It’s Time to Shift the Paradigm: Translation and Clinical Application of Non-αvβ3 Integrin Targeting Radiopharmaceuticals

For almost the entire period of the last two decades, translational research in the area of integrin-targeting radiopharmaceuticals was strongly focused on the subtype αvβ3, owing to its expression on endothelial cells and its well-established role as a biomarker for, and promoter of, angiogenesis. Despite a large number of translated tracers and clinical studies, a clinical value of αvβ3-integrin imaging could not be defined yet. The focus of research has, thus, been moving slowly but steadily towards other integrin subtypes which are involved in a large variety of tumorigenic pathways. Peptidic and non-peptidic radioligands for the integrins α5β1, αvβ6, αvβ8, α6β1, α6β4, α3β1, α4β1, and αMβ2 were first synthesized and characterized preclinically. Some of these compounds, targeting the subtypes αvβ6, αvβ8, and α6β1/β4, were subsequently translated into humans during the last few years. αvβ6-Integrin has arguably attracted most attention because it is expressed by some of the cancers with the worst prognosis (above all, pancreatic ductal adenocarcinoma), which substantiates a clinical need for the respective theranostic agents. The receptor furthermore represents a biomarker for malignancy and invasiveness of carcinomas, as well as for fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), and probably even for Sars-CoV-2 (COVID-19) related syndromes. Accordingly, the largest number of recent first-in-human applications has been reported for radiolabeled compounds targeting αvβ6-integrin. The results indicate a substantial clinical value, which might lead to a paradigm change and trigger the replacement of αvβ3 by αvβ6 as the most popular integrin in theranostics.

There is a world beyond αvβ3-integrin: Multimeric ligands for imaging of the integrin subtypes αvβ6, αvβ8, αvβ3, and α5β1 by positron emission tomography

Background In the context of nuclear medicine and theranostics, integrin-related research and development was, for most of the time, focused predominantly on 'RGD peptides' and the subtype αvβ3-integrin. However, there are no less than 24 known integrins, and peptides without the RGD sequence as well as non-peptidic ligands play an equally important role as selective integrin ligands. On the other hand, multimerization is a well-established method to increase the avidity of binding structures, but multimeric radiopharmaceuticals have not made their way into clinics yet. In this review, we describe how these aspects have been interwoven in the framework of the German Research Foundation's multi-group interdisciplinary funding scheme CRC 824, yielding a series of potent PET imaging agents for selective imaging of various integrin subtypes. Results The gallium-68 chelator TRAP was utilized to elaborate symmetrical trimers of various peptidic and non-peptidic integrin ligands. Preclinical data suggested a high potential of the resulting Ga-68-tracers for PET-imaging of the integrins α5β1, αvβ8, αvβ6, and αvβ3. For the first three, we provide some additional immunohistochemistry data in human cancers, which suggest several future clinical applications. Finally, application of αvβ3- and αvβ6-integrin tracers in pancreatic carcinoma patients revealed that unlike αvβ3-targeted PET, αvβ6-integrin PET is not characterized by off-target uptake and thus, enables a substantially improved imaging of this type of cancer. Conclusions Novel radiopharmaceuticals targeting a number of different integrins, above all, αvβ6, have proven their clinical potential and will play an increasingly important role in future theranostics.

IFIT3 (Interferon Induced Protein with Tetratricopeptide Repeats 3) Modulates STAT1 Expression in small Extracellular Vesicles

We have previously shown that the αvβ6 integrin plays a key role in promoting prostate cancer (PrCa) as it can be transferred to recipient cells via small extracellular vesicles (sEVs). Furthermore, we have reported in a proteomic analysis that αvβ6 integrin downregulation increases the expression of IFIT3 (interferon induced protein with tetratricopeptide repeats 3) in PrCa cells and their derived sEVs. IFIT3 is a protein well known for being an antiviral effector, but recently its role in cancer has also been elucidated. To study the relationship between IFIT3 and STAT1 (signal transducer and activator of transcription 1), an upstream regulator of IFIT3, in PrCa cells and their released sEVs, we used CRISPR/Cas9 techniques to downregulate the expression of the β6 integrin subunit, IFIT3 or STAT1. Our results show that IFIT3 and STAT1 are highly expressed in PrCa cells devoid of the β6 integrin subunit. However, IFIT3 but not STAT1, is present in sEVs derived from PrCa cells lacking the β6 integrin subunit. We demonstrate that loss of IFIT3 generates sEVs enriched in STAT1 but reduces the levels of STAT1 in the cells. As expected, IFIT3 is not detectable in STAT1 negative cells or sEVs. We thus propose that the observed STAT1 enrichment in sEVs is a compensatory mechanism for the loss of IFIT3. Overall, these results provide new insights into the intrinsic role of IFIT3 as a regulator of STAT1 expression in PrCa derived sEVs and in intercellular communication in PrCa.

PET/CT imaging of head-and-neck and pancreatic cancer in humans by targeting the “Cancer Integrin” αvβ6 with Ga-68-Trivehexin

Purpose To develop a new probe for the αvβ6-integrin and assess its potential for PET imaging of carcinomas. Methods Ga-68-Trivehexin was synthesized by trimerization of the optimized αvβ6-integrin selective cyclic nonapeptide Tyr2 (sequence: c[YRGDLAYp(NMe)K]) on the TRAP chelator core, followed by automated labeling with Ga-68. The tracer was characterized by ELISA for activities towards integrin subtypes αvβ6, αvβ8, αvβ3, and α5β1, as well as by cell binding assays on H2009 (αvβ6-positive) and MDA-MB-231 (αvβ6-negative) cells. SCID-mice bearing subcutaneous xenografts of the same cell lines were used for dynamic (90 min) and static (75 min p.i.) µPET imaging, as well as for biodistribution (90 min p.i.). Structure–activity-relationships were established by comparison with the predecessor compound Ga-68-TRAP(AvB6)3. Ga-68-Trivehexin was tested for in-human PET/CT imaging of HNSCC, parotideal adenocarcinoma, and metastatic PDAC. Results Ga-68-Trivehexin showed a high αvβ6-integrin affinity (IC50 = 0.047 nM), selectivity over other subtypes (IC50-based factors: αvβ8, 131; αvβ3, 57; α5β1, 468), blockable uptake in H2009 cells, and negligible uptake in MDA-MB-231 cells. Biodistribution and preclinical PET imaging confirmed a high target-specific uptake in tumor and a low non-specific uptake in other organs and tissues except the excretory organs (kidneys and urinary bladder). Preclinical PET corresponded well to in-human results, showing high and persistent uptake in metastatic PDAC and HNSCC (SUVmax = 10–13) as well as in kidneys/urine. Ga-68-Trivehexin enabled PET/CT imaging of small PDAC metastases and showed high uptake in HNSCC but not in tumor-associated inflammation. Conclusions Ga-68-Trivehexin is a valuable probe for imaging of αvβ6-integrin expression in human cancers.

PET/CT Imaging of Head-and-Neck and Pancreatic Cancer in Humans by Targeting the "Cancer Integrin" αvβ6 with Ga-68-Trivehexin

PurposeTo develop a new probe for the αvβ6-integrin and assess its potential for PET imaging of carcinomas.MethodsGa-68-Trivehexin was synthesized by trimerization of an optimized αvβ6-integrin selective cyclicnonapeptide on the TRAP chelator core and automated labeling with Ga-68. The tracer wascharacterized by ELISA for activities towards integrin subtypes αvβ6, αvβ8, αvβ3, and α5β1, as well asby cell binding assays on H2009 (αvβ6-positive) and MDA-MB-231 (αvβ6-negative) cells. SCID micebearing subcutaneous xenografts of the same cell lines were used for dynamic (90 min) and static(75 min p.i.) μPET imaging, as well as for biodistribution (90 min p.i.). Structure-activity-relationshipswere established by comparison with the predecessor compound Ga-68-TRAP(AvB6)3. Ga-68-Trivehexin was tested for in-human PET/CT imaging of HNSCC, parotideal adenocarcinoma, andPDAC.ResultsGa-68-Trivehexin showed a high αvβ6-integrin affinity (IC50 = 0.033 nM), selectivity over othersubtypes (IC50-based factors: αvβ8, 188; αvβ3, 82; α5β1, 667), blockable uptake in H2009 cells, andnegligible uptake in MDA-MB-231 cells. Biodistribution and preclinical PET imaging confirmed a hightarget-specific uptake in tumor and a low non-specific uptake in other organs and tissues except theexcretory organs (kidneys and urinary bladder). Preclinical PET corresponded well to in-human results,showing high and persistent uptake in metastatic PDAC and HNSCC (SUVmax = 10–13) as well as inkidneys/urine. Ga-68-Trivehexin enabled PET/CT imaging of small PDAC metastases and showed highuptake in HNSCC but not in tumor-associated inflammation.ConclusionsGa-68-Trivehexin is a valuable probe for imaging of αvβ6-integrin expression in human cancers.

Frischer Wind für Integrine

ZusammenfassungSelektive PET- oder SPECT- Radiopharmaka sind inzwischen nicht nur für αvβ3, sondern auch weitere der 24 verschiedenen Integrine verfügbar, zum Beispiel α5β1, αvβ6, αvβ8 und α6. Da diese unter anderem auch von verschiedenen Karzinomen und im Zuge von Fibrose exprimiert werden, ist die Vorstellung, dass Integrine nur als Zielstrukturen für die Bildgebung von Angiogenese in Betracht kommen, endgültig überholt. Die derzeit besten Aussichten auf eine breite klinische Anwendung, sowohl diagnostisch als auch therapeutisch, haben derzeit αvβ6-Integrin-Radiopharmaka, da αvβ6 von vielen malignen Krebsarten (v. a. Pankreas-, Plattenepithel-, Basalzell-, Lungen- und Colonkarzinom) überexprimiert wird.

Efficient Intravenous Tumor Targeting Using the αvβ6 Integrin-Selective Precision Virotherapy Ad5NULL-A20

We previously developed a refined, tumor-selective adenovirus, Ad5NULL-A20, harboring tropism ablating mutations in each major capsid protein, to ablate all native means of infection. We incorporated a 20-mer peptide (A20) in the fiber knob for selective infection via αvβ6 integrin, a marker of aggressive epithelial cancers. Methods: To ascertain the selectivity of Ad5NULL-A20 for αvβ6-positive tumor cell lines of pancreatic and breast cancer origin, we performed reporter gene and cell viability assays. Biodistribution of viral vectors in mice harboring xenografts with low, medium, and high αvβ6 levels was quantified by qPCR for viral genomes 48 h post intravenous administration. Results: Ad5NULL-A20 vector transduced cells in an αvβ6-selective manner, whilst cell killing mediated by oncolytic Ad5NULL-A20 was αvβ6-selective. Biodistribution analysis following intravenous administration into mice bearing breast cancer xenografts demonstrated that Ad5NULL-A20 resulted in significantly reduced liver accumulation coupled with increased tumor accumulation compared to Ad5 in all three models, with tumor-to-liver ratios improved as a function of αvβ6 expression. Conclusions: Ad5NULL-A20-based virotherapies efficiently target αvβ6-integrin-positive tumors following intravenous administration, validating the potential of Ad5NULL-A20 for systemic applications, enabling tumor-selective overexpression of virally encoded therapeutic transgenes.