Preclinical evaluation of [¹⁸F]FB-A20FMDV2 as a selective marker for measuring α_Vβ₆ integrin occupancy using positron emission tomography in rodent lung

Integrins are a class of receptors that play an important role in cell-cell and cell-matrix interactions in all higher organisms. There are 24 known heterodimeric receptors composed of an alpha subunit and a beta subunit. In a subset of eight integrins, the Arg-Gly-Asp (RGD) sequence was identified as the minimal integrin-binding motif [1]. Integrin α_vβ₆ belongs to this RGD subset of the integrin family and is expressed principally on epithelial cells [2]. The similarity between the RGD binding regions presents a challenge to finding a ligand that binds with high selectivity and affinity to α_Vβ₆, as most RGD ligands previously described showed residual yet significant affinity to other RGD integrins as well [3, 4].

With the exception of the gastrointestinal tract, expression of α_Vβ₆ is not usually detected in healthy adult tissues by immunohistochemistry; its expression increases to detectable levels on injured epithelial cells. Increased expression of α_Vβ₆ contributes to fibrosis, tumorigenesis and metastasis [5‐9]. Kaplan-Meier analysis of integrin α_Vβ₆ mRNA expression in 488 colorectal carcinomas revealed a striking reduction in median survival time of patients with high integrin α_Vβ₆ expression [10]. Similarly, Kaplan-Meier analysis of integrin α_Vβ₆ immunohistochemistry of lung tissue sections from 43 subjects with idiopathic pulmonary fibrosis revealed that the extent of integrin α_Vβ₆ immunostaining was associated with increased mortality [11]. These data suggest that high levels of integrin α_Vβ₆ may identify subjects with progressive malignancy or fibrotic disease [10‐12]. However, assessment of integrin α_Vβ₆ expression has to date only been possible through focused and invasive biopsy of diseased tissue.

A non-invasive imaging method to assess integrin α_Vβ₆ expression such as PET/CT would have prognostic applications in clinical practice. In addition, α_Vβ₆ PET/CT could be used during early clinical development to assess target engagement of a new therapeutic inhibitor of α_Vβ_6. In fact, the work presented in this manuscript forms the preclinical basis for a recently completed clinical study: a First Time in Human (FTIH) study of inhaled GSK3008348 (an α_Vβ₆ inhibitor) in Healthy Volunteers and Idiopathic Pulmonary Fibrosis Patients (NCT02612051). In addition, such a non-invasive technique could be used in clinical assessment of disease severity, disease activity and prognosis. Thus, there is an increasing interest in developing a positron emission tomography (PET) ligand for non-invasive imaging of α_Vβ₆ expression for preclinical and clinical applications.

Several α_Vβ₆-targeting peptides have been identified to date, and their properties as ligands for PET and single photon emission computed tomography (SPECT) imaging have been investigated [4, 13‐15]. The synthetic 20-amino acid peptide A20FMDV2 (NAVPNLRGDLQVLAQKVART) is derived from foot-and-mouth disease virus (FMDV) and has been reported as a selective inhibitor of α_Vβ₆ in a pancreatic cancer xenograft model [16]. A20FMDV2 shows high binding affinity and good selectivity towards the α_vβ₆ integrin compared with the other members of the RGD integrin family, namely, α_vβ₃, α_vβ₅, α₅β₁ and α_IIbβ₃ [16, 17]. A20FMDV2 was initially labelled with fluorine-18, using 4-[¹⁸F]fluorobenzoic acid ([¹⁸F]FBA) and developed as a preclinical PET tracer for in vivo cancer imaging [16]. High-specific binding to α_Vβ₆ was demonstrated in in vitro cell binding assays, and [¹⁸F]FB-A20FMDV2 (otherwise known as [¹⁸F]IMAFIB and [¹⁸F]GSK2634673) was shown to selectively image α_Vβ₆-positive tumours in vivo in mice-bearing human melanoma xenografts [16]. Indium-111-labelled A20FMDV2 peptide is able to detect increased levels of α_vβ₆ integrin in the lungs of mice in the bleomycin-induced model of pulmonary fibrosis [18, 19]. This has been confirmed independently using radioligand binding assays where [³H]A20FMDV2 was shown to bind to α_Vβ₆ with high affinity (K_D: 0.22 nmol/l) and selectivity (at least 85-fold) for α_Vβ₆ over the other members of the RGD integrin family [20].

More recently, attempts have been made to improve the imaging properties of [¹⁸F]FB-A20FMDV2 as an α_Vβ₆ ligand by using different prosthetic groups and chelators for radiolabelling and by introducing spacers [17, 21‐27]. Furthermore, A20FMDV2 has been labelled with other PET and SPECT nuclides, and the effects of those on pharmacokinetics, metabolism and tumour uptake have also been investigated [17, 18, 21‐27]. While moderate improvements in pharmacokinetics were observed, [¹⁸F]FB-A20FMDV2 remains one of the most potent and selective α_Vβ₆ ligands reported to date [4]. The availability of a specific and selective PET ligand to delineate α_Vβ₆ integrin in humans in vivo would allow exploration of the role of this integrin receptor in disease and provide a means to support drug development activities aimed at targeting this integrin. To date, animal models of disease have involved the use of bleomycin to induce lung fibrosis. This model leads to significant weight loss in the animals and highly variable levels of fibrosis and requires significant resource investment to ensure optimal results. Initial evidence through our own efforts suggested that, despite the low tissue density and high blood compartment in the lung, sufficient α_Vβ₆ integrin may be expressed in healthy animals to allow determination of drug-associated occupancy. The ability to do so without the need for the bleomycin model would significantly improve the applicability of the technology and provide further confidence for clinical translation.

Here we report the translational preclinical characterisation and GMP-compliant manufacture of [¹⁸F]FB-A20FMDV2 in support of future clinical studies.

Details on materials including the precursor A20FMDV2 and the reference standard FB-A20FMDV2 (alternative identifiers: IMAFIB, GSK2634673) can be found in the Supplementary Information.

All experiments were carried out in accordance with the Animals (Scientific Procedures) Act 1986, in line with EU directive 2010/63/EU and approved by the Animal Welfare and Ethical Review Board of Imperial College London. Details can be found in the Supplementary Information.

The peptide NAVPNLRGDLQVLAQKVART (A20FMDV2), derived from the foot-and-mouth disease virus, has been identified as a potent and selective binder of α_vβ₆ [16, 20]. This has been confirmed in the current manuscript by evaluating affinity, selectivity and specificity of radiolabelled A20FMDV2 by in vitro competition binding assays, rodent in vivo heterologous block and both in vivo and ex vivo homologous block rodent studies. Furthermore, this work further demonstrated the high selectivity of radiolabelled A20FMDV2 for the α_vβ₆ integrin over the seven other RGD integrins.

Additionally, we have adapted the previously reported manual synthesis of [¹⁸F]FB-A20FMDV2 [16] to enable the successful implementation of an automated and EU GMP-compliant synthesis. This challenging radiosynthesis, to our knowledge, represents the first example of fully automated process for solid-phase [¹⁸F] radiolabelling of a peptide in a GMP setting.

Several challenges had to be overcome due to the use of a resin-bound precursor. To prevent blockages of the Modular-Lab™ valves and tubing by the rink amide resin and facilitate its separation from the crude reaction mixture, a fritted cartridge was mounted directly onto a three-way solenoid valve. This cartridge/valve assembly was then placed directly over a magnetic stirrer. To allow good reagent penetration and enhance the yield and efficiency of coupling, the resin beads were pre-swelled in a small volume of solvent during the set-up procedure [27, 36].

The use of TFA for resin cleavage and deprotection commonly results in peptides being delivered as trifluoroacetate salts. No permissible daily exposure (PDE) limit exists for TFA within ICH Q3C guidelines [32], and, consequently, there was a need to limit the amount of TFA in the final product. Generally, TFA can be removed by lyophilisation or in vacuo for an extended period. However, these options are not available within the time constraints for PET chemistry. Consequently, TFA was removed by performing anion exchange using a solid phase extraction (SPE) cartridge [37].

The quality control methods of [¹⁸F]FB-A20FMDV2 were implemented according to EU GMP guidelines, and a method for the determination of residual TFA in the final dose of [¹⁸F]FB-A20FMDV2 was implemented to demonstrate its successful removal (< 2 ppm in final product). In order to support translation of [¹⁸F]FB-A20FMDV2 to the clinic, an HPLC radiometabolite analysis method was also developed to allow generation of the parent arterial input function.

In order to estimate the magnitude and specificity of displaceable component for α_vβ₆ in vivo in rats, [¹⁸F]FB-A20FMDV2 scans were performed under baseline conditions and following administration of a pharmacological dose of either FB-A20FMDV2 or the anti-α_vβ₆ antibody (8G6). All three subjects in the homologous competition study exhibited a decrease in lung SUVR_30–60 compared to baseline conditions. Similarly, in the heterologous competition study, a decrease in lung SUVR_30–60 was observed in the three subjects with measurable levels of the 8G6 antibody in blood. A greater decrease in SUVR was observed for subject 6, which may be explained by the fact that the post-dose scan was performed 49 h after i.p. injection of 8G6 compared to the five other subjects which were scanned 24 h after i.p. injection. Subjects 2 and 5 did not have measurable levels of 8G6 antibody in blood and subsequently did not demonstrate the expected large reduction in signal following heterologous block. An interesting anomaly in this dataset is the decrease of ~45% was observed in subject 4 (which did not have quantifiable blood levels of the antibody, 8G6). The reason for this is unclear at this stage, but it is worth noting that subject 4 had a higher than expected SUVR_30–60 value of 1.89 at baseline. If this measurement were spuriously high, this may partially explain the larger than expected decrease in PET signal for this subject.

The ex vivo homologous block study demonstrated a heterogeneous uptake of the [¹⁸F]FB-A20FMDV2 radioligand throughout the rodent which was reduced in all organs under review following administration of unlabelled FB-A20FMDV2.

Taken as whole, the in vitro, ex vivo and in vivo datasets provide evidence to suggest that [¹⁸F]FB-A20FMDV2 can specifically and selectively label α_vβ₆ in healthy rat lung. These data further suggest that [¹⁸F]FB-A20FMDV2 may be used to demonstrate target engagement even in healthy tissues without the need for upregulation of integrin α_vβ_{6 in} an animal model of disease. This has two significant benefits: it reduces the burden on animals from this severe model (bleomycin induced lung fibrosis), and it avoids the very significant variability that is seen from this model [38].

Rodent dosimetry was conducted to estimate the human radiation exposure of [¹⁸F]FB-A20FMDV2. Analysis using OLINDA/EXM showed the bladder wall as the organ with the highest absorbed dose. The resulting calculated human effective dose for [¹⁸F]FB-A20FMDV2 was 33.5 μSv/MBq, which enables repeat scans in patients and healthy volunteers for occupancy and longitudinal studies. This has recently been confirmed in a first-in-human PET dosimetry study performed as part of the clinical translation of [¹⁸F]FB-A20FMDV2 where the effective dose was determined to be 0.022 mSv/MBq [39]. These findings also demonstrate that initial rodent dosimetry provides a safe estimation of human effective dose to support initial clinical studies.

This work provides the foundation for a series of ongoing clinical applications for the detection of integrin α_vβ₆ using [¹⁸F]FB-A20FMDV2 as radioligand in PET/CT studies.

The results reported in this paper demonstrate the utility of [¹⁸F]FB-A20FMDV2 to specifically and selectively delineate α_vβ₆ integrins in healthy and diseased lung tissue. Taken together, these results form the basis of ongoing clinical studies using [¹⁸F]FB-A20FMDV2 to measure α_vβ₆ integrin levels in fibrotic disease such as idiopathic lung fibrosis and liver fibrosis as well as the determination of occupancy from novel drug candidates. The ability to measure drug-target interaction of novel drug candidates in healthy animals using [¹⁸F]FB-A20FMDV2 provides a significant improvement over existing approaches using animal models of disease. Therefore, [¹⁸F]FB-A20FMDV2 may be used as a selective, specific and reversible PET ligand for the α_vβ₆ integrin and provides an imaging tool that can be utilised in humans to track clinical benefit of emerging therapeutics in debilitating and life-limiting diseases such as lung fibrosis.