
developments are very promising, the definition of high-yielding is disputable when
comparing against enzyme-mediated conjugations, where rate constants of 2.7x106 M-1s-1 and
yields of > 90 % are typical [29]. Nevertheless, the impetus in the field of APP radiolabelling
with 18F is encouraging and the concerted efforts alongside the afforded results provide
assurance for the long awaited appearance of [18F]APPs in routine clinical use.
Disclosure statement
The authors declare no potential sources of conflict of interest or competing financial interest.
Acknowledgements
OM gratefully acknowledges the CRUK/EPSRC Imaging Centre in Cambridge &
Manchester (CRUK/EPSRC [C8742/A18097]) and would like to thanks AM and JG for
critically reviewing this article.
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