The aluminium-[18F]fluoride revolution: simple radiochemistry with a big impact for radiolabelled biomolecules
The aluminium-[18F]fluoride ([18F]AlF) radiolabelling method combines the favourable decay characteristics of fluorine-18 with the convenience and familiarity of metal-based radiochemistry and has been used to parallel gallium-68 radiopharmaceutical developments. As such, the [18F]AlF method is popular and widely implemented in the development of radiopharmaceuticals for the clinic. In this review, we capture the current status of [18F]AlF-based technology and reflect upon its impact on nuclear medicine, as well as offering our perspective on what the future holds for this unique radiolabelling method.
<!>Introduction<!><!>Imaging prostate specific membrane antigen (PSMA)<!><!>Imaging prostate specific membrane antigen (PSMA)<!>Imaging the immune system<!><!>Imaging the immune system<!><!>Imaging fibroblast activation protein (FAP)<!>Imaging the epidermal growth factor (EGF) receptor family<!><!>Imaging the epidermal growth factor (EGF) receptor family<!><!>Imaging integrins<!><!>Imaging the gastrin-releasing peptide receptor (GRPR)<!><!>Current and future perspectives on the [18F]AlF method<!><!>The future of [18F]AlF chelator design<!>Improving accessibility to [18F]AlF-based radioconjugates<!>Conclusions<!>
<p>A schematic of general PET radiolabelling methods</p><p>Schematic representation of the process for efficient [18F]AlF2+ complex formation and subsequent chelation from nucleophilic [18F]F− in 18O water</p><p>Examples of [18F]F− purification by QMA SPE cartridge</p><p>Elution of [18F]F− from a QMA is invariably efficient (ca. 99% recovery) but the choice of eluent can modulate radioactive concentration. This was demonstrated by Kersemans et al. (2018) and may be considered as a parameter for optimisation when developing new [18F]AlF labelling protocols (Kersemans et al. 2018)</p><p>Structures of chelators evaluated for [18F]AlF and their proposed complexes. R = bioconjugation handle. 3D models were created in ChemBio3D (Cambridgesoft, UK) with MM2 energy minimization applied. Atom colours: carbon = light grey, hydrogen = white, oxygen = red, nitrogen = blue, fluorine = yellow, aluminium = dark grey. RT = room temperature. *Optional 1:1 (v/v) co-solvent included in the reaction mixture to improve RCY</p><!><p>As the [18F]AlF method has been applied to peptides previously radiolabelled with 68Ga, the relatively high temperatures (100–120 °C) required to chelate [18F]AlF2+ in N3O2 configured chelators is not problematic as small peptides have been proven withstand these temperatures. However, large proteins where biological activity is derived from a precise tertiary structure can denature at these temperatures, which has encouraged the development of [18F]AlF chelators for ambient temperature radiolabelling. Cleeren et al. (2016) described two promising acyclic chelators (Fig. 3, 5 and 6) for [18F]AlF complexation at moderate temperatures (40 ºC) in good RCY (> 90%). While [18F]AlF-6 showed stability in rat plasma at 2 h (ca 90%), a reasonable timeframe for PET imaging, it was poorly stable at 4 h (66%); [18F] AlF-5 was unstable in vitro (Cleeren et al. 2016). This was improved by the N2O3 configured derivative (±)-H3RESCA (Fig. 3, 7) which utilised a cyclohexyl moiety to impart structural rigidity into the molecule (Cleeren et al. 2017). (±)-H3RESCA was radiolabelled at room temperature and was stable in rat plasma for at least 4 h, comparable to NODA derivatives. [18F]AlF-(±)-H3RESCA conjugates of human serum albumin (RCY: 52–63%), nanobody NbV4m119 targeting CRIg (RCY: 35–53%) and an affibody molecule ZHER2:2891 targeting HER2 (RCY: 20 ± 7%) were synthesised, demonstrating the utility of this new chelator (Cleeren et al. 2017). Russelli et al. (2020) developed three acyclic chelators based around the 2-aminomethylpiperidine (AMP) group (Fig. 3, 8–10). All chelators radiolabelled efficiently at room temperature and pH 5 (RCY: 55–81%) with 2-AMPTA-HB (10) showing the greatest stability at 240 min post radiosynthesis in human serum (87 ± 5%), PBS (93 ± 1%) and saline (92 ± 2%). The in vivo evaluation of [18F]AlF-2-AMPDA-HB showed low bone uptake at 2 h p.i. (1.63 ± 0.73%ID/g) (Russelli et al. 2020). The synthesis of a bifunctional derivative is now underway.</p><p>These new chelators provide an elegant solution to radiolabelling heat-sensitive biomolecules and will benefit from a full evaluation in the clinic; we predict that this will be achieved within the next five years. It is our opinion that the commercial availability and affordability of pentadentate NODA and NOTA derivatives mean they are unlikely to be replaced by acyclic chelators, at least for the time being, for instances where ambient temperature radiochemistry is a convenience rather than a necessity. If the research community adopt these chelators (or future derivatives) for use in projects, their commercialisation may be expedited. The development of ambient temperature kit-based production protocols, akin to 68Ga-trishydroxypyridinone (THP) which aims to be simple to use in the radiopharmacy setting (Young et al. 2017), may also increase the demand and implementation of alternative chelators. Nevertheless, it is important to remember that multistep synthesise of chelators can present a barrier to their use in projects which aim to focus on radioconjugate development.</p><!><p>Prominent examples of [18F]AlF radioconjugates discussed in this review</p><p>Structure of A [18F]AlF-NOTA-octreotide and B [68Ga]Ga-DOTA-TATE/[68Ga]Ga-DOTA-TOC</p><p>Clinical PET studies using [18F]AlF-NOTA-octreotide</p><p>Healthy: 3</p><p>Patients: 22</p><p>Healthy: 6</p><p>Patients: 6</p><p>Healthy: 3</p><p>Patients: 8</p><!><p>Prostate specific membrane antigen (PSMA) is highly expressed in prostate cancer (PCa) and, when imaged by targeted PET radioligands, provides a crucial biomarker to assess disease burden with the potential for patient stratification to receive PRRT (Werner et al. 2020; Rahbar et al. 2017; Sathekge et al. 2019). Many radiopharmaceuticals have been developed around the simple Lys-Urea-Glu binding motif and linker strategies have been adapted to modulate binding affinity and PK. 68Ga-based PSMA radiopharmaceuticals have been extensively evaluated in the clinic, leading to FDA approval for [68Ga]Ga-PSMA-11 in December 2020 for PET imaging of PSMA positive lesions in men with prostate cancer. Fluorine-18 PSMA radioligands have been developed and evaluated clinically, including [18F]DCFPyL, [18F]PSMA-1007, [18F]JK-PSMA-7 and [18F]CTT1057; while their structures differ, they share a similar radiolabelling approach based around the 18F-fluorination of an aromatic moiety (Werner et al. 2020; Bouvet et al. 2016; Naka et al. 2020; Zlatopolskiy et al. 2019; Behr et al. 2019).</p><!><p>Structures of A [18F]AlF-PSMA-11, B [18F]AlF-PSMA-BCH, C [18F]AlF-NOTA(5)-DUPA-Pep, D [18F]AlF-Glu-urea-Lys(Ahx)L3 and E [18F]AlF-P16-093</p><p>Clinical PET studies using</p><!><p>Two NOTA derivatives containing the Lys-Urea-Glu PSMA binding motif have been developed ([18F]AlF-NOTA(5)-DUPA-Pep and [18F]AlF-PSMA-BCH, Figs. 5B, 5C) which differ in their linker strategy. The radioconjugates were synthesised in a RCY of 79 ± 0.7% and 32.2 ± 4.5%, respectively (Liu et al. 2019; Malik et al. 2012). While [18F]AlF-NOTA(5)-DUPA-Pep is yet to be preclinically evaluated, [18F]AlF-PSMA-BCH entered a small clinical trial of 11 newly diagnosed PCa patients; the radioconjugate was well tolerated and visualised tumour lesions at 1 and 2 h p.i. (Liu et al. 2019). Cleeren et al. (2016) developed [18F]AlF-Glu-urea-Lys(Ahx)L3 to exemplify the application of their new [18F]AlF chelator that reacts efficiently at lower temperatures (Fig. 5D). The radioconjugate was produced in a large batch (8.14 GBq) from a 12 min reaction at 40 ºC; the RCY was 25% (unoptimized) and the molar activity was 27 GBq/µmol (Cleeren et al. 2016). No significant bone uptake was observed at 60 min p.i. (0.74 ± 0.07% %ID/g), encouraging the further development of more efficient chelators that react at lower temperatures, ultimately leading to (±)-H3RESCA (Cleeren et al. 2016, 2017). Zha et al. (2021) described the synthesis and pre-clinical evaluation of [18F]AlF-P16-093, derived from [68Ga]Ga-P16-093, a PSMA targeted radioligand currently in phase II clinical trial. [18F]AlF-P16-093 showed high in vivo tumour uptake in mouse PIP-PC3 xenografts (18.8 ± 5.14%ID/g at 60 min p.i.) with some uptake in the bone (2.82 ± 0.49%ID/g), higher than observed for [68Ga]Ga-P16-093 (0.26 ± 0.07%ID/g), indicating demetallation (Zha et al. 2021).</p><!><p>Immune checkpoint inhibitors are an important therapeutic intervention for tumours evading the immune system, but patient response is variable (Postow et al. 2015; Royal et al. 2010; Marrone et al. 2016). Immune checkpoints PD-1 and CTLA-4 are often targeted in combination to improve response rates but can lead to toxicity and immune-related side effects (Khoja et al. 2017; Cousin and Italiano 2016). Radiopharmaceuticals to measure treatment response are of great interest and the [18F]AlF method has been used to develop radioconjugates for imaging T-cell activation.</p><!><p>Structure of A [18F]AlF-mNOTA-GZP and B [18F]AlF-RESCA-IL2</p><!><p>The interleukin-2 receptor (IL2R) is overexpressed on activated T cells and PET radioligands have been developed using recombinant IL2 as a targeting molecule. Gialleonardo et al. (2012) first labelled IL2 with fluorine-18 via the [18F]SFB prosthetic group ([18F]FB-IL2) and the radiosynthesis was translated into a GMP compliant clinical production to evaluate [18F]FB-IL2 in human trials (Veen et al. 2019). The complexity of automating the radiosynthesis of [18F]FB-IL2 encouraged the simplification of the radiolabelling process by employing the [18F]AlF method and the RESCA chelator to produce [18F]AlF-RESCA-IL2 (Fig. 6B) (Veen et al. 2020). The radioconjugate was synthesised in a 2.4 ± 1.6% RCY with a molar activity of 910 ± 927 GBq/µmol in multipatient doses (1375 ± 791 MBq from < 50 GBq fluorine-18) in 90 min (Veen et al. 2020). [18F]AlF-RESCA-IL2 showed high in vitro uptake in activated peripheral blood mononuclear cells (PBMC), and in vivo uptake in PBMC xenografts and lymphoid tissue, supporting further evaluation of this radioligand.</p><p>An [18F]AlF-based radioconjugate was developed to target programmed death ligand 1 (PD-L1), which is expressed in tumours responding to anti-PD-1 therapy (e.g. pembrolizumab). The affibody molecule PD-L1 (ZPD-L1_1) was conjugated to NOTA via its unique cystine and radiolabelled with the [18F]AlF complex ([18F]AlF-NOTA-ZPD-L1_1 in a radiochemical yield of 15.1 ± 5.6% and a molar activity of 14.6 ± 6.5 GBq/µmol; the radioconjugate differentiated between PD-L1 positive and negative tumours in vivo (González Trotter et al. 2017).</p><!><p>Structure of A [18F]AlF-NOTA-FAPI-74 and B [18F]AlF-NOTA-FAPI-04</p><!><p>The centralised production of [18F]AlF-based radiopharmaceuticals requires automated radiosynthesis and Jiang et al. (2021) describe the production of [18F]AlF-NOTA-FAPI-04 on a cassette-based Trasis AIO™ platform (Fig. 7B). Multipatient doses of radiopharmaceutical were synthesised (9.1 ± 0.6 GBq) in an excellent RCY of 26.4 ± 1.5% (n.d.c) which enabled both preclinical in vivo evaluation and clinical scan (Jiang et al. 2021).</p><!><p>The epidermal growth factor (EGF) receptor family are overexpressed in many cancers and therapeutics have been developed towards these targets, including Cetuximab targeting EGFR and Trastuzumab targeting HER2 (Wang 2017; Xu et al. 2017b; Pernas and Tolaney 2019). Stratifying patients based on EGF receptor expression using PET imaging is of great interest and while zirconium-89 labelled monoclonal antibodies (mAb) with long-lived radioisotopes (t1/2 = 3.3 days) have been developed, smaller biomolecule fragments with fast PK and radiolabelled short half-life isotopes are of great interest (Tolmachev and Orlova 2020).</p><p>The first Affibody molecule to be radiolabelled with the [18F]AlF complex was ZHER2:2395 which targeted the HER2 receptor and showed specific tumour uptake (T:B of 7.4 ± 1.8 and tumour uptake of 4.4 ± 0.8%ID/g at 1 h p.i.) in SK-OV-3 xenografts (Heskamp et al. 2012). A NOTA-maleimide chelator was conjugated to the unique cystine residue of the Affibody molecule and radiolabelled within 30 min to produce [18F]AlF-NOTA-ZHER2:2395 in a RCY of 21.0 ± 5.7% and molar activity of 7.7 ± 3.0 GBq/µmol; the affinity of the radioconjugate for HER2 was Kd = 6.2 nM (Heskamp et al. 2012). A similar approach was taken to produce [18F]AlF-NOTA-MAL-MZHER2:342 and [18F]AlF-NOTA-ZHER2:2891 (Xu et al. 2017a; Glaser et al. 2013).</p><!><p>Three different approaches to radiolabelling Affibody molecules with the [18F]AlF complex: A maleimide-thiol conjugation; B IEDDA "click" approach and C PODS conjugation</p><!><p>An EGFR targeted Affibody molecule [18F]AlF-NOTA-ZEGFR:1907 has been developed and produced in a radiochemical yield of 15% (Su et al. 2014). As all of these examples show, Affibody molecules are typically functionalised at their unique cystine residue via a maleimide-thiol Michael addition; however, instability has been observed during radiosynthesis and in vivo studies (Pieve et al. 2020; Ponte et al. 2016). Da Pieve et al. (2020) have recently described phenyloxadiazolyl methylsulfone (PODS) derivatives of NOTA and NODAGA chelators which provides an alternative to maleimide chemistry and may overcome instability associated with the maleimide-thiol ligation at later time points. [18F]AlF-NOTA-PODS-ZEGFR:03115 and [18F]AlF-NODAGA-PODS-ZEGFR:03115 radioconjugates were synthesised and showed specific tumour uptake 14.1 ± 5.3 and 16.7 ± 4.5%ID/g (1 h p.i.), respectively (Pieve et al. 2020).</p><p>Despite implementing the [18F]AlF method in the development of numerous Affibody molecule radioconjugates targeting the EGF family, the clinical translation of these probes is yet to be reported. However, given that 68Ga-labelled Affibody molecules targeting HER2 have been evaluated in phase I/II clinical trials, we are likely to see [18F]AlF derivatives in the near future (Sandström et al. 2016; Sörensen et al. 2016).</p><!><p>Structure of [18F]AlF labelled peptides targeting integrins</p><p>Clinical PET studies using [18F]AlF-NOTA-PRGD2 (18F-Alfatide) and [18F]AlF-NOTA-E[PEG4-c(RGDfK)]2 (18F-Alfatide II)</p><!><p>Guo et al. (2014) compared two novel dimeric RGD peptides, [18F]AlF-NOTA-PEG4-E[c(RGDfK)]2 and [18F]AlF-NOTA-E[PEG4-c(RGDfK)]2 which included PEG4 linker strategies aimed to optimise radiolabelling and PK performance of the probes and compared these to [18F]AlF-NOTA-RGD2. All probes were synthesised in a RCY of 40–60%, with molar activities ranging from 14.8 to 37 GBq/µmol. All three probes exhibited favourable in vivo performance with high tumour uptake and good target-to-background ratios, but [18F]AlF-NOTA-E[PEG4-c(RGDfK)]2 (also known as 18F-Alfatide II) was highlighted as a promising candidate for clinical translation owed to the lowest liver uptake and highest tumour uptake (2.92 ± 0.40%ID/g) (Guo et al. 2014). First-in-human studies of 18F-Alfatide II commenced in 2015 in healthy volunteers and patients with brain metastases; the radioconjugate was well tolerated in all healthy volunteers and successfully visualised all 20 brain lesions (Yu et al. 2015). A preliminary clinical study using 18F-Alfatide II to identify breast cancer was performed in 2018, concluding its suitability for clinical use in breast cancer patients and its good performance, albeit not superior to [18F]FDG in this study (Wu et al. 2018); a range of clinical studies evaluating [18F]AlF-NOTA-E[PEG4-c(RGDfK)]2 have been summarised in Table 5. Dijkgraaf et al. (2013) evaluated NODAGA-E[c(RGDfK)]2 for radiolabelling with multiple isotopes including [18F]AlF, 68Ga and 111In, but low radiolabelling yields were obtained for [18F]AlF owed to the N3O3 donor set; despite this, tumours could be visualised with [18F]AlF-NODAGA-E[c(RGDfK)]2 (3.44 ± 0.20%ID/g at 2 h p.i.) but the uptake was significantly lower than its 68Ga (6.23 ± 0.76%ID/g) and 111In (4.99 ± 0.64%ID/g) counterparts (Dijkgraaf et al. 2013). Integrin ⍺vβ6 is expressed on the surface of epithelial cells in a variety of cancer types and can be targeted by the peptide A20FMDV2 (Bandyopadhyay and Raghavan 2009). Hausner et al. (2014) modified peptide with a NOTA-PEG28 moiety and radiolabelled with [18F]AlF complex ([18F]AlF-NOTA-PEG28-A20FMDV2) in a RCY (d.c.) of 19.3 ± 5.4% and a molar activity of 158 ± 36 GBq/µmol; the radioconjugate exhibited high cellular internalisation in ⍺vβ6 positive cells and specific in vivo uptake.</p><!><p>Structures of [18F]AlF BBN analogs targeting GRPR</p><p>A summary of GRPR targeted radiopharmaceuticals and their serum stabilities, tumour-to-blood (T:B) and tumour-to-muscle (T:M) ratios at a given time point post-injection</p><p>aIntact parent radioconjugate in mouse serum after 1 h incubation, unless stated otherwise</p><p>bThe timepoint post-injection where T:B and T:M data is reported. T:B = tumour-to-blood ratio and T:M = tumour-to-muscle ratio calculated from ex vivo biodistribution data presented in the original manuscript and represented as a mean value</p><p>cEstimated from graphical data in the original manuscript</p><!><p>Dijkgraaf et al. (2012) first described an [18F]AlF labelled NOTA-conjugated BBN analogue ([18F]AlF-NOTA-8-Aoc-BBN(7–14)NH2) which was produced in a RCY > 50% and molar activity > 10 GBq/µmol within 45 min. Uptake in PC-3 tumours at 1 h p.i. was 2.15 ± 0.55%ID/g and the signal was confirmed to be specific by co-injection of a blocking dose of NOTA-8-Aoc-BBN(7–14)NH2 (Dijkgraaf et al. 2012). Liu et al. (2013) synthesised [18F]AlF-NODAGA-RM1 and [18F]AlF-NODAGA-AMBA analogues. The [18F]AlF-NODAGA-RM1 and [18F]AlF-NODAGA-AMBA peptides were labelled in 40 min with a radiochemical yield of 5.6 ± 1.1% and 4.9 ± 1.3%, respectively; the molar activity was > 1.85 GBq/µmol (Liu et al. 2013). The [18F]AlF-NODAGA-RM1 radioligand was most stable in serum whereas [18F]AlF-NODAGA-AMBA degraded. Both [18F]AlF-NODAGA-RM1 and [18F]AlF-NODAGA-AMBA exhibited excellent in vivo tumour uptake (3.70 ± 0.70 and 4.60 ± 1.50%ID/g at 0.5 h) with [18F]AlF-NODAGA-RM1 showing significantly higher uptake than [18F]AlF-NODAGA-AMBA at 1 and 2 h p.i. time points (Liu et al. 2013). The authors conclude that [18F]AlF-NODAGA-RM1 is the most promising radioligand. Full-length BBN peptides are poorly stable in vivo so efforts were made by Carlucci et al. (2015) to stabilise the peptide using peptidase resistant lanthionine thioether crosslinked amino acids. Two radioconjugates were synthesised, [18F]AlF-NOTA-4,7-lanthionine-BBN and [18F]AlF-NOTA-2,6-lanthionine-BBN, which were produced in a RCY of 50–60% (d.c) with a molar activity > 63 GBq/µmol and > 88 GBq/µmol, respectively (Carlucci et al. 2015). Tumour uptake of both [18F]AlF-NOTA-4,7-lanthionine-BBN and [18F]AlF-NOTA-2,6-lanthionine-BBN in PC-3 xenografts at 120 min p.i. was 0.82 ± 0.23 and 1.40 ± 0.81%ID/g, respectively; moreover, both radioconjugates exhibited high in vivo stability with an average of 87–88% intact tracer in the tumour, confirming that lanthionine crosslinkers were advantageous (Carlucci et al. 2015). Introducing a hydrophilic linker into BBN analogues, as demonstrated by Pan et al. (2014) in their report of [18F]AlF-NOTA(6)-MATBBN can improve the PK profile of the radioconjugate and lead to high tumour uptake (4.59 ± 0.40%ID/g at 60 min p.i.) and excellent T:M contrast (6.77 ± 1.10) by image analysis. [18F]AlF-NOTA(6)-MATBBN was produced in a RCY of 62.5 ± 2.1% with a molar activity of at least 30 GBq/µmol (Pan et al. 2014).</p><p>The antagonist [18F]AlF-JMV5132 was produced in a RCY of 88% (n.d.c) with a molar activity of 40 ± 4 GBq/µmol which showed high in vivo uptake in PC3 tumours at 60 min p.i. (4.96 ± 1.20%ID/g) (Chatalic et al. 2014). Varasteh et al. (2013) developed [18F]AlF-NOTA-P2-RM26 a antagonist BBN analogue produced in a RCY of 60–65% (d.c.) and with a molar activity of 55 GBq/µmol (Varasteh et al. 2013); tumour uptake was high at 3 h p.i. (5.5 ± 0.7%ID/g) along with excellent T:B (87 ± 42) and T:M (159 ± 47) contrast (Varasteh et al. 2013).</p><!><p>Structures of A [18F]AlF-pentixather derivatives and B [18F]AlF-NOTA-T140</p><p>A "complex" relationship with gallium-68 Generator-produced gallium-68 (t1/2 = 68 min, βem+ = 89%) is used primarily to produce doses of PET radiopharmaceuticals from the local hospital radiopharmacy via a decentralised production model (Fig. 12). This is in contrast to the centralised production model exemplified by 18F-radiopharmaceuticals like [18F]FDG, which are produced in a large scale to serve several hospitals and imaging facilities from a single radiopharmaceutical production facility (Fig. 12). Gallium-68 metal-based radiochemistry is convenient and depending upon the age of the 68Ge/68Ga generator, can produce up to 3 patient doses per elution; increasing for high-capacity generators and cyclotron produced 68Ga. Although some future developments may increase this capacity the shorter half-life of gallium-68 remains an issue for a centralised supply model. As we implement personalised medicine into routine clinical practice and use PET biomarkers to underpin patient stratification and monitor treatment response, we need expedient solutions to scale radiopharmaceutical production to meet future clinical demand. The dose capacity of 68Ge/68Ga generators diminishes over time and is limited, which may be a concern if more 68Ga-radiopharmaceuticals gain approval for routine clinical use. Additionally, while it has not been fully evaluated and is somewhat context dependent, the variable and diminishing molar activity (Am) of 68Ga over the lifespan of a generator may confound PET imaging of some biological targets. Finally, the availability of generators has been a challenge in recent years with long lead-times on delivery and rising cost, both of which tighten the already slim financial margin for operating a sustainable radiopharmacy (Mueller et al. 2019). To futureproof the throughput for producing a repertoire of 68Ga-radiopharmaceuticals for the clinic, there is an option to commit resource to cyclotron produced gallium-68 where viable and effective methods have been described, producing > 3.5 GBq of 68Ga within 60 min from a liquid target with a 14.3 MeV beam energy (Rodnick et al. 2020); or, alternatively, we could develop 18F derivatives of commonly used 68Ga radiopharmaceuticals to harness the abundance of nucleophilic [18F]fluoride from existing radiopharmaceutical networks already producing [18F]FDG; this allows [18F]AlF derivatives to modulate tracer demand and would allow decentralised 68Ga production to focus on smaller batch runs for less frequent speciality tracer scans for lower numbers of patients. The [18F]AlF method promotes the latter without significant reimagination of existing radiopharmaceuticals, as would be necessary for most 18F-fluorination chemistries. It is promising that early data suggests 68Ga and [18F]AlF radiopharmaceuticals exhibit similar pharmacokinetic profiles (PK) although in some cases, differences have been observed, particularly in biodistribution (Hou et al. 2020).</p><p>Fluorine-18 radiochemistry is challenging! If we are to seek a centralised production model to produce 18F-peptides previously labelled with 68Ga, then a lot of development work is required. As we alluded to, developing fluorine-18 radiochemistry to radiolabel complex and sensitive molecules like proteins and peptides is challenging. The radiochemistry can be laborious and as the overall pharmacokinetic (PK) and metabolic profile is influenced by the radiolabelling and linker strategy, an iterative approach to developing suitable radioconjugates may be required which adds complexity. Several late-stage fluorination methods and 18F-prosthetic group strategies suitable for radiolabelling peptides have been described and some evaluated in the clinic, but none as close in characteristics to the existing 68Ga radiochemistry as the [18F]AlF method (Allott et al. 2020; Narayanam et al. 2020; Kee et al. 2020; Yuan et al. 2018; Krishnan et al. 2017; Cole et al. 2014). Therefore, if the research experience lies with gallium-68 radiochemistry, the parallels offered by the [18F]AlF method will be a facile adjustment.</p><p>Whether [18F]AlF radiopharmaceuticals are produced in a decentralised radiopharmacy using kit-based procedures alongside 68Ga-radiopharmaceuticals, or a centralised fluorine-18 radiopharmaceutical production facility will depend on radiopharmaceutical demand and flexibility</p><!><p>Conventional fluorine-18 radiopharmaceuticals require automated radiosynthesis procedures for their GMP production, which adds additional complexity to the translational pathway (Allott and Aboagye 2020). However, automated procedures are convenient for [18F]AlF-based radiopharmaceuticals and will be necessary where a centralized production model produces multi-patient doses for transportation to hospitals from a remote facility (Fig. 12). Automation is not a necessity for low level production runs to access a couple of patient doses and therefore [18F]AlF is also amendable to decentralised, manual kit-based production akin to 68Ga-radiopharmaceuticals (Fig. 12). We often consider [18F]AlF as a replacement for 68Ga; the question "Will [18F]AlF replace 68Ga for metal chelate labelling?" was presented in the title of an excellent review by Fersing et al. (2019) which provided a comprehensive overview of [18F]AlF radioconjugates, stopping short of speculating an answer to the question. The radiolabelling protocols for [18F]AlF and 68Ga are comparable and we have seen many examples where [18F]AlF-derivatives of 68Ga-peptides have been developed in order to address the limitations of 68Ga (i.e. short half-life and small dose batch sizes); but our opinion is that we should not consider [18F]AlF-radiopharmaceuticals as a replacement for 68Ga-radipharmaceuticals. 68Ga is, and always will be, a very important isotope that is unlikely to vanish from the clinic and actually, the flexibility that 68Ga will afford the clinic is yet to come into its own. The more personalised treatments we implement, the greater range of imaging radiopharmaceuticals we will require to stage and monitor disease, treatment efficacy and progression. This will inevitably lead to a large toolbox of radiopharmaceuticals, some of which will be used every day—as we see currently with [18F]FDG—where many patients benefit from a centralised production model which sets out to achieve multipatient and multihospital doses, allowing high patient throughput and in turn, lowers the cost-per-dose to healthcare services (Fig. 12); however, some radiopharmaceuticals may be used more infrequently, but their access is vital for patients with equivocal disease.</p><!><p>The screwdriver analogy. All tools are essential for the task they perform irrespective of how frequently that task is performed; the same is true for radiopharmaceuticals and therefore [18F]AlF and 68Ga radioconjugates both have an important role to play in modulating supply to meet the clinical demand for a range of imaging probes to offer patients the best possible care</p><!><p>We have an excellent variety of chelators to sequester [18F]AlF (Fig. 3); however, further expansion of ambient temperature chelators with improved stability is an area of research that could be further developed. Interestingly, a computational approach to calculate the thermodynamic stability of zirconium-89 chelates has been reported and we propose that this approach could be used to prospectively optimise the design of [18F]AlF chelators to focus synthesis efforts and experiment with underexplored functional groups (Holland 2020). The idea of developing more chelators can be supported or dismissed by the evaluation and clinical translation of [18F]AlF-PSMA-11, which presents an interesting conundrum! The HBED chelator is a poor choice for [18F]AlF chelation and results in a degree of in vivo demetallation; but, [18F]AlF-PSMA-11 is being pursued clinically for prostate cancer imaging, a disease renowned for osteoblastic lesions where the potential for the mischaracterisation of metastatic disease by off-target bone uptake may be high; based on this, one may argue that we can "make do" with the chelators we already have. On the other hand, perhaps [18F]AlF-PSMA-11 represents a scenario where we have placed the "horse before the cart" because the demand for [18F]AlF-based PSMA radiopharmaceuticals is such that we are willing to take the path of least resistance and accept the limitations of sub-optimal chelation because the necessary radiochemistry precursors are commercially available at this moment in time, to support clinical studies. We don't think that this detracts from the implementation of optimal [18F]AlF chelators in our radioconjugates, but rather supports the idea of rapid commercialisation of clinically important precursors for the community to implement and evaluate.</p><p>With ever increasing interest in radionuclide therapy and theranostic isotope pairs, for example 68Ga and 177Lu which both form stable chelates with DOTA, should we now focus our attention to develop chelators to seamlessly support [18F]AlF and 177Lu isotope pairs from a single precursor molecule? There is a precedent for this approach in the literature (Lepage et al. 2020). Again, consideration needs to be given to future isotope requirements and, in this case, the impact of switching from an imaging isotope to a therapeutic isotope (where there may be future changes in demand).</p><!><p>Automated methods are crucial if [18F]AlF-based radiopharmaceuticals are to be implemented within a centralized production model (Tshibangu et al. 2020; Allott et al. 2017). Despite extensive work in developing a raft of [18F]AlF-based radioligands for various targets, they are still not mainstream; an interesting communication by Hassan et al. (2019) discusses the challenge of implementing [18F]AlF-based radiopharmaceuticals in 'normal' research institutions or university hospitals even when NOTA-peptide conjugates are commercially available. They correctly highlight that the expertise required to implement the radiochemistry, and the manual nature [18F]AlF-radiochemistry reserves these radiopharmaceuticals for well-trained radiochemists and radiopharmacists in dedicated research institutions (Hassan et al. 2019). In time to come, we will see the commercialization of cassettes to produce [18F]AlF-radiopharmaceuticals on automated radiosynthesis platforms, simplifying the production of these products. This may not be the answer all production facilities are looking for, as automated radiochemistry required a large initial financial investment with full systems costing upwards of £100 k ($140 k), and continual maintenance and consumable overheads; however, automated radiochemistry has been significantly cost-reduced by the emergence of smaller platforms specifically designed for metal-based radiochemistry. Gallium-68 radioconjugates can now be produced from commercially available cassettes and we envisage that [18F]AlF-radioconjugates will follow suit. If production scale is not a concern, then commercial and validated kits may provide a convenient solution. New technologies like microfluidic "lab-on-a-chip" devices which have been exemplified for metal-based radiochemistry may provide automated radiochemistry and quality control on a single, inexpensive device for clinical use (He et al. 2017, 2016; Zhang et al. 2020). These devices may also serve to improve the issues of fluoride concentration and efficiency of [18F]AlF labelling (He et al. 2014; Wang and Dam 2020). While these are not fully established and are not yet commercially available, they may serve to bring safe, standardised and automated GMP production to facilities with less extensive infrastructure, staffed by nuclear medicine technologists.</p><!><p>The [18F]AlF method has undoubtedly changed the landscape of fluorine-18 radiopharmaceutical development and adds another facet to metal-based radiolabelling that is relatively simple to implement and complements existing radiometal-based imaging agents. Over the last decade, the [18F]AlF method has risen to the status of an effective tool in radiopharmaceutical design and has all the characteristic hallmarks of longevity (i.e. implementation in new radiopharmaceutical development projects for a range of disease phenotypes, automated methods, extensive translation into clinical trials); while we cannot predict the future landscape of PET with any certainty, we hope to see [18F]AlF-based radiopharmaceuticals produced for the clinic on scale within the next 5–10 years.</p><!><p>Radiochemical yield</p><p>Specific activity</p><p>Molar activity</p><p>Pharmacokinetic</p><p>Decay corrected</p><p>Non-decay corrected</p><p>The authors identified an error in Table 2 and Figure 3. The The original article has been corrected.</p><p>Publisher's Note</p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p><p>Change history</p><p>9/24/2021</p><p>A Correction to this paper has been published: 10.1186/s41181-021-00148-7</p>