A Dimeric FAP-Targeting Small-Molecule Radioconjugate with High and Prolonged Tumor Uptake

Abstract

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 [ 177 Lu]Lu-OncoFAP-DOTAGA ( 177 Lu-OncoFAP) and [ 177 Lu]Lu-BiOncoFAP-DOTAGA ( 177 Lu-BiOncoFAP) were performed in mice bearing FAP-positive HT-1080 tumours.Results: 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, 177 Lu-BiOncoFAP exhibited a more stable and prolonged tumour uptake than 177 Lu-OncoFAP (~20% ID/g vs ~4% ID/g, at 24h p.i., respectively). Notably, 177 Lu-BiOncoFAP showed favorable tumour-to-organ ratios with low kidney uptake. Both 177 Lu-OncoFAP and 177 Lu-BiOncoFAP displayed potent anti-tumour efficacy when administered at therapeutic doses in tumour bearing mice. Conclusions:177 Lu-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.

Mass Spectrometry-Based Method for the Determination of the Biodistribution of Tumor-Targeting Small Molecule–Metal Conjugates

“…Uptake values in tumor and healthy organs (in %ID/g) measured by the LC-MS methodology were comparable to values obtained with radioactivity-based quantification. 11 No significant differences were observed between the two analytical techniques (p > 0.05 multiple t-test, Table S5) (Figure 3). Orthogonal validation further confirms the reliability of the newly developed MS quantification method, thereby opening new opportunities for nonradioactive LC-MS analysis of SMMCs.…”

“…More recently, we developed a dimeric derivative owning two targeting moieties (i.e., BiOncoFAP), showing higher tumor uptake and residence time than OncoFAP in preclinical models of cancer. 11 The biodistribution properties of novel tumor-targeting SMRCs are of key importance for the success of the diagnosis and therapeutic outcome. radiopharmaceuticals in tumor-bearing animals is typically evaluated by direct measurement of the radioactivity present in each organ after sacrificing of experimental animals.…”

“…radiopharmaceuticals in tumor-bearing animals is typically evaluated by direct measurement of the radioactivity present in each organ after sacrificing of experimental animals. 10,11 To date, these radioactivity-based analyses are still crucial to evaluate tumor accumulation and to obtain spatial biodistribution data of the compounds in the animal/human body. On the contrary, for most of the other nonradioactive pharmaceuticals, liquid chromatography-mass spectrometry (LC-MS) analysis represents the analytical method of choice when it comes to accurately quantifying the analyte of interest in biological specimens.…”

“…OncoFAP and BiOncoFAP, as well as their metal chelator conjugates and "cold"-labeled derivatives, were synthesized through established protocols. 6,10,11 For the synthesis of internal standards (ISs), we used isotopically labeled building blocks. In particular, 13 C 4 -succinic anhydride was exploited for OncoFAP derivatives, while 13 C 6 15 N 2 -L-lysine was used for BiOncoFAP conjugates.…”

“…Our results further confirm the versatility of this class of molecules as targeted radiopharmaceuticals. 6,10,11 Biodistribution of [ nat F]AlF-OncoFAP-NODAGA and [ nat F]AlF-OncoFAP-NOTA. In addition to gallium-68 and lutetium-177, fluorine-18 is also frequently used for nuclear medicine applications in cancer patients.…”