Radiolabelling an 18F biologic via facile IEDDA "click" chemistry on the GE FASTLab™ platform.
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ABSTRACT:
SUBMITTER: Allott L
PROVIDER: S-EPMC8167423 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We have exemplified a pretargeted approach to interrogate hypoxia in live cells using radioactive bioorthogonal inverse electron demand Diels-Alder (IEDDA) "click" chemistry. Our novel 18F-tetrazine probe ([18F]FB-Tz) and 2-nitroimidazole-based TCO targeting molecule (8) showed statistically significant (P < 0.0001) uptake in hypoxic cells (ca. 90 %ID per mg) vs. normoxic cells (<10 %ID per mg) in a 60 min incubation of [18F]FB-Tz. This is the first time that an intracellularly targeted small-molecule for IEDDA "click" has been used in conjunction with a radioactive reporter molecule in live cells and may be a useful tool with far-reaching applicability for a variety of applications.
| S-EPMC8182949 | biostudies-literature
Project description:Purpose(S)-4-(3-18F-Fluoropropyl)-ʟ-Glutamic Acid ([18F]FSPG) is a radiolabeled non-natural amino acid that is used for positron emission tomography (PET) imaging of the glutamate/cystine antiporter, system xC-, whose expression is upregulated in many cancer types. To increase the clinical adoption of this radiotracer, reliable and facile automated procedures for [18F]FSPG production are required. Here, we report a cassette-based method to produce [18F]FSPG at high radioactivity concentrations from low amounts of starting activity.ProceduresAn automated synthesis and purification of [18F]FSPG was developed using the GE FASTlab. Optimization of the reaction conditions and automated manipulations were performed by measuring the isolated radiochemical yield of [18F]FSPG and by assessing radiochemical purity using radio-HPLC. Purification of [18F]FSPG was conducted by trapping and washing of the radiotracer on Oasis MCX SPE cartridges, followed by a reverse elution of [18F]FSPG in phosphate-buffered saline. Subsequently, the [18F]FSPG obtained from the optimized process was used to image an animal model of non-small cell lung cancer.ResultsThe optimized protocol produced [18F]FSPG in 38.4 ± 2.6 % radiochemical yield and >96 % radiochemical purity with a molar activity of 11.1 ± 7.7 GBq/μmol. Small alterations, including the implementation of a reverse elution and an altered Hypercarb cartridge, led to significant improvements in radiotracer concentration from <10 MBq/ml to >100 MBq/ml. The improved radiotracer concentration allowed for the imaging of up to 20 mice, starting with just 1.5 GBq of [18F]Fluoride.ConclusionsWe have developed a robust and facile method for [18F]FSPG radiosynthesis in high radiotracer concentration, radiochemical yield, and radiochemical purity. This cassette-based method enabled the production of [18F]FSPG at radioactive concentrations sufficient to facilitate large-scale preclinical experiments with a single prep of starting activity. The use of a cassette-based radiosynthesis on an automated synthesis module routinely used for clinical production makes the method amenable to rapid and widespread clinical translation.
| S-EPMC8578107 | biostudies-literature
Project description:A novel 7-((4-(3-((2-[18F]fluoropyridin-3-yl)oxy)propyl)-1H-1,2,3-triazol-1-yl)methyl)-1H-benzo[d]imidazole derivative of the angiotensin II type-1 receptor (AT1R) blocker candesartan, [18F]fluoropyridine-candesartan, was synthesized via the copper-catalyzed azide-alkyne cycloaddition click reaction between 2-[18F]fluoro-3-(pent-4-yn-1-yloxy)pyridine ([18F]FPyKYNE) and the tetrazole-protected azido-candesartan derivative, followed by acid deprotection. This three-step, two-pot, and two-step purification synthesis was done within 2 h. The use of tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine (THPTA) as a Cu(I) stabilizing agent increased the overall radiochemical yield by 4-fold (10 ± 2%, n = 13) compared to the reaction without THPTA (2.4 ± 0.2%, n = 3; decay-corrected from 18F produced at the end-of-beam). Complete separation of [18F]FPyKYNE from its nitro precursor and [18F]fluoropyridine-candesartan from the deprotected azido-candesartan allowed for high molar activities (>380 GBq/μmol) of the tracer. The use of 0.1% trifluoroacetic acid in water for reformulation and the addition of sodium ascorbate to the final formulation (1.6 ± 0.2 GBq/mL, n = 3) prevented tracer radiolysis with >97% radiochemical purity for a period of up to 10 h after the end-of-synthesis. A significant reduction in the uptake (86 ± 3%, n = 8) of the tracer was observed ex vivo in rats (at 20 min postinjection) in the AT1R-rich kidney cortex following pretreatment with saturating doses of the AT1R antagonist candesartan or losartan. This specific binding to AT1R was confirmed in vitro in the rat renal cortex (autoradiography) by a reduction of 26 ± 5% (n = 12) with losartan coincubation (10 μM). These favorable binding properties support further studies to assess the potential of [18F]fluoropyridine-candesartan as a tracer for the positron emission tomography imaging of renal AT1R.
| S-EPMC7439361 | biostudies-literature
Project description:Radiopharmaceuticals targeting the prostate-specific membrane antigen (PSMA) has become the gold standard for PET imaging of prostate cancer. [68Ga]Ga-PSMA-11 has been the forerunner but a [18F]F-PSMA ligand has been developed because of the intrinsic advantages of Fluorine-18. Fluorine-18 labelled compounds are usually prepared in centers with an on-site cyclotron. Since our center has not an on-site cyclotron, we decided to verify the feasibility of producing the experimental 18F-labelled radiopharmaceutical [18F]F-PSMA-1007 with [18F]F- from different external suppliers. A quality agreement has been signed with two different suppliers, and a well-established and correctly implemented quality assurance protocol has been followed. The [18F]F- was produced with cyclotrons, on Nb target, but with different beam energy and current. Extensive validation of the [18F]F-PSMA-1007 synthesis process has been performed. The aim of this paper was the description of all the quality documentation which allowed the submission and approval of the Investigational Medicinal Product Dossier (IMPD) to the Competent Authority, addressing the quality problems due to different external suppliers. The result indicates that no significant differences have been found between the [18F]F- from the two suppliers in terms of radionuclidic and radiochemical purity and [18F]F- impacted neither the radiochemical yield of the labelling reaction nor the quality control parameters of the IMP [18F]F-PSMA-1007. These results prove how a correct quality assurance system can overcome some Regulatory Authorities issue that may represent an obstacle to the clinical use of F-18-labelled radiopharmaceuticals without an on-site cyclotron.
| S-EPMC8308622 | biostudies-literature
Project description:Click reaction is a very fast, high yield with no by-product, biocompatible, tolerant to surrounded medium, and very specific cycloaddition reaction between azides and alkynes to form triazole. They are widely being employed in the synthesis of various polymeric materials. Here, the design, fabrication and characterization of hydrogel prepared using click reaction have been reported. At first, telechelic acetylene precursor for click reaction is prepared from diisocyanatohexane and propargyl alcohol in the presence of triethylamine. The azide derivatives of poly(hydroxyethylmethacrylate), i.e. poly(HEMA), are successfully prepared following two different routes. In route 1, esterification of bromopropionic acid is performed with HEMA monomer using N,N'-dicyclohexylcarbodiimide/4-dimethylaminopyridine (DCC/DMAP) as a catalyst followed by replacing bromide by azide moiety. Free radical polymerization of the fabricated monomer is then performed under N2 atmosphere using azobisisobutyronitrile (AIBN) as an initiator. In route 2, polymerization of HEMA has been carried out first, then modification of the polymer with azide group via successive steps to obtain azide derivative polymer for click reaction. The hydrogel is prepared by a very fast, highly specific, and simple click reaction between azide derivative polymer and telechelic acetylene precursor using copper as a catalyst. The structures of derivatives of azide-functionalized HEMA, acetylene precursors and hydrogels are confirmed by FTIR and 1H-NMR spectroscopy. The optimized structure of each precursor is determined, and their chemical and thermodynamic parameters are computationally studied in detail.
| S-EPMC8074938 | biostudies-literature
Project description:The facile conjugation of three azido modified functionalities, namely a therapeutic drug (methotrexate), a targeting moiety (folic acid), and an imaging agent (fluorescein) with a G5 PAMAM dendrimer scaffold with cyclooctyne molecules at the surface through copper-free click chemistry is reported. Mono-, di-, and tri-functional PAMAM dendrimer conjugates can be obtained via combinatorial mixing of different azido modified functionalities simultaneously or sequentially with the dendrimer platform. Preliminary flow cytometry results indicate that the folic acid targeted nanoparticles are efficiently binding with KB cells.
| S-EPMC3331967 | biostudies-literature
Project description:A first-of-its-kind (18)F pretargeted PET imaging approach based on the bioorthogonal inverse electron demand Diels-Alder (IEDDA) reaction between tetrazine (Tz) and trans-cyclooctene (TCO) is presented. As proof-of-principle, a TCO-bearing immunoconjugate of the anti-CA19.9 antibody 5B1 and an Al[(18)F]NOTA-labeled tetrazine radioligand were harnessed for the visualization of CA19.9-expressing BxPC3 pancreatic cancer xenografts. Biodistribution and (18)F-PET imaging data clearly demonstrate that this methodology effectively delineates tumor mass with activity concentrations up to 6.4 %ID/g at 4 h after injection of the radioligand.
| S-EPMC4759614 | biostudies-literature
Project description:Bispecific antibodies (BsAbs) for T cell engagement have shown great promise in cancer immunotherapy, and their clinical applications have been proven in treating hematological malignance. Bispecific antibody binding fragment (BiFab) represents a promising platform for generating non-Fc bispecific antibodies. However, the generation of BiFab is still challenging, especially by means of chemical conjugation. More conjugation strategies, e.g., enzymatic conjugation and modular BiFab preparation, are needed to improve the robustness and flexibility of BiFab preparation. We successfully used chemo-enzymatic conjugation approach to generate bispecific antibody (i.e., BiFab) with Fabs from full-length antibodies. Paired click handles (e.g., N3 and DBCO) was introduced to the C-terminal LPETG tag of Fabs via sortase A mediated transpeptidation, followed by site-specific conjugation between two click handle-modified Fabs for BiFab generation. Both BiFabCD20/CD3 (EC50 = 0.26 ng/mL) and BiFabHer2/CD3 exhibited superior efficacy in mediating T cells, from either PBMC or ATC, to kill target tumor cell lines while spared antigen-negative tumor cells in vitro. The BiFabCD20/CD3 also efficiently inhibited CD20-positive tumor growth in mouse xenograft model. We have established a facile sortase A-mediated click handle installation to generate homogeneous and functional BiFabs. The exemplary BiFabs against different targets showed superior efficacy in redirecting and activating T cells to specifically kill target tumor cells, demonstrating the robustness of sortase A-mediated "bio-click" chemistry in generating various potent BiFabs. This approach also holds promise for further efficient construction of a Fab derivative library for personalized tumor immunotherapy in the future.
| S-EPMC8467688 | biostudies-literature
Project description:PurposeInterest in targeted alpha-therapy has surged due to α-particles' high cytotoxicity. However, the widespread clinical use of this approach could be limited by on-/off-target toxicities. Here, we investigated the inverse electron-demand Diels-Alder ligation between an 225Ac-labeled tetrazine radioligand and a trans-cyclooctene-bearing anti-CA19.9 antibody (5B1) for pretargeted α-radioimmunotherapy (PRIT) of pancreatic ductal adenocarcinoma (PDAC). This alternative strategy is expected to reduce nonspecific toxicities as compared with conventional radioimmunotherapy (RIT).Experimental Design: A side-by-side comparison of 225Ac-PRIT and conventional RIT using a directly 225Ac-radiolabeled immunoconjugate evaluates the therapeutic efficacy and toxicity of both methodologies in PDAC murine models.ResultsA comparative biodistribution study of the PRIT versus RIT methodology underscored the improved pharmacokinetic properties (e.g., prolonged tumor uptake and increased tumor-to-tissue ratios) of the PRIT approach. Cerenkov imaging coupled to PRIT confirmed the in vivo biodistribution of 225Ac-radioimmunoconjugate but-importantly-further allowed for the ex vivo monitoring of 225Ac's radioactive daughters' redistribution. Human dosimetry was extrapolated from the mouse biodistribution and confirms the clinical translatability of 225Ac-PRIT. Furthermore, longitudinal therapy studies performed in subcutaneous and orthotopic PDAC models confirm the therapeutic efficacy of 225Ac-PRIT with the observation of prolonged median survival compared with control cohorts. Finally, a comparison with conventional RIT highlighted the potential of 225Ac-PRIT to reduce hematotoxicity while maintaining therapeutic effectiveness.ConclusionsThe ability of 225Ac-PRIT to deliver a radiotherapeutic payload while simultaneously reducing the off-target toxicity normally associated with RIT suggests that the clinical translation of this approach will have a profound impact on PDAC therapy.
| S-EPMC6343144 | biostudies-literature
Project description:Glutamine (Gln) and its analogues may serve as imaging agents for tumor diagnosis using positron emission tomography (PET), especially for tumors with negative [(18)F]FDG scan. We report the first automated synthesis of [(18)F](2S,4R)-4-fluoroglutamine ([(18)F]FGln) on a GE TRACERlab™ FX-N Pro module. [(18)F]FGln was obtained in 80±3min with a radiochemical yield of 21±3% (n=5, uncorrected). The radiochemical purity was >98%, and optical purity 90±5%. The synthesis is highly reproducible with good chemical purity, radiochemical yield, and is suitable for translation to cGMP production.
| S-EPMC4853275 | biostudies-literature