Explore the vast range of titles available.

PurposeFibroblast activation protein-α (FAP)-targeting radioligands have recently demonstrated high diagnostic potential. However, their therapeutic value is impaired by the short tumor residence time. Several strategies have been tested to overcome this limitation, but a head-to-head comparison has never been done. With the aim to identify strengths and limitations of the suggested strategies, we compared the monomer FAPI-46 versus (a) its dimer (FAPI-46-F1D), (b) two albumin binders conjugates (FAPI-46-Ibu (ibuprofen) and FAPI-46-EB (Evans Blue)), and (c) cyclic peptide FAP-2286.Methods177Lu-labeled ligands were evaluated in vitro in cell lines with low (HT-1080.hFAP) and high (HEK-293.hFAP) humanFAP expression. SPECT/CT imaging and biodistribution studies were conducted in HT-1080.hFAP and HEK-293.hFAP xenografts. The areas under the curve (AUC) of the tumor uptake and tumor-to-critical-organs ratios and the absorbed doses were estimated.ResultsRadioligands showed IC50 in the picomolar range. Striking differences were observed in vivo regarding tumor uptake, residence, specificity, and total body distribution. All [177Lu]Lu-FAPI-46-based radioligands showed similar uptake between the two tumor models. [177Lu]Lu-FAP-2286 showed higher uptake in HEK-293.hFAP and the least background. The AUC of the tumor uptake and absorbed dose was higher for [177Lu]Lu-FAPI-46-F1D and the two albumin binder conjugates, [177Lu]Lu-FAPI-46-Ibu and [177Lu]Lu-FAPI-46-EB, in HT1080.hFAP xenografts and for [177Lu]Lu-FAPI-46-EB and [177Lu]Lu-FAP-2286 in HEK293.hFAP xenografts. The tumor-to-critical-organs AUC values and the absorbed doses were in favor of [177Lu]Lu-FAP-2286, but tumor-to-kidneys.ConclusionThe study indicated dimerization and cyclic peptide structures as promising strategies for prolonging tumor residence time, sparing healthy tissues. Albumin binding strategy outcome depended on the albumin binding moiety. The peptide showed advantages in terms of tumor-to-background ratios, besides tumor-to-kidneys, but its tumor uptake was FAP expression–dependent.

We describe the development of OncoFAP, an ultra-high-affinity ligand of fibroblast activation protein (FAP) for targeting applications with pan-tumoral potential. OncoFAP binds to human FAP with affinity in the subnanomolar concentration range and cross-reacts with the murine isoform of the protein. We generated various fluorescent and radiolabeled derivatives of OncoFAP in order to study biodistribution properties and tumor-targeting performance in preclinical models. Fluorescent derivatives selectively localized in FAP-positive tumors implanted in nude mice with a rapid and homogeneous penetration within the neoplastic tissue. Quantitative in vivo biodistribution studies with a lutetium-177–labeled derivative of OncoFAP revealed a preferential localization in tumors at doses of up to 1,000 nmol/kg. More than 30% of the injected dose had already accumulated in 1 g of tumor 10 min after intravenous injection and persisted for at least 3 h with excellent tumor-to-organ ratios. OncoFAP also served as a modular component for the generation of nonradioactive therapeutic products. A fluorescein conjugate mediated a potent and FAP-dependent tumor cell killing activity in combination with chimeric antigen receptor (CAR) T cells specific to fluorescein. Similarly, a conjugate of OncoFAP with the monomethyl auristatin E-based Vedotin payload was well tolerated and cured tumor-bearing mice in combination with a clinical-stage antibody-interleukin-2 fusion. Collectively, these data support the development of OncoFAP-based products for tumor-targeting applications in patients with cancer.

The encoding of chemical compounds with amplifiable DNA tags facilitates the discovery of small-molecule ligands for proteins. To investigate the impact of stereo- and regiochemistry on ligand discovery, we synthesized a DNA-encoded library of 670,752 derivatives based on 2-azido-3-iodophenylpropionic acids. The library was selected against multiple proteins and yielded specific ligands. The selection fingerprints obtained for a set of protein targets of pharmaceutical relevance clearly showed the preferential enrichment of ortho-, meta- or para-regioisomers, which was experimentally verified by affinity measurements in the absence of DNA. The discovered ligands included novel selective enzyme inhibitors and binders to tumour-associated antigens, which enabled conditional chimeric antigen receptor T-cell activation and tumour targeting.A DNA-encoded chemical library based on regio- and stereoisomers of phenylalanine has been synthesized and used for affinity-based selections against multiple target proteins. This approach led to the isolation and validation of potent ligands capable of CAR T-cell activation and tumour targeting.

FAP-targeted radiopharmaceuticals represent a breakthrough in cancer imaging and a viable option for therapeutic applications. OncoFAP is an ultra-high-affinity ligand of FAP with a dissociation constant of 680 pM. OncoFAP has been recently discovered and clinically validated for PET imaging procedures in patients with solid malignancies. While more and more clinical validation is becoming available, the need for scalable and robust procedures for the preparation of this new class of radiopharmaceuticals continues to increase. In this article, we present the development of automated radiolabeling procedures for the preparation of OncoFAP-based radiopharmaceuticals for cancer imaging and therapy. A new series of [68Ga]Ga-OncoFAP, [177Lu]Lu-OncoFAP and [18F]AlF-OncoFAP was produced with high radiochemical yields. Chemical and biochemical characterization after radiolabeling confirmed its excellent stability, retention of high affinity for FAP and absence of radiolysis by-products. The in vivo biodistribution of [18F]AlF-NOTA-OncoFAP, a candidate for PET imaging procedures in patients, was assessed in mice bearing FAP-positive solid tumors. The product showed rapid accumulation in solid tumors, with an average of 6.6% ID/g one hour after systemic administration and excellent tumor-to-healthy organs ratio. We have developed simple, quick, safe and robust synthetic procedures for the preparation of theranostic OncoFAP-compounds based on Gallium-68, Lutetium-177 and Fluorine-18 using the commercially available FASTlab synthesis module.

Background Fibroblast activation protein (FAP)-targeting radioligands have gained attention for the ability to image multiple tumor types. Current FAP-targeting radioligands are labeled with 68 Ga and 18 F, but their short half-lives limit distribution range after production and later time-point imaging. This study describes the development Kalios, a novel class of NODAGA-conjugated FAP-targeting radioligands labeled with the cyclotron-produced Copper-61 (t 1/2  = 3.33 h), for greater temporal range for FAP-targeted imaging. Results Four Kalios ligands were synthesized and radiolabeled with [ 61 Cu]CuCl 2 in high yield and radiochemical purity within 5 min at room temperature. All radioligands demonstrated high hydrophilicity and strong affinity for FAP, and were primarily internalized after incubation with FAP-positive cells. PET/CT images obtained at 0–1 h and 4 h post-injection (p.i.) illustrated accumulation of all radioligands in FAP-positive tumors. Biodistribution studies of [ 61 Cu]Cu-Kalios-02 demonstrated stable tumor uptake between 1 and 4 h p.i., with washout from normal tissues at 4 h, resulting in improved tumor-to-background ratios. Conclusions Kalios ligands represent a new class of FAP-targeting 61 Cu-labeled radioligands. The half-life of 61 Cu allowed delayed 4-h imaging with improved tumor-to-background ratios. The improved delayed imaging and greater distribution range of these 61 Cu-labeled FAP-targeting radioligands demonstrates their clear potential for clinical translation, while combination with the therapeutic twin 67 Cu allows for truly paired Kalios theranostics.

Fibroblast activation protein (FAP)-targeting radioligands have gained attention for the ability to image multiple tumor types. Current FAP-targeting radioligands are labeled with Ga and F, but their short half-lives limit distribution range after production and later time-point imaging. This study describes the development Kalios, a novel class of NODAGA-conjugated FAP-targeting radioligands labeled with the cyclotron-produced Copper-61 (t  = 3.33 h), for greater temporal range for FAP-targeted imaging. Four Kalios ligands were synthesized and radiolabeled with [ Cu]CuCl in high yield and radiochemical purity within 5 min at room temperature. All radioligands demonstrated high hydrophilicity and strong affinity for FAP, and were primarily internalized after incubation with FAP-positive cells. PET/CT images obtained at 0-1 h and 4 h post-injection (p.i.) illustrated accumulation of all radioligands in FAP-positive tumors. Biodistribution studies of [ Cu]Cu-Kalios-02 demonstrated stable tumor uptake between 1 and 4 h p.i., with washout from normal tissues at 4 h, resulting in improved tumor-to-background ratios. Kalios ligands represent a new class of FAP-targeting Cu-labeled radioligands. The half-life of Cu allowed delayed 4-h imaging with improved tumor-to-background ratios. The improved delayed imaging and greater distribution range of these Cu-labeled FAP-targeting radioligands demonstrates their clear potential for clinical translation, while combination with the therapeutic twin Cu allows for truly paired Kalios theranostics.

Fibroblast activation protein (FAP)-targeting radioligands have gained attention for the ability to image multiple tumor types. Current FAP-targeting radioligands are labeled with 68Ga and 18F, but their short half-lives limit distribution range after production and later time-point imaging. This study describes the development Kalios, a novel class of NODAGA-conjugated FAP-targeting radioligands labeled with the cyclotron-produced Copper-61 (t1/2 = 3.33 h), for greater temporal range for FAP-targeted imaging.BACKGROUNDFibroblast activation protein (FAP)-targeting radioligands have gained attention for the ability to image multiple tumor types. Current FAP-targeting radioligands are labeled with 68Ga and 18F, but their short half-lives limit distribution range after production and later time-point imaging. This study describes the development Kalios, a novel class of NODAGA-conjugated FAP-targeting radioligands labeled with the cyclotron-produced Copper-61 (t1/2 = 3.33 h), for greater temporal range for FAP-targeted imaging.Four Kalios ligands were synthesized and radiolabeled with [61Cu]CuCl2 in high yield and radiochemical purity within 5 min at room temperature. All radioligands demonstrated high hydrophilicity and strong affinity for FAP, and were primarily internalized after incubation with FAP-positive cells. PET/CT images obtained at 0-1 h and 4 h post-injection (p.i.) illustrated accumulation of all radioligands in FAP-positive tumors. Biodistribution studies of [61Cu]Cu-Kalios-02 demonstrated stable tumor uptake between 1 and 4 h p.i., with washout from normal tissues at 4 h, resulting in improved tumor-to-background ratios.RESULTSFour Kalios ligands were synthesized and radiolabeled with [61Cu]CuCl2 in high yield and radiochemical purity within 5 min at room temperature. All radioligands demonstrated high hydrophilicity and strong affinity for FAP, and were primarily internalized after incubation with FAP-positive cells. PET/CT images obtained at 0-1 h and 4 h post-injection (p.i.) illustrated accumulation of all radioligands in FAP-positive tumors. Biodistribution studies of [61Cu]Cu-Kalios-02 demonstrated stable tumor uptake between 1 and 4 h p.i., with washout from normal tissues at 4 h, resulting in improved tumor-to-background ratios.Kalios ligands represent a new class of FAP-targeting 61Cu-labeled radioligands. The half-life of 61Cu allowed delayed 4-h imaging with improved tumor-to-background ratios. The improved delayed imaging and greater distribution range of these 61Cu-labeled FAP-targeting radioligands demonstrates their clear potential for clinical translation, while combination with the therapeutic twin 67Cu allows for truly paired Kalios theranostics.CONCLUSIONSKalios ligands represent a new class of FAP-targeting 61Cu-labeled radioligands. The half-life of 61Cu allowed delayed 4-h imaging with improved tumor-to-background ratios. The improved delayed imaging and greater distribution range of these 61Cu-labeled FAP-targeting radioligands demonstrates their clear potential for clinical translation, while combination with the therapeutic twin 67Cu allows for truly paired Kalios theranostics.

Nuclear medicine plays a key role in modern diagnosis and cancer therapy. The development of tumor targeting radionuclide conjugates (also named Small Molecule-Radio Conjugates - SMRCs) represents a significant improvement over the clinical use of metabolic radiotracers (e.g., [18F]-Fluorodeoxyglucose) for imaging and over the application of biocidal external beam radiations for therapy. During the discovery of SMRCs, molecular candidates must be carefully evaluated typically by performing biodistribution assays in preclinical tumor models. Quantification methodologies based on radioactive counts are typically demanding due to safety concerns, availability of radioactive material, and infrastructures. In this article, we report the development of a mass spectrometry (MS)-based method for the detection and quantification of small molecule-metal conjugates (SMMCs) as cold surrogates of SMRCs. We applied this methodology for the evaluation of the biodistribution of a particular class of tumor-targeting drug candidates based on natLu, natGa, natF and directed against Fibroblast Activation Protein (FAP). The reliability of the LC-MS analysis was validated by direct comparison of MS-based and radioactivity-based biodistribution data. Results show that MS biodistribution of stable isotope metal conjugates is an orthogonal tool for the preclinical characterization of different classes of radiopharmaceuticals. Competing Interest Statement D.N. is a cofounder and shareholder of Philogen S.p.A. (http://www.philogen.com/en/), a Swiss-Italian Biotech company that operates in the field of ligand-based pharmacodelivery. E.G., A.Z, A.G., T.S., J.M, S.C, and R.S. are employees of Philochem AG, the daughter company of Philogen, that owns and has patented OncoFAP, BiOncoFAP and their derivatives. No other potential conflicts of interest relevant to this article exist.

Imaging procedures based on small molecule-radio conjugates (SMRCs) targeting fibroblast activation protein (FAP) have recently emerged as a powerful tool for the diagnosis of a wide variety of tumours. However, the therapeutic potential of radiolabeled FAP-targeting agents is limited by their short residence time in neoplastic lesions. In this work, we present the development and in vivo characterization of BiOncoFAP, a new dimeric FAP-binding motif with extended tumour residence time and favorable tumour-to-organ ratio. Methods: The binding properties of BiOncoFAP and its monovalent OncoFAP analogue were assayed against recombinant hFAP. Preclinical experiments with [177Lu]Lu-OncoFAP-DOTAGA (177Lu-OncoFAP) and [177Lu]Lu-BiOncoFAP-DOTAGA (177Lu-BiOncoFAP) were performed in mice bearing FAP-positive HT-1080 tumours. OncoFAP and BiOncoFAP displayed comparable sub-nanomolar dissociation constants towards hFAP in solution, but the bivalent BiOncoFAP bound more avidly to the target immobilized on solid supports. In a comparative biodistribution study, 177Lu-BiOncoFAP exhibited a more stable and prolonged tumour uptake than 177Lu-OncoFAP (~20% ID/g vs ~4% ID/g, at 24h p.i., respectively). Notably, 177Lu-BiOncoFAP showed favorable tumour-to-organ ratios with low kidney uptake. Both 177Lu-OncoFAP and 177Lu-BiOncoFAP displayed potent anti-tumour efficacy when administered at therapeutic doses in tumour bearing mice. 177Lu-BiOncoFAP is a promising candidate for radioligand therapy of cancer, with favorable in vivo tumour-to-organ ratio, long tumour residence time and potent anti-cancer efficacy. Competing Interest Statement D.N. is a cofounder and shareholder of Philogen (http://www.philogen.com/en/), a Swiss-Italian Biotech company that operates in the field of ligand-based pharmacodelivery. A.G., A.Z., M.B., J. Millul, A.E., J. Mock and S.C. are employees of Philochem AG, the daughter company of Philogen that owns and has patented OncoFAP (PCT/EP2021/053494) and BiOncoFAP (PCT/EP2022/053404). No other potential conflicts of interest relevant to this article exist.