Evaluation of [¹⁸F]AlF NOTA-5G, an Aluminum [¹⁸F]fluoride Labeled Peptide Targeting the Cell Surface Receptor Integrin Alpha(v)beta(6) for PET Imaging

Abstract

Purpose

Peptide-based probes targeting integrin α_vβ₆ have shown promise in clinical trials for cancer imaging based on the high over-expression of this epithelial-specific cell surface receptor in many cancerous tissues. Recently, the α_vβ₆-targeting gallium-68 labeled DOTA-5G peptide, [⁶⁸Ga]Ga DOTA-5G, demonstrated diagnostic value in patients with metastatic pancreatic cancer. To facilitate adoption at sites without access to gallium-68 and take advantage of the characteristics of fluorine-18 through convenient [¹⁸F]fluoride chelation chemistry, this study evaluated the fluorine-18 labeled analog, [¹⁸F]AlF NOTA-5G, in vitro and in vivo in a tumor mouse model, and compared it to [⁶⁸Ga]Ga DOTA-5G.

Procedures

NOTA-5G was synthesized on solid phase and radiolabeled with aluminum [¹⁸F]fluoride to generate [¹⁸F]AlF NOTA-5G. Cell binding and internalization of [¹⁸F]AlF NOTA-5G were evaluated in paired DX3puroβ6 (α_vβ₆ +) and DX3puro (α_vβ₆ −), and pancreatic BxPC-3 (α_vβ₆ +) cells. Imaging (1–6 h) and biodistribution were performed in BxPC-3 tumor-bearing mice.

Results

[¹⁸F]AlF NOTA-5G was obtained in > 93% radiochemical purity. Cell binding was α_vβ₆-targeted (1 h: 66% bound to DX3puroβ6, vs 2% to DX3puro), and ≥ 50% of bound activity was internalized; analogous to [⁶⁸Ga]Ga DOTA-5G, PET imaging showed clearly delineated tumors. Excretion remained primarily renal (1 to 4 h: 18.6 to 12.5% ID/g). Tumor uptake remained relatively steady (1 to 4 h: 2.3 ± 0.4 to 1.8 ± 0.6% ID/g – closely matching [⁶⁸Ga]Ga DOTA-5G with 2.6 ± 0.8 and 2.0 ± 0.6% ID/g at 1 and 2 h), resulting in tumor/pancreas, tumor/liver, and tumor/blood ratios of 18/1, 24/1, and 162/1, respectively (4 h); by comparison, for [⁶⁸Ga]Ga DOTA-5G the values were 21/1, 20/1, and 22/1 (2 h).

Conclusions

[¹⁸F]AlF NOTA-5G demonstrated selective α_vβ₆-targeting and tumor uptake similar to [⁶⁸Ga]Ga DOTA-5G. The tumor-to-background ratio resulted high-contrast PET images, with an extended imaging window compared to [⁶⁸Ga]Ga DOTA-5G. The synthesis of [¹⁸F]AlF NOTA-5G is currently being optimized for clinical production.

Introduction

Peptide-based radiopharmaceuticals are successfully targeting cancer-associated cell surface receptors with high affinity and selectivity [1, 2]. The epithelial-specific cell surface receptor integrin α_vβ₆ has been identified as significantly up-regulated in a range of malignancies, along with generally no to low expression in healthy adult tissues. It has been found to promote progression and metastasis, and expression levels correlate negatively with outcome in several malignancies, including breast, cervical, lung, gastric, colorectal, prostate, and pancreatic cancer; making the integrin α_vβ₆ a valuable target for both detection and treatment of such malignancies [3–8]. For example, over 50% of cervical and lung cancer samples have been shown to express integrin α_vβ₆ [6], and over 80% of the pancreatic cancer samples [7, 8]. Most recently, the integrin α_vβ₆-targeting gallium-68 labeled DOTA-5G peptide has been validated pre-clinically [9] and demonstrated diagnostic value in patients with metastatic pancreatic cancer as a positron emission tomography (PET) imaging agent, identifying patients that are more likely to benefit from the theranostic β⁻-radiotherapy partner [¹⁷⁷Lu]Lu DOTA-ABM-5G (NCT04665947, NCT06228482, and NCT06389123).

While radiolabeling with positron emission tomography (PET)-radiometals is generally rapid, simple, and convenient, large scale or on-demand preparations can be impeded by practical issues, such as requiring either costly on-site radiometal generators or long distance shipping from a central manufacturer. Gallium-68 in particular, while providing on-demand availability, at present still generally requires a gallium-68 generator, which, at best, can deliver sufficient radiometal amounts (in the range of a few gigabecquerels) to prepare only a few PET imaging doses at a time that must be used within a short time after preparation (typically 2 h) due to the short half-life of 68 min (Table 1) [10, 11]. Furthermore, particularly when compared to fluorine-18, the decay energy of gallium-68 is relatively high which leads to comparatively lower-resolution PET images [12]. Fluorine-18 with its 110-min half-life is widely available through a vast network of academic and industrial sites in 10s-to-100s of gigabecquerel amounts on all six populated continents [13], thus covering many geographic areas where gallium-68 is not readily available. Furthermore, over the last decade simplified fluorine-18 labeling strategies have matured, including the radiometal-like capture of cationic fluorine-18 species by macrocyclic chelators such as NOTA (Fig. 1) [14–17].

Table 1.

Comparison of key characteristics of [¹⁸F]AlF NOTA-5G and the previously reported [⁶⁸Ga]Ga DOTA-5G [9], and the fluorine-18 and gallium-68 radioisotopes used [11–13]

Key characteristics		[18F]AlF NOTA-5G	[68Ga]Ga DOTA-5G
Stage		Preclinical (this study)	Clinical trials (NCT04665947, NCT06228482, NCT06389123)
Radiolabeling		[18F]AlF: a simple F-18 labeling	Simple radiometal chelation
Isotope decay properties	T1/2 = Mean β+ range = Positron yield =	109.7 min0.27 mm (soft tissue) 96.9%	67.7 min1.05 mm (soft tissue) 89.1%
Typical isotope production scale	Per synthesis: Availability:	10 s to 100 s of gigabecquerels (100 s of millicuries to curies) Many sites & regional shipping	Few gigabecquerels (10 s of millicuries) On-site, limited number of sites
Additional considerations		NOTA compatible with narrower range of radioisotopes	DOTA compatible with many therapeutic radioisotopes (e.g.: Lu-177)

Fig. 1.

(A) Radiosynthesis and structure of [¹⁸F]AlF NOTA-5G, and comparison with [⁶⁸Ga]Ga DOTA-5G. (B) Analytical radio-HPLC trace of purified [¹⁸F]AlF NOTA-5G (X-axis: elution time, min; Y-axis: radioactive signal, V, arbitrary scale; Rt = 16.5 min)

In order to facilitate adoption at sites without a gallium-68 generator and take advantage of availability and decay characteristics of fluorine-18 through fast and straightforward aluminum [¹⁸F]fluoride chelation chemistry, this study evaluated the fluorine-18 labeled [¹⁸F]AlF NOTA-5G in vitro and in vivo. Following cell binding and internalization experiments, PET/computed tomography (CT) imaging and biodistribution studies were done with [¹⁸F]AlF NOTA-5G in mice bearing integrin α_vβ₆-expressing BxPC-3 cell xenograft tumors, and the results compared to those previously obtained for [⁶⁸Ga]Ga DOTA-5G [9].

Materials and Methods

General

Reagents and solvents were purchased from Millipore Sigma (St. Louis, MO, USA), Fisher Scientific (Pittsburgh, PA, USA), or Acros/VWR (Radnor, PA, USA) unless stated otherwise. NovaSyn TGR R resin was used with 9-Fluorenylmethoxycarbonyl (Fmoc)-protected L-amino acids bearing the following amino-acid side-chain protections: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine, tert-butyl ester (OtBu) for aspartic acid, tertbutyl (tBu) for threonine, trityl (Trt) for asparagine and glutamine. Monodisperse Fmoc-amino-PEG-propionic acid (Fmoc-PEG-COOH; FW = 1544.8 g/mol) was purchased from Polypure (Oslo, Norway); 2-(4,7-bis(2-(tertbutoxy)-2-oxoethyl)-1,4,7-triazonan-1-yl)acetic acid (NOTA-bis(tert-butyl ester)) was purchased from Macrocyclics (Plano, TX, USA). Biotinylated latency associated protein (Bt-LAP) was purchased from G&P Biosciences (Santa Clara, CA, USA) and Bt-Vitronectin from ACRObiosystems (Newark, DE, USA). [¹⁸F]fluoride ion in ¹⁸O-water was either produced in-house on a Siemens RDS111 cyclotron (Siemens Medical Solutions, Knoxville, TN, USA) or supplied by PETNET Solutions (Sacramento, CA, USA). CHROMAFIX 30-PS-HCO3 cartridges were purchased from ABX (Radeberg, Germany) and Sep-Pak C18 Plus cartridges were purchased from Waters (Milford, MA, USA). Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), Penicillin–Streptomycin-Glutamine (PSG), and phosphate buffered saline (PBS) were purchased from Gibco/Thermo Fisher (Waltham, MA, USA). Growth factor reduced (GFR) Matrigel was purchased from Corning (item #354230; Corning, NY, USA). A Wizard 1470 or 2470 γ-counter (Perkin-Elmer, Waltham, MA, USA) was used to count radioactivity in solution, cell-binding, and biodistribution samples.

Mass spectrometry analysis was performed at the UC Davis Mass Spectrometry Facility using a matrix assisted laser desorption ionization time of flight (MALDI TOF) spectrometer (UltraFlextreme; Bruker, Billerica, MA, USA; 4700 Mass Spectrometer; Applied Biosystems/Thermo Fisher) in positive ionization mode using an α-cyano-4-hydroxycinnamic acid or sinapinic acid matrix.

The cell lines have been described previously [18, 19]. Cells were cultured at 37 °C and 5% CO₂ in medium containing 10% fetal bovine serum and 1% PSG; DMEM containing puromycin (2 mg/mL) was used for the DX3puro and DX3puroβ6 cells, and RPMI was used for the BxPC-3 cells. Integrin expression was confirmed prior to experiments by flow cytometry using a FACS Canto instrument (BD, Franklin Lakes, NJ, USA) and the data processed using FlowJo v10.8.1 (BD).

Flow cytometry and chromatography methods are provided in the electronic supplementary material.

Chemistry and Radiochemistry

The NOTA-5G peptide was synthesized manually following previously described solid-phase peptide synthesis Fmoc chemistry [20, 21]. The non-radioactive AlF NOTA-5G was prepared using aluminum chloride and potassium fluoride, and was used as non-radioactive reference spike for HPLC [22]. [¹⁸F]fluoride was eluted with aqueous sodium acetate (0.5 M, pH 4.1, 0.5 mL) from a CHROMAFIX 30-PS-HCO3 cartridge, and [¹⁸F]AlF NOTA-5G was prepared, purified, and formulated following previously described methods (Fig. 1) [21, 22].

In vitro Studies (logD_7.4, ELISA, Cell Binding and Internalization)

The lipophilicity of [¹⁸F]AlF NOTA-5G was determined as the partition coefficient logD_7.4 in an octanol/PBS system as previously described [22]. Competitive binding Enzyme Linked Immunosorbent Assays (ELISAs) for integrin α_vβ₆ and α_vβ₃ followed previously described methods [19] using Bt-LAP (for α_vβ₆-ELISA) or Bt-Vitronectin (for α_vβ₃-ELISA), and were analyzed using Prism software (GraphPad, Boston, MA, USA) by performing a sigmoidal fit of the experimental binding data over the range of peptide concentrations to determine the half maximal inhibitory concentration (IC₅₀) against the respective biotinylated natural ligands. Binding of [¹⁸F]AlF NOTA-5G to and internalization into DX3puroβ6 (α_vβ₆ +) and DX3puro (α_vβ₆ −), and pancreatic BxPC-3 (α_vβ₆ +) cells were determined as previously described [21, 23] using quadruplicate experiments with 3.75 × 10⁶ cells/experiment for each cell line, phosphate buffered saline with calcium and magnesium, and non-fat dry milk powder for blocking [22].

In vivo Studies

All animal procedures conformed to the Animal Welfare Act and were approved by the University of California Davis Institutional Animal Care and Use Committee. Female athymic nude mice (Charles River Laboratories; Wilmington, MA, USA) were inoculated subcutaneously with 5 × 10⁶ BxPC-3 cells in RPMI/Matrigel (1/1 v/v). Studies commenced once tumors reached a maximum diameter of ~ 0.5 cm, at approx. 3 weeks post inoculation.

The radiotracer [¹⁸F]AlF NOTA-5G formulated in PBS was injected into the tail vein of mice anesthetized with isoflurane in medical grade oxygen (imaging: 7–8 MBq/animal; biodistribution: 2.2–3.3 MBq/animal) while the injected mass was kept constant at 0.175 nmol per injection (corresponding to 7 nmol/kg for a 25-g mouse) through addition of nonmetallated compound to the radiotracer solution [22]. For imaging, the animals were placed in a prone position (triplicate; anesthesia: approx. 1.5–2.0% isoflurane) and scanned concurrently using a GNEXT PET/CT scanner (SOFIE, Dulles, VA, USA; PET scans: 15-min at 1, 2, and 4 h p.i., 30-min at 6 h p.i.) Imaging data were analyzed as previously described using the AMIDE Medical Image Data Examiner (v1.0.6; [24]).

For biodistribution studies mice were euthanized and dissected 1, 2, and 4 h p.i. (quadruplicate/time point). Tissues were collected, rinsed, and radioactivity measured in a γ-counter; calibrated, decay-corrected radioactivity concentrations are expressed as percent of injected dose per gram of sample (% ID/g). For blocking studies 8.36 μmol/kg NOTA-5G peptide was injected intravenously (n = 2; 110 μL saline solution) 10 min before administration of [¹⁸F]AlF NOTA-5G, and animals were sacrificed 1 h p.i. and tissues handled and analyzed as described above.

Statistical analysis

Quantitative data are reported as mean ± standard deviation (SD). Statistical analysis was done using a paired two-tailed Student’s t-tests to evaluate statistical significance, where P < 0.05 was considered statistically significant.

Results

Chemistry and Radiochemistry

The NOTA-5G peptide was obtained in ≥ 98% purity (UV – 220 nm) after HPLC purification (MALDI mass spec: calculated M + H⁺ (C₂₂₂H₄₁₅N₄₀O₉₁) = 5097.9076, found = 5098.9114). The non-radioactive AlF NOTA-5G was obtained in ≥ 98% purity (UV – 220 nm) after HPLC purification (MALDI mass spec: calculated M + Na⁺ (C₂₂₂H₄₁₂AlFN₄₀NaO₉₁) = 5163.8538, found = 5163.3629). [¹⁸F]AlF NOTA-5G was produced > 93% radiochemical purity after HPLC purification.

In vitro Studies (logD_7.4, ELISA, Cell Binding and Internalization)

The lipophilicity, determined by the octanol/PBS partitioning method, was measured as logD_7.4 = −2.34 ± 0.08. In vitro affinity and selectivity were determined by competitive ELISA against the biotinylated natural integrin ligands Bt-LAP and Bt-Vitronectin. Affinity (IC₅₀) for α_vβ₆ was 5.2 ± 1.9 nmol/L; whereas the IC₅₀ for integrin α_vβ₃ was > 40 μmol/L (Fig. 2A), resulting in an IC₅₀-ratio of > 7700/1. Cell-binding studies with [¹⁸F]AlF NOTA-5G for the matched pair of DX3puroβ6 and DX3puro cell lines showed 66.2 ± 0.8% of total radioactivity bound to α_vβ₆( +) DX3puroβ6 cells, compared to 2.0 ± 0.2% for α_vβ₆(−) DX3puro cells (ratio = 33/1; Fig. 2B); for pancreatic α_vβ₆( +) BxPC-3 cells 29.1 ± 3.7% binding were observed (Fig. 2C). These observations are in good correlation with the different integrin α_vβ₆ expression levels of these cell lines, namely a receptor density of ~ 40,000 and < 100 receptors/cell, respectively, for DX3puroβ6 and DX3puro cells [25], and ~ 12,000 receptors/cell for BxPC-3 [26]. For the α_vβ₆( +) DX3puroβ6 and BxPC-3 cells, ≥ 50% of radioactivity was internalized at 1 h at 37 °C, and the ratio of internalization for DX3puroβ6 vs DX3puro was 96/1.

Fig. 2.

(A) Competitive ELISA towards integrin α_vβ₆ and α_vβ₃ against biotinylated LAP and Vitronectin, respectively. The IC₅₀s were 5.18 ± 1.85 nM and 40.3 ± 18 μM, respectively. (B & C) Cell binding (black) and internalization (gray) of [¹⁸F]AlF NOTA-5G at 1 h for the pair of α_vβ₆( +) DX3puroβ6 and α_vβ₆(−) DX3puro cell lines, and the α_vβ₆( +) BxPC-3 cell line. Data are shown as the fraction of total radioactivity in the experiment for binding (black) and internalization (gray) as mean ± standard deviation (n = 4/experiment)

In vivo Studies

The PET images of [¹⁸F]AlF NOTA-5G showed clearly delineated tumors as early as 1 h p.i. and throughout the studied time frame (up to 6 h p. i.), along with notable uptake in the kidneys and the gastro-intestinal (GI) tract (Figs. 3, S4). Biodistribution data confirmed the PET imaging observations, including primarily renal excretion (Fig. 4). Radioactivity levels in the kidneys dropped from 18.6 ± 6.4 to 12.5 ± 1.8% ID/g (1 to 4 h p. i., p > 0.05), and bladder voiding resulted in elimination of over 60% of injected radioactivity (decay corrected) from the body as early as 1 h p. i. (Figs. 3B & S3). By 4 h p.i., 80% of injected radioactivity had been excreted. This coincided with rapid clearing from the blood, where radioactivity levels dropped from a low 0.16 ± 0.03% ID/g at 1 h p.i. further to 0.01 ± 0.01% ID/g at 4 h p.i. For the later time points, excretion through the GI tract played an increasing role, with radioactivity in the feces increasing significantly from 0.7 ± 0.1 to 5.0 ± 2.1% ID/g from 1 to 4 h p. i., respectively (p = 0.007). Aside from the kidneys and the GI tract, tissues, including the bone, had radioactivity levels ≤ 1.6% ID/g at all time points.

Fig. 3.

(A) Representative PET/CT maximum intensity projections of mice bearing BxPC-3 tumors injected with 7–8 MBq [¹⁸F]AlF NOTA-5G at 1, 2, 4, and 6 h p. i., and comparison with [⁶⁸Ga]Ga DOTA-5G in the same mouse model (2 h p. i.; coronal PET/CT cross section; K = kidneys; used with permission from Ganguly et al. originally published in The Journal of Nuclear Medicine [9]; tumors are indicated by arrows; PET data are shown in red, CT data in gray). (B) Radioactivity remaining in the body at various times post injection determined by whole-body measurement. Data, expressed as decay-corrected percentage of injected dose (dc %ID), are shown for individual animals during the PET/CT study immediately prior to the PET/ CT scans (x-axis: time p. i. (h), y-axis: remaining radioactivity (dc % ID))

Fig. 4.

Biodistribution data of [¹⁸F]AlF NOTA-5G in mice bearing BxPC-3 tumors injected with 2.2–3.3 MBq [¹⁸F]AlF NOTA-5G/animal. Uptake is expressed as decay corrected percentage of injected dose per gram of tissue (dc % ID/g) ± standard deviation (n = 4/time point)

The biodistribution data showed that tumor uptake remained relatively steady at 2.3 ± 0.4 to 1.8 ± 0.6% ID/g from 1 to 4 h p. i., respectively (p > 0.05), resulting in a tumor/blood ratio of 162/1, while the ratios of tumor/muscle, tumor/pancreas, and tumor/liver were 6/1, 18/1, and 24/1, respectively at 4 h. Tumor uptake could be blocked by 82% at 1 h p.i. by pre-administration of non-radioactive peptide (Table S1).

Discussion

Widespread availability and convenient manufacturing are both key factors to consider during the development of radiopharmaceuticals to enable them to reach their full potential. For peptides, radiolabeling with gallium-68 for clinical PET imaging is particular popular and very convenient [27], but at present generally requires access to an on-site bench-top gallium-68 generator [10, 11]. A typical generator can deliver sufficient gallium-68 for at best only a few doses at a time, and the doses must be used within a short time after preparation (typically 2 h) due to the short half-life of 68 min. While several PET radiometal isotopes can be considered to complement gallium-68, they also come with certain limitations of on-demand availability, production sites, or sub-optimal decay characteristics (including low positron yield and high-energy decay) [28].

Meanwhile, fluorine-18 is very widely available in large quantities, and its decay characteristics include a favorable low-energy decay and a 110 min half-life [12, 13]. Over the past several years, [¹⁸F]fluoride radiolabeling chemistries have been developed, simplified, and refined based on the capture of ionic [¹⁸F]fluoride in aqueous solution [14, 16, 29]; they have proven themselves to be convenient and fast routes to synthesize fluorine-18 radiolabeled peptides. Among them, the aluminum [¹⁸F]fluoride-based approach is particularly similar to radiometallation because the aluminum [¹⁸F]fluoride species can be considered a pseudo-radiometal [15, 17, 30, 31]. Therefore, aluminum [¹⁸F]fluoride labeled peptides offer the promise of being twins of the matched gallium-68 labeled peptides [30, 32]. This, together with the aforementioned need for wider availability, has led the molecular imaging community to prepare a range of aluminum [¹⁸F]fluoride labeled peptides targeting clinically important receptors, and compare them to their gallium-68 labeled analogs in preclinical and clinical studies with good results [17]. The present study evaluated the peptide [¹⁸F]AlF NOTA-5G and compared it to [⁶⁸Ga]Ga DOTA-5G which has previously been validated as targeting the cancer-associated cell surface receptor integrin α_vβ₆ in preclinical studies [9]; based on these studies, [⁶⁸Ga]Ga DOTA-5G was subsequently advanced to clinical trials in patients with metastatic cancer as a positron emission tomography (PET) imaging agent, identifying patients that are more likely to benefit from the theranostic β⁻-radiotherapy partner [¹⁷⁷Lu]Lu DOTA-ABM-5G (NCT04665947, NCT06228482, and NCT06389123).

Prepared by established radiolabeling methods, [¹⁸F]AlF NOTA-5G was obtained in > 93% radiochemical purity, and maintained the anticipated high integrin α_vβ₆-selectivity in vitro. It was then evaluated in a mouse model bearing subcutaneous BxPC-3 tumors. Analogous to [⁶⁸Ga]Ga DOTA-5G in the same α_vβ₆( +) pancreatic cancer model, PET images obtained with [¹⁸F]AlF NOTA-5G clearly showed the tumors as early as 1 h p.i. and throughout the study (Fig. 3). For [¹⁸F]AlF NOTA-5G in the present study, this included the last time point at 6 h p.i. which maintained high image quality for the tumor – along with continued clearing from background tissues, kidneys, and the GI tract – thus effectively highlighting the various benefits of fluorine-18 and its favorable decay characteristics, including the expanded imaging time window.

Owing to the relatively hydrophilic nature as determined by the logD_7.4 experiment, circulating [¹⁸F]AlF NOTA-5G was cleared rapidly from the blood stream, primarily through the kidneys. In fact, bladder voiding was by far the major contributor to eliminating nearly two-thirds of the injected radioactivity as early as 1 h p.i. (Fig. S3). Overall, the pharmacokinetic profile of [¹⁸F]AlF NOTA-5G was generally similar to that of [⁶⁸Ga]Ga DOTA-5G, [9], i.e., good uptake and retention in the tumor, and elimination primarily through kidneys and secondarily through the GI tract. For both radiotracers, this resulted in tumor-to-organ ratios of greater than 15/1 for blood, liver, and pancreas at 2 h p.i. (and for [¹⁸F]AlF NOTA-5G the values further improved at 4 h p.i.). Importantly for [¹⁸F]AlF NOTA-5G, the bone uptake of < 0.2% ID/g at these time points demonstrated the in vivo stability of the aluminum [¹⁸F]fluoride chelation.

Overall, these preclinical results demonstrated that [¹⁸F]AlF NOTA-5G can be viewed as a fluorine-18 based equivalent of [⁶⁸Ga]Ga DOTA-5G. It is anticipated that [¹⁸F]AlF NOTA-5G will provide a practical and valuable alternative to [⁶⁸Ga]Ga DOTA-5G for imaging of the cancer-associated integrin α_vβ₆ at clinical sites where gallium-68 is not readily available or where multiple daily scans are likely, thus making [¹⁸F]AlF NOTA-5G an alternative companion diagnostic to the theranostic β⁻-radiotherapy [¹⁷⁷Lu]Lu DOTA-ABM-5G. To facilitate GMP manufacturing and the deployment of [¹⁸F]AlF NOTA-5G, work is currently underway to optimize the radiochemistry, notably by further simplifying the procedure by eliminating the need for HPLC and switching to an entirely cartridge-based purification.

Conclusion

[¹⁸F]AlF NOTA-5G was easily synthesized utilizing aluminum [¹⁸F]fluoride chelation chemistry, demonstrated integrin α_vβ₆ selective binding in vitro, and α_vβ₆-selective tumor uptake similar to [⁶⁸Ga]Ga DOTA-5G in vivo. These data suggest that [¹⁸F]AlF NOTA-5G complements [⁶⁸Ga]Ga DOTA-5G as a diagnostic imaging agent for the detection of a wide range of malignancies. Based on these results the synthesis is currently being adapted for GMP production for clinical deployment.

Supplementary Material

The online version contains supplementary material available at https://doi.org/10.1007/s11307-025-01989-3.

Data Availability

Data are available from the corresponding authors on reasonable request.