UNASSIGNED: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease of unknown cause with a dismal prognosis. Nintedanib and Pirfenidone are approved worldwide for the treatment of IPF, but they only slow the rate of functional decline and disease progression. Therefore, there is an urgent need for more efficacious and better tolerated drugs.
UNASSIGNED: αvβ6 and αvβ1 are two integrins overexpressed in fibrotic tissue, which play a critical role in the development of lung fibrosis. They act by converting transforming growth factor (TGF)-β, one of the most important profibrotic cytokine, in its active form. Here, we summarize and critically discuss the potential of a dual αvβ6/αvβ1 integrin inhibitor for the treatment of IPF.
UNASSIGNED: Bexotegrast, a dual αvβ6/αvβ1 integrin inhibitor, has the potential to slow or even halt disease progression in IPF. Indeed, the strong pre-clinical rationale and promising early phase clinical trial data have raised expectations.

UNASSIGNED: Head and neck squamous cell carcinoma (HNSCC) has a particularly poor prognosis. Improving the surgical resection boundary, reducing local recurrence, and ultimately ameliorating the overall survival rate are the treatment goals.
UNASSIGNED: To obtain a complete surgical resection (R0 resection), we investigated the use of a fluorescent imaging probe that targets the integrin subtype αvβ6, which is upregulated in many kinds of epithelial cancer, using animal models.
UNASSIGNED: αvβ6 expression was detected using polymerase chain reaction (PCR) and immunoprotein blotting of human tissues for malignancy. Protein expression localization was observed. αvβ6 and epidermal growth factor receptor (EGFR) were quantified by PCR and immunoprotein blotting, and the biosafety of targeting the αvβ6 probe material was examined using Cell Counting Kit-8 assays. Indocyanine green (ICG) was used as a control to determine the localization of the probe at the cellular level. In vivo animal experiments were conducted through tail vein injections to evaluate the probe\'s imaging effect and to confirm its targeting in tissue sections.
UNASSIGNED: αvβ6 expression was higher than EGFR expression in HNSCC, and the probe showed good targeting in in vivo and in vitro experiments with a good safety profile.
UNASSIGNED: The ICG-αvβ6 peptide probe is an exceptional and sensitive imaging tool for HNSCC that can distinguish among tumor, normal, and inflammatory tissues.

Integrins are necessary for cell adhesion, migration, and positioning. Essential for inducing signalling events for cell survival, proliferation, and differentiation, they also trigger a variety of signal transduction pathways involved in mediating invasion, metastasis, and squamous-cell carcinoma. Several recent studies have demonstrated that the up- and down-regulation of the expression of αv and other integrins can be a potent marker of malignant diseases and patient prognosis. This review focuses on an arginine-glycine-aspartic acid (RGD)-dependent integrin αVβ6, its biology, and its role in healthy humans. We examine the implications of αVβ6 in cancer progression and the promotion of epithelial-mesenchymal transition (EMT) by contributing to the activation of transforming growth factor beta TGF-β. Although αvβ6 is crucial for proper function in healthy people, it has also been validated as a target for cancer treatment. This review briefly considers aspects of targeting αVβ6 in the clinic via different therapeutic modalities.

The integrin αvβ6 is expressed at low levels in most normal healthy tissue but is very often upregulated in a disease context including cancer and fibrosis. Integrins use endocytosis and trafficking as a means of regulating their surface expression and thus their functions, however little is known of how this process is regulated in the context of αvβ6. As αvβ6 is a major target for the development of therapeutics in cancer and fibrosis, understanding these dynamics is critical in the development of αvβ6-targeted therapies. Following development of a flow cytometry-based assay to measure ligand (A20FMDV2 or LAP)-bound αvβ6 endocytosis, an siRNA screen was performed to identify which genes were responsible for internalising αvβ6. These data identified 15 genes (DNM2, CBLB, DNM3, CBL, EEA1, CLTC, ARFGAP3, CAV1, CYTH2, CAV3, CAV2, IQSEC1, AP2M1, TSG101) which significantly decreased endocytosis, predominantly within dynamin-dependent pathways. Inhibition of these dynamin-dependent pathways significantly reduced αvβ6-dependent migration (αvβ6-specific migration was 547 ± 128 under control conditions, reduced to 225 ± 73 with clathrin inhibition, and 280 ± 51 with caveolin inhibition). Colocalization studies of αvβ6 with endosome markers revealed that up to 6 h post-internalisation of ligand, αvβ6 remains in Rab11-positive endosomes in a perinuclear location, with no evidence of αvβ6 degradation up to 48 h post exposure to A20FMDV2. Additionally, 60% of ligand-bound αvβ6 was recycled back to the surface by 6 h. With studies ongoing using conjugated A20FMDV2 to therapeutically target αvβ6 in cancer and fibrosis, these data have important implications. Binding of A20FMDV2 seemingly removes much of the αvβ6 from the cell membrane, and upon its recycling, a large fraction appears to still be in the ligand-bound state. While these results are observed with A20FMDV2, these data will be of value in the design of αvβ6-specific therapeutics and potentially the types of therapeutic load.

A20FMDV2 is a 20-mer peptide that exhibits high selectivity and affinity for the tumour-related αvβ6 integrin that can compete with extracellular ligands for the crucial RGD binding site, playing a role as a promising αvβ6-specific inhibitor for anti-cancer therapies. Unfortunately, the clinical value of A20FMDV2 is limited by its poor half-life in blood caused by rapid renal excretion and its reported high susceptibility to serum proteases. The incorporation of poly (ethylene glycol) chains, coined PEGylation, is a well-established approach to improve the pharmacokinetic properties of drug molecules. Here, we report a systematic study on the incorporation of a varying number of ethylene glycol units (1-20) into the A20FMDV2 peptide to establish the effects of PEGylation size on the peptide stability in both rat serum and human plasma. In addition, the effect of acetyl and propionyl PEGylation handles on peptide stability is also described. Selected peptide analogues were assessed for integrin-αvβ6-targeted binding, showing good specificity and activity in vitro. Stability studies in rat serum established that all of the PEGylated peptides displayed good stability, and an A20FMDV2 peptide containing twenty ethylene glycol units (PEG20) was the most stable. Surprisingly, the stability testing in human plasma identified shorter PEGs (PEG2 and PEG5) as more resistant to degradation than longer PEGs, a trend which was also observed with affinity binding to integrin αvβ6.

The outbreak of SARS-CoV-2 has been an extraordinary event that constituted a global health emergency. As the novel coronavirus is continuing to spread over the world, the need for therapeutic agents to control this pandemic is increasing. αV β6 Integrin may be an intriguing target not only for the inhibition of SARS-CoV-2 entry, but also for the diagnosis/treatment of COVID-19 related fibrosis, an emerging type of fibrotic disease which will probably affect a significant part of the recovered patients. In this short article, the possible role of this integrin for fighting COVID-19 is discussed on the basis of recently published evidence, showing how its underestimated involvement may be interesting for the development of novel pharmacological tools.

Integrins have been extensively investigated as therapeutic targets over the last decades, which has been inspired by their multiple functions in cancer progression, metastasis, and angiogenesis as well as a continuously expanding number of other diseases, e.g., sepsis, fibrosis, and viral infections, possibly also Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2). Although integrin-targeted (cancer) therapy trials did not meet the high expectations yet, integrins are still valid and promising targets due to their elevated expression and surface accessibility on diseased cells. Thus, for the future successful clinical translation of integrin-targeted compounds, revisited and innovative treatment strategies have to be explored based on accumulated knowledge of integrin biology. For this, refined approaches are demanded aiming at alternative and improved preclinical models, optimized selectivity and pharmacological properties of integrin ligands, as well as more sophisticated treatment protocols considering dose fine-tuning of compounds. Moreover, integrin ligands exert high accuracy in disease monitoring as diagnostic molecular imaging tools, enabling patient selection for individualized integrin-targeted therapy. The present review comprehensively analyzes the state-of-the-art knowledge on the roles of RGD-binding integrin subtypes in cancer and non-cancerous diseases and outlines the latest achievements in the design and development of synthetic ligands and their application in biomedical, translational, and molecular imaging approaches. Indeed, substantial progress has already been made, including advanced ligand designs, numerous elaborated pre-clinical and first-in-human studies, while the discovery of novel applications for integrin ligands remains to be explored.

The integrin αvβ6 is an antigen expressed at low levels in healthy tissue but upregulated during tumorigenesis, which makes it a promising target for cancer imaging and therapy. A20FMDV2 is a 20-mer peptide derived from the foot-and-mouth disease virus that exhibits nanomolar and selective affinity for αvβ6 versus other integrins. Despite this selectivity, A20FMDV2 has had limited success in imaging and treating αvβ6+ tumors in vivo because of its poor serum stability. Here, we explore the cyclization and modification of the A20FMDV2 peptide to improve its serum stability without sacrificing its affinity and specificity for αvβ6. Using cysteine amino acid substitutions and cyclization by perfluoroarylation with decafluorobiphenyl, we synthesized six cyclized A20FMDV2 variants and discovered that two retained binding to αvβ6 with modestly improved serum stability. Further d-amino acid substitutions and C-terminal sequence optimization outside the cyclized region greatly prolonged peptide serum stability without reducing binding affinity. While the cyclized A20FMDV2 variants exhibited increased nonspecific integrin binding compared with the original peptide, additional modifications with the non-natural amino acids citrulline, hydroxyproline, and d-alanine were found to restore binding specificity, with some modifications leading to greater αvβ6 integrin selectivity than the original A20FMDV2 peptide. The peptide modifications detailed herein greatly improve the potential of utilizing A20FMDV2 to target αvβ6 in vivo, expanding opportunities for cancer targeting and therapy.

Rectal cancer patients with a complete response after neoadjuvant therapy can be monitored with a watch-and-wait strategy. However, regrowth rates indicate that identification of patients with a pathological complete response (pCR) remains challenging. Targeted near-infrared fluorescence endoscopy is a potential tool to improve response evaluation. Promising tumor targets include carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), integrin αvβ6, and urokinase-type plasminogen activator receptor (uPAR). To investigate the applicability of these targets, we analyzed protein expression by immunohistochemistry and quantified these by a total immunostaining score (TIS) in tissue of rectal cancer patients with a pCR. CEA, EpCAM, αvβ6, and uPAR expression in the diagnostic biopsy was high (TIS > 6) in, respectively, 100%, 100%, 33%, and 46% of cases. CEA and EpCAM expressions were significantly higher in the diagnostic biopsy compared with the corresponding tumor bed (p < 0.01). CEA, EpCAM, αvβ6, and uPAR expressions were low (TIS < 6) in the tumor bed in, respectively, 93%, 95%, 85%, and 62.5% of cases. Immunohistochemical evaluation shows that CEA and EpCAM could be suitable targets for response evaluation after neoadjuvant treatment, since expression of these targets in the primary tumor bed is low compared with the diagnostic biopsy and adjacent pre-existent rectal mucosa in more than 90% of patients with a pCR.

High levels of reactive oxygen species (ROS) in tumors have been shown to exert anti-tumor activity, leading to the concept of ROS induction as therapeutic strategy. The organometallic compound ferrocene (Fc) generates ROS through a reversible one-electron oxidation. Incorporation of Fc into a tumor-targeting, bioactive molecule can enhance its therapeutic activity and enable tumor specific delivery. Therefore, we conjugated Fc to five synthetic, Arg-Gly-Asp (RGD)-based integrin binding ligands to enable targeting of the cell adhesion and signaling receptor integrin subtypes αvβ3, α5β1, or αvβ6, which are overexpressed in various, distinct tumors. We designed and synthesized a library of integrin-ligand-ferrocene (ILF) derivatives and showed that ILF conjugates maintained the high integrin affinity and selectivity of their parent ligands. A thorough biological characterization allowed us to identify the two most promising ligands, an αvβ3 (L2b) and an αvβ6 (L3b) targeting ILF, which displayed selective integrin-dependent cell uptake and pronounced ferrocene-mediated anti-tumor effects in vitro, along with increased ROS production and DNA damage. Hence, ILFs are promising candidates for the selective, tumor-targeted delivery of ferrocene to maximize its anti-cancer efficacy and minimize systemic toxicity, thereby improving the therapeutic window of ferrocene compared to currently used non-selective anti-cancer drugs.