Preclinical evaluation of peptide-based radiotracers for integrin αvβ6-positive pancreatic carcinoma

Abstract

Introduction Integrin αvβ6 shows a high expression rate in several cancer entities. As it is absent in most healthy adult tissues, it represents a promising target for tumor targeting with peptidic radiotracers. This study was performed to pave the way of the recently published αvβ6-binding peptide SFLAP3 for the clinical application in patients with pancreatic cancer.

Methods The expression of integrin αvβ6 on several pancreatic cancer cell lines was assessed using flow cytometry and cell binding assays. The affinity was determined in competition binding assays followed by internalization and efflux studies. To increase the affinity, the binding sequence was modified and trimerization of the SFLAP3 peptide was achieved by oxime ligation. PET and biodistribution assays were conducted in Capan-2 tumor bearing mice. Finally, a first pancreatic tumor patient was examined with ⁶⁸Ga-DOTA-SFLAP3.

Results Flow cytometric analysis and in vitro cell binding revealed high expression of integrin αvβ6 on most pancreatic tumor cell lines. Modification of SFLAP3 led to compounds with improved in vitro binding properties. Unfortunately, these superior properties could not be transferred into improved pharmacokinetics. Consequently, the first pancreatic tumor patient was examined with ⁶⁸Ga-DOTA-SFLAP3. The PET revealed specific accumulation (with SUV_(max) values in the metastases ranging from 5 to 10) and a long retention in the tumor.

Conclusion SFLAP3 showed high affinity to integrin αvβ6 on pancreatic cancer cell lines. The in vitro performance could be confirmed in tumor bearing mice and by PET imaging. These data suggest that DOTA-SFLAP3 is a promising tracer for targeting αvβ6-expressing pancreatic tumors.

Zusammenfassung

Einleitung Die Expressionsrate von Integrin αvβ6 ist in vielen Krebsarten stark erhöht. Da es in gesunden adulten Geweben fast nicht vorhanden ist, stellt es ein vielversprechendes Zielprotein für zielgerichtete Krebsdiagnostik mit peptidischen Radiotracern dar. Diese Arbeit wurde durchgeführt, um den Weg des kürzlich veröffentlichten αvβ6-bindenden Peptids SFLAP3 für die klinische Anwendung bei Patienten mit Pankreaskarzinom zu ebnen.

Methoden Die Integrin αvβ6-Expression verschiedener Pankreaskarzinomzelllinien wurde mittels Durchflusszytometrie und Zellbindungsassays untersucht. Die Affinität wurde in kompetitiven Bindungsassays bestimmt und Internalisierungs- und Efflux-Studien durchgeführt. Um die Affinität zu erhöhen, wurde die Bindungssequenz modifiziert und das SFLAP3-Peptid durch Oximligation trimerisiert. PET- und Bioverteilungsversuche wurden in Capan-2-tumortragenden Mäusen durchgeführt. Letztendlich wurde ein erster Pankreas-Tumor-Patient mit ⁶⁸Ga-DOTA-SFLAP3 untersucht.

Ergebnisse Die durchflusszytometrische Analyse und die in vitro-Zellbindungen zeigten auf den meisten Pankreas-Tumorzelllinien, eine hohe Expression von Integrin αvβ6. Modifikationen von SFLAP3 führten zu Verbindungen mit verbesserten in vitro-Bindungseigenschaften. Leider führten diese Verbesserungen im Tiermodell zu keiner verbesserten Pharmakokinetik. Folglich wurde für die Untersuchung eines ersten Pankreas-Tumor-Patienten SFLAP3, das Peptid mit den ausgewogensten Eigenschaften, ausgewählt. Die PET-Studie mit ⁶⁸Ga-DOTA-SFLAP3 zeigte eine spezifische Tracerakkumulation (mit SUV_(max)-Werten in den Metastasen im Bereich von 5 bis 10) mit langer Tumor-Retention.

Schlussfolgerung SFLAP3 zeigte eine hohe Affinität zu Integrin αvβ6 auf Pankreas-Tumorzelllinien. Die in vitro Eigenschaften konnten in tumortragenden Mäusen und durch PET-Bildgebung eines Pankreaskarzinom-Patienten bestätigt werden. Diese Daten legen nahe, dass DOTA-SFLAP3 ein vielversprechender Tracer für αvβ6-exprimierende Pankreastumore ist.

Key words

Integrins - αvβ6 - peptides - pancreatic cancer - PET imaging - RGD motif

Schlüsselwörter

Integrin - αvβ6 - Peptide - Pankreaskarzinom - PET - RGD-Sequenz

• References

• 1 Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA: a cancer journal for clinicians 2017; 67: 7-30 doi:10.3322/caac.21387
• 2 Mayo SC, Nathan H, Cameron JL. et al. Conditional survival in patients with pancreatic ductal adenocarcinoma resected with curative intent. Cancer 2012; 118: 2674-2681 doi:10.1002/cncr.26553
• 3 Oettle H, Post S, Neuhaus P. et al. Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curative-intent resection of pancreatic cancer: a randomized controlled trial. Jama 2007; 297: 267-277 doi:10.1001/jama.297.3.267
• 4 Bandyopadhyay A, Raghavan S. Defining the role of integrin alphavbeta6 in cancer. Current drug targets 2009; 10: 645-652
• 5 Steiger K, Schlitter AM, Weichert W. et al. Perspective of alphavbeta6-Integrin Imaging for Clinical Management of Pancreatic Carcinoma and Its Precursor Lesions. Molecular imaging 2017; 16: 1536012117709384 doi:10.1177/1536012117709384
• 6 van der Flier A, Sonnenberg A. Function and interactions of integrins. Cell and tissue research 2001; 305: 285-298
• 7 Breuss JM, Gillett N, Lu L. et al. Restricted distribution of integrin beta 6 mRNA in primate epithelial tissues. The journal of histochemistry and cytochemistry: official journal of the Histochemistry Society 1993; 41: 1521-1527
• 8 Breuss JM, Gallo J, DeLisser HM. et al. Expression of the beta 6 integrin subunit in development, neoplasia and tissue repair suggests a role in epithelial remodeling. Journal of cell science 1995; 108 Pt [phone]
• 9 Giancotti FG, Ruoslahti E. Integrin signaling. Science (New York. NY) 1999; 285: 1028-1032
• 10 Wipff PJ, Hinz B. Integrins and the activation of latent transforming growth factor beta1 – an intimate relationship. European journal of cell biology 2008; 87: 601-615 doi:10.1016/j.ejcb.2008.01.012
• 11 Tod J, Hanley CJ, Morgan MR. et al. Pro-migratory and TGF-beta-activating functions of alphavbeta6 integrin in pancreatic cancer are differentially regulated via an Eps8-dependent GTPase switch. The Journal of pathology 2017; 243: 37-50 doi:10.1002/path.4923
• 12 Hazelbag S, Kenter GG, Gorter A. et al. Overexpression of the alpha v beta 6 integrin in cervical squamous cell carcinoma is a prognostic factor for decreased survival. The Journal of pathology 2007; 212: 316-324 doi:10.1002/path.2168
• 13 Zhang ZY, Xu KS, Wang JS. et al. Integrin alphanvbeta6 acts as a prognostic indicator in gastric carcinoma. Clinical oncology (Royal College of Radiologists (Great Britain)) 2008; 20: 61-66 doi:10.1016/j.clon.2007.09.008
• 14 Bates RC, Bellovin DI, Brown C. et al. Transcriptional activation of integrin beta6 during the epithelial-mesenchymal transition defines a novel prognostic indicator of aggressive colon carcinoma. The Journal of clinical investigation 2005; 115: 339-347 doi:10.1172/jci23183
• 15 Niu J, Li Z. The roles of integrin alphavbeta6 in cancer. Cancer letters 2017; 403: 128-137 doi:10.1016/j.canlet.2017.06.012
• 16 Zhu X, Li J, Hong Y. et al. 99mTc-labeled cystine knot peptide targeting integrin alphavbeta6 for tumor SPECT imaging. Molecular pharmaceutics 2014; 11: 1208-1217 doi:10.1021/mp400683q
• 17 Notni J, Reich D, Maltsev OV. et al. In Vivo PET Imaging of the Cancer Integrin alphavbeta6 Using (68)Ga-Labeled Cyclic RGD Nonapeptides. Journal of nuclear medicine: official publication. Society of Nuclear Medicine 2017; 58: 671-677 doi:10.2967/jnumed.116.182824
• 18 Hausner SH, DiCara D, Marik J. et al. Use of a peptide derived from foot-and-mouth disease virus for the noninvasive imaging of human cancer: generation and evaluation of 4-[18F]fluorobenzoyl A20FMDV2 for in vivo imaging of integrin alphavbeta6 expression with positron emission tomography. Cancer research 2007; 67: 7833-7840 doi:10.1158/0008-5472.Can-07-1026
• 19 Keat N, Kenny J, Chen K. et al. A Microdose PET Study of the Safety, Immunogenicity, Biodistribution, and Radiation Dosimetry of (18)F-FB-A20FMDV2 for Imaging the Integrin alphavbeta6. Journal of nuclear medicine technology 2018; 46: 136-143 doi:10.2967/jnmt.117.203547
• 20 Altmann A, Sauter M, Roesch S. et al. Identification of a Novel ITGalphavbeta6-Binding Peptide Using Protein Separation and Phage Display. Clinical cancer research: an official journal of the American Association for Cancer Research 2017; 23: 4170-4180 doi:10.1158/1078-0432.Ccr-16-3217
• 21 Roesch S, Lindner T, Sauter M. et al. Comparative study of the novel RGD motif-containing ITGalphavbeta6 binding peptides SFLAP3 and SFITGv6 for diagnostic application in HNSCC. Journal of nuclear medicine: official publication. Society of Nuclear Medicine 2018; 59: 1679-1685 doi:10.2967/jnumed.118.210013
• 22 Dong X, Hudson NE, Lu C. et al. Structural determinants of integrin beta-subunit specificity for latent TGF-beta. Nature structural & molecular biology 2014; 21: 1091-1096 doi:10.1038/nsmb.2905
• 23 Bottger R, Hoffmann R, Knappe D. Differential stability of therapeutic peptides with different proteolytic cleavage sites in blood, plasma and serum. PloS one 2017; 12: e0178943 doi:10.1371/journal.pone.0178943
• 24 Gotthardt M, van Eerd-Vismale J, Oyen WJ. et al. Indication for different mechanisms of kidney uptake of radiolabeled peptides. Journal of nuclear medicine: official publication. Society of Nuclear Medicine 2007; 48: 596-601
• 25 Choy CJ, Ling X, Geruntho JJ. et al. (177)Lu-Labeled Phosphoramidate-Based PSMA Inhibitors: The Effect of an Albumin Binder on Biodistribution and Therapeutic Efficacy in Prostate Tumor-Bearing Mice. Theranostics 2017; 7: 1928-1939 doi:10.7150/thno.18719
• 26 Patsenker E, Popov Y, Stickel F. et al. Inhibition of integrin alphavbeta6 on cholangiocytes blocks transforming growth factor-beta activation and retards biliary fibrosis progression. Gastroenterology 2008; 135: 660-670 doi:10.1053/j.gastro.2008.04.009
• 27 Korsinczky ML, Schirra HJ, Craik DJ. Sunflower trypsin inhibitor-1. Current protein & peptide science 2004; 5: 351-64