Summary

The development of diagnostic and/or theranostic radiotracers targeting deregulated extra-cellular matrix (ECM) proteins has been growing increasingly attractive in the last years due to the high abundance of targets, easy accessibility and, most importantly, extensive involvement in many pathological conditions. In contrast to conventional antibodies that specifically target proteins of interest, fibronectin-binding peptides (FnBPs) consist of 3–4 kDa bacterial adhesins-derived peptides capable of distinguishing between different stretch-regulated conformations of fibronectin (Fn). The authors had previously discovered that certain FnBPs preferentially bind to relaxed Fn fibrils, therefore constituting highly attractive strain-sensitive tools for visualizing altered ECM tensional architecture within pathological tissues. In a previous study of the authors, they successfully developed [¹¹¹In]In-FnBPA5 as a Fn-targeting radiotracer for SPECT imaging of prostate cancer in xenografted mice. However, despite the Fn-specific uptake of [¹¹¹In]In-FnBPA5 in tumors as detected by SPECT/CT, the radiotracer also heavily accumulated in the kidneys.
Profound retention of radioactivity in the kidneys is a common drawback of low-molecular-weight antibody constructs (LMW-Abs) and peptide-based radiopharmaceuticals undergoing renal clearance, thus posing limitations to their in vivo applications. The undesired renal accumulation of radioactivity is mainly caused by the long renal residence time of radiometabolites generated upon proteolytic degradation of the parent radiotracer. Following unspecific glomerular filtration and subsequent reabsorption at the proximal tubular cells via the megalin receptor, the radiotracers are transported into the lysosomal compartment, where they persist until decayed or otherwise processed. Several strategies have been attempted to address this issue, including the co-infusion of competitive blockers of tubular reabsorption, such as cocktails of cationic amino acids, poly-glutamic acid chains or plasma expanders such as Gelofusine. Indeed, co-infusion of radioprotectors proved effective in clinics in lowering renal accumulation of radiotracers from early post-injection onwards. However, besides featuring the advantage of not requiring chemical modifications of the targeting agent, co-infusion of such pharmacological agents is unfortunately not universally efficacious and not exempt from side effects. Besides these, the majority of the strategies aiming to reduce renal retention of radiopharmaceuticals rely on chemical modifications of the parent radiotracers. Remarkably, modifications of the tracers net charge or charge distribution can be effective in achieving a partial blockage of tubular reabsorption, whereas covalent attachment of polyethylene glycol chains or albumin binding moieties have proved to extend their in vivo half-life, consequently affecting renal uptake. Aside from these strategies, the interposition of specific renal enzyme-metabolizable linkers between the radiometal-carrying prosthetic moiety and the targeting vector constitutes an elegant way to address this issue. Indeed, brush border membrane (BBM) enzyme-cleavable linkers, specifically designed to be cleaved off in the kidneys at the level of the proximal tubular BBM and to release the radiometal-chelator complex from the vehicle, have been successfully employed for reducing renal uptake and retention of radiolabeled LMW-Abs as well as peptides. With respect to this, the tripeptide MVK was identified by Arano et al. as the most effective cleavable linkage for LMW-Abs labelled with gallium-67/68 by means of p-SCN-Bn-NOTA. Notably, their ^67/68Ga-labelled MVK-featuring construct revealed a striking reduction of the renal retention already at early post-injection times, without any impairments in terms of tumor uptake with respect to the control. The effectiveness of the strategy is based on the selective recognition and cleavage of MVK at the Met-Val bond by neprilysin (NEP), a metalloendopeptidase abundantly expressed on the BBM proximal convoluted tubules. Upon recognition of the substrate, NEP leads to the generation of the radiometabolite [^67/68Ga]Ga-NOTA-Bn-M, displaying a fast elimination rate from the coated vesicle of the renal cells into the urinary tract. Besides Abs-based constructs, the same approach was also found to be effective for peptides, such as Exendin-4, as demonstrated by Zheng et al. in a subsequent study.
Inspired by these exciting findings and given the identical coordination chemistry between gallium-67/68 and indium-111 to the macrocyclic chelator NOTA, the authors applied the MVK strategy to the ¹¹¹In-labelled FnBPA5.1. In the present study, they evaluated the effect of single or dual renal cleavable MVK sequence(s) on the biodistribution of ¹¹¹In-labelled FnBPs. Upon applying slight modifications to the original design of the NOTA-MVK-featuring cleavable linker, a rapid synthetic procedure granting access to the scaffold almost entirely via solid-phase peptide synthesis (SPPS) was established in the authors' laboratories. Furthermore, two additional linkers equipped with a second MVK-based cleavable site were included for further enhancing the cleavage effect. The authors' hypothesis was that the dual MVK-based linkers would lead to an enhanced washout of radioactivity due to an enhanced local concentration of substrate at the BBM. The applicability of single and dual MVK strategies to ¹¹¹In-labelled FnBPs was first assessed in vitro, by means of a BBM enzyme mediated cleavage assay, then in vivo through SPECT/CT studies and finally by investigating the ex-vivo biodistribution of PC-3 tumor-bearing mice.

Results from the nanoScan SPECT/CT

For the SPECT-CT imaging, eleven-weeks-old female CD1 nu/nu mice were injected into the tail vein with 10–15 MBq of ¹¹¹In-labelled radiotracers, previously diluted with PBS to a concentration of 100–150 MBq/mL (n = 2 animals per group). At 24 h p.i., mice were subjected to a SPECT scan of 40 min, following an initial CT scan of 7.5 min with the NanoSPECT/CT camera. During scans, mice were kept under anesthesia with 1.5 %. isoflurane (Attane, Piramal Enterpises, India). Data were reconstructed with HiSPECT software (version 1.4.3049, SciVis GmbH, Göttingen, Germany) and analyzed using VivoQuant (version 3.0, inviCRO Imaging Services and Software, Boston USA). Scale of activity was set to 0 – 20 Bq/voxel.
The in vivo behavior of the ¹¹¹In-labelled MVK-based FnBPA5.1 conjugates A-C in comparison to the reference compound [¹¹¹In]In-FnBPA5.1 was at first evaluated in non-tumor bearing mice by means of single-photon emission computed tomography (SPECT) scans at 24 h p.i. (Fig. 3). To our delight, all MVK-based FnBPA5.1 radioconjugates A-C revealed reduced renal retention compared to the control peptide [¹¹¹In]In-FnBPA5.1, providing a proof-of-concept. Specifically, the lowest signal intensity within the kidneys was observed with the dual MVK featuring radioconjugates [¹¹¹In]In-FnBPA5.1-B (Fig. 3B) and [¹¹¹In]In-FnBPA5.1-C (Fig. 3C), with the former performing better than the latter. On the other hand, the radioconjugate equipped with a single MVK sequence [¹¹¹In]In-FnBPA5.1-A revealed the poorest reduction of signal intensity with respect to the control (Fig. 3A).

• The installation of dual MVK-based cleavable linkers between the Fn-targeting moiety and the chelating unit led to an enhanced washout of radioactivity from the kidneys and overall increased tumor-to-kidney ratio, thus resulting in the improved diagnostic probe [¹¹¹In]In-FnBPA5.1-B ([¹¹¹In]In-NOTA-Bn-MV-amBn-MVK(hex-FnBPA5.1)).
• Having proved effective on lowering ¹¹¹In-labelled FnBPs renal radioactivity levels, the authors demonstrated that their dual-MVK strategy is a crucial step towards the clinical translation of the mechano-sensory FnBPs.
• In addition, the authors are convinced that the herein presented dual MVK-based approach may be extended to other radiopharmaceuticals suffering from high renal retention.

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