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Dianthin-30 or gelonin versus monomethyl auristatin E, each configured with an anti-calcitonin receptor antibody, are differentially potent in vitro in high-grade glioma cell lines derived from glioblastoma

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Abstract

We have reported that calcitonin receptor (CTR) is widely expressed in biopsies from the lethal brain tumour glioblastoma by malignant glioma and brain tumour-initiating cells (glioma stem cells) using anti-human CTR antibodies. A monoclonal antibody against an epitope within the extracellular domain of CTR was raised (mAb2C4) and chemically conjugated to either plant ribosome-inactivating proteins (RIPs) dianthin-30 or gelonin, or the drug monomethyl auristatin E (MMAE), and purified. In the high-grade glioma cell line (HGG, representing glioma stem cells) SB2b, in the presence of the triterpene glycoside SO1861, the EC50 for mAb2C4:dianthin was 10.0 pM and for mAb2C4:MMAE [antibody drug conjugate (ADC)] 2.5 nM, 250-fold less potent. With the cell line U87MG, in the presence of SO1861, the EC50 for mAb2C4:dianthin was 20 pM, mAb2C4:gelonin, 20 pM, compared to the ADC (6.3 nM), which is >300 less potent. Several other HGG cell lines that express CTR were tested and the efficacies of mAb2C4:RIP (dianthin or gelonin) were similar. Co-administration of the enhancer SO1861 purified from plants enhances lysosomal escape. Enhancement with SO1861 increased potency of the immunotoxin (>3 log values) compared to the ADC (1 log). The uptake of antibody was demonstrated with the fluorescent conjugate mAb2C4:Alexa Fluor 568, and the release of dianthin-30:Alexa Fluor488 into the cytosol following addition of SO1861 supports our model. These data demonstrate that the immunotoxins are highly potent and that CTR is an effective target expressed by a large proportion of HGG cell lines representative of glioma stem cells and isolated from individual patients.

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Abbreviations

ADC:

Antibody–drug conjugate

BTIC:

Brain tumour initiating cell

CTR:

Calcitonin receptor

DTT:

Dithiothreitol

EDTA:

Ethylene diamine tetra acetate

EGF:

Epidermal growth factor

EGFR:

Epidermal growth factor receptor

FGF:

Fibroblast growth factor

GBM:

Glioblastoma multiforme

hCTR:

Human calcitonin receptor

HGG:

High-grade glioma

LDH:

Lactate dehydrogenase

MMAE:

Monomethyl auristatin E

NTA:

Nitrilotriacetate

RIP:

Ribosome-inactivating protein

VEGF:

Vascular endothelial growth factor

References

  1. Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, Scheithauer BW, Kleihues P (2007) The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol 114:97–109

    Article  PubMed  PubMed Central  Google Scholar 

  2. Galli R, Binda E, Orfanelli U, Cipelletti B, Gritti A, De Vitis S, Fiocco R, Foroni C, Dimeco F, Vescovi A (2004) Isolation and characterization of tumorigenic, stem-like neural precursors from human glioblastoma. Cancer Res 64:7011–7021

    Article  CAS  PubMed  Google Scholar 

  3. Reya T, Morrison SJ, Clarke MF, Weissman IL (2001) Stem cells, cancer, and cancer stem cells. Nature 414:105–111

    Article  CAS  PubMed  Google Scholar 

  4. Fomchenko EI, Holland EC (2005) Stem cells and brain cancer. Exp Cell Res 306:323–329

    Article  CAS  PubMed  Google Scholar 

  5. Das S, Marsden PA (2013) Angiogenesis in glioblastoma. N Engl J Med 369:1561–1563

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Behin A, Hoang-Xuan K, Carpentier AF, Delattre JY (2003) Primary brain tumours in adults. Lancet 361:323–331

    Article  PubMed  Google Scholar 

  7. Hamilton GS (2015) Antibody–drug conjugates for cancer therapy: the technological and regulatory challenges of developing drug-biologic hybrids. Biologicals 43:318–332. doi:10.1016/j.biologicals.2015.05.006

    Article  CAS  PubMed  Google Scholar 

  8. de Goeij BE, Lambert JM (2016) New developments for antibody–drug conjugate-based therapeutic approaches. Curr Opin Immunol 40:14–23

    Article  PubMed  Google Scholar 

  9. Ledford H (2016) Weaponized antibodies use new tricks to fight cancer. Nature 540:19–20

    Article  CAS  PubMed  Google Scholar 

  10. Reardon DA, Lassman AB, van den Bent M, Kumthekar P, Merrell R, Scott AM, Fichtel L, Sulman EP, Gomez E, Fischer J, Lee HJ, Munasinghe W, Xiong H, Mandich H, Roberts-Rapp L, Ansell P, Holen KD, Gan HK (2016) Efficacy and safety results of ABT-414 in combination with radiation and temozolomide in newly diagnosed glioblastoma. Neuro Oncol. doi:10.1093/neuonc/now257

    Google Scholar 

  11. Fermani S, Falini G, Ripamonti A, Bolognesi A, Polito L, Stirpe F (2003) Crystallization and preliminary X-ray diffraction analysis of two ribosome-inactivating proteins: lychnin and dianthin 30. Acta Crystallogr D Biol Crystallogr 59:1227–1229

    Article  PubMed  Google Scholar 

  12. Hosur MV, Nair B, Satyamurthy P, Misquith S, Surolia A, Kannan KK (1995) X-ray structure of gelonin at 1.8 A resolution. J Mol Biol 250:368–380

    Article  CAS  PubMed  Google Scholar 

  13. von Mallinckrodt B, Thakur M, Weng A, Gilabert-Oriol R, Durkop H, Brenner W, Lukas M, Beindorff N, Melzig MF, Fuchs H (2014) Dianthin-EGF is an effective tumor targeted toxin in combination with saponins in a xenograft model for colon carcinoma. Future Oncol 10:2161–2175

    Article  Google Scholar 

  14. Thakur M, Mergel K, Weng A, von Mallinckrodt B, Gilabert-Oriol R, Durkop H, Melzig MF, Fuchs H (2013) Targeted tumor therapy by epidermal growth factor appended toxin and purified saponin: an evaluation of toxicity and therapeutic potential in syngeneic tumor bearing mice. Mol Oncol 7:475–483

    Article  CAS  PubMed  Google Scholar 

  15. Weng A, Manunta MD, Thakur M, Gilabert-Oriol R, Tagalakis AD, Eddaoudi A, Munye MM, Vink CA, Wiesner B, Eichhorst J, Melzig MF, Hart SL (2015) Improved intracellular delivery of peptide- and lipid-nanoplexes by natural glycosides. J Control Release 206:75–90. doi:10.1016/j.jconrel.2015.03.007

    Article  CAS  PubMed  Google Scholar 

  16. Furness SG, Liang YL, Nowell CJ, Halls ML, Wookey PJ, Dal Maso E, Inoue A, Christopoulos A, Wootten D, Sexton PM (2016) Ligand-dependent modulation of g protein conformation alters drug efficacy. Cell 167(739–749):e11. doi:10.1016/j.cell.2016.09.021

    Google Scholar 

  17. Wookey P, Zulli A, Lo C, Hare D, Schwarer A, Darby I, Leung A (2010) Calcitonin receptor (CTR) expression in embryonic, foetal and adult tissues: developmental and pathophysiological implications. In: Hay D, Dickerson I (eds) The calcitonin gene-related peptide family; form, function and future perspectives. Springer, The Netherlands, pp 199–233

    Chapter  Google Scholar 

  18. Wookey PJ, Turner K, Furness JB (2012) Transient expression of the calcitonin receptor by enteric neurons of the embryonic and early post-natal mouse. Cell Tissue Res 347:311–317

    Article  CAS  PubMed  Google Scholar 

  19. Marx SJ, Aurbach GD, Gavin JR 3rd, Buell DW (1974) Calcitonin receptors on cultured human lymphocytes. J Biol Chem 249:6812–6816

    CAS  PubMed  Google Scholar 

  20. Body JJ, Glibert F, Nejai S, Fernandez G, Van Langendonck A, Borkowski A (1990) Calcitonin receptors on circulating normal human lymphocytes. J Clin Endocrinol Metab 71:675–681

    Article  CAS  PubMed  Google Scholar 

  21. Jagger C, Chambers T, Pondel M (2000) Transgenic mice reveal novel sites of calcitonin receptor gene expression during development. Biochem Biophys Res Commun 274:124–129

    Article  CAS  PubMed  Google Scholar 

  22. Tikellis C, Xuereb L, Casley D, Brasier G, Cooper ME, Wookey PJ (2003) Calcitonin receptor isoforms expressed in the developing rat kidney. Kidney Int 63:416–426

    Article  CAS  PubMed  Google Scholar 

  23. Tolcos M, Tikellis C, Rees S, Cooper M, Wookey P (2003) Ontogeny of calcitonin receptor mRNA and protein in the developing central nervous system of the rat. J Comp Neurol 456:29–38

    Article  CAS  PubMed  Google Scholar 

  24. Furness SGB, Hare DL, Kourakis A, Turnley AM, Wookey PJ (2016) A novel ligand of calcitonin receptor reveals a potential new sensor that modulates programmed cell death. Cell Death Discov 2:16062. doi:10.1038/cddiscovery.2016.62 (eCollection 2016)

    Article  PubMed  PubMed Central  Google Scholar 

  25. Wookey PJ, McLean CA, Hwang P, Furness SG, Nguyen S, Kourakis A, Hare DL, Rosenfeld JV (2012) The expression of calcitonin receptor detected in malignant cells of the brain tumour glioblastoma multiforme and functional properties in the cell line A172. Histopathology 60:895–910. doi:10.1111/j.1365-2559.2011.04146.x

    Article  PubMed  Google Scholar 

  26. Silvestris F, Cafforio P, De Matteo M, Quatraro C, Dammacco F (2008) Expression and function of the calcitonin receptor by myeloma cells in their osteoclast-like activity in vitro. Leuk Res 32:611–623

    Article  CAS  PubMed  Google Scholar 

  27. Venkatanarayan A, Raulji P, Norton W, Chakravarti D, Coarfa C, Su X, Sandur SK, Ramirez MS, Lee J, Kingsley CV, Sananikone EF, Rajapakshe K, Naff K, Parker-Thornburg J, Bankson JA, Tsai KY, Gunaratne PH, Flores ER (2015) IAPP-driven metabolic reprogramming induces regression of p53-deficient tumours in vivo. Nature 517:626–630

    Article  CAS  PubMed  Google Scholar 

  28. Nicholson GC, Horton MA, Sexton PM, D’Santos CS, Moseley JM, Kemp BE, Pringle JA, Martin TJ (1987) Calcitonin receptors of human osteoclastoma. Horm Metab Res 19:585–589

    Article  CAS  PubMed  Google Scholar 

  29. Gorn AH, Rudolph SM, Flannery MR, Morton CC, Weremowicz S, Wang TZ, Krane SM, Goldring SR (1995) Expression of two human skeletal calcitonin receptor isoforms cloned from a giant cell tumor of bone. The first intracellular domain modulates ligand binding and signal transduction. J Clin Invest 95:2680–2691

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gillespie M, Thomas R, Pu Z, Zhou H, Martin T, Findlay D (1997) Calcitonin receptors, bone sialoprotein and osteopontin are expressed in primary breast cancers. Int J Cancer 73:812–815

    Article  CAS  PubMed  Google Scholar 

  31. Wu G, Burzon D, di Sant’Agnese P, Schoen S, Deftos L, Gershagen S, Cockett A (1996) Calcitonin receptor mRNA expression in human prostate. Urology 47:376–381

    Article  CAS  PubMed  Google Scholar 

  32. Thomas S, Muralidharan A, Shah GV (2007) Knock-down of calcitonin receptor expression induces apoptosis and growth arrest of prostate cancer cells. Int J Oncol 31:1425–1437

    CAS  PubMed  Google Scholar 

  33. Frendo JL, Delage-Mourroux R, Cohen R, Pichaud F, Pidoux E, Guliana JM, Jullienne A (1998) Calcitonin receptor mRNA is expressed in human medullary thyroid carcinoma. Thyroid 8:141–147

    Article  CAS  PubMed  Google Scholar 

  34. Scott AM, Wolchok JD, Old LJ (2012) Antibody therapy of cancer. Nat Rev Cancer 12:278–287

    Article  CAS  PubMed  Google Scholar 

  35. Verhaak RG, Hoadley KA, Purdom E, Wang V, Qi Y, Wilkerson MD, Miller CR, Ding L, Golub T, Mesirov JP, Alexe G, Lawrence M, O’Kelly M, Tamayo P, Weir BA, Gabriel S, Winckler W, Gupta S, Jakkula L, Feiler HS, Hodgson JG, James CD, Sarkaria JN, Brennan C, Kahn A, Spellman PT, Wilson RK, Speed TP, Gray JW, Meyerson M, Getz G, Perou CM, Hayes DN, Cancer Genome Atlas Research Network (2010) Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17:98–110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Day BW, Stringer BW, Al-Ejeh F, Ting MJ, Wilson J, Ensbey KS, Jamieson PR, Bruce ZC, Lim YC, Offenhauser C, Charmsaz S, Cooper LT, Ellacott JK, Harding A, Leveque L, Inglis P, Allan S, Walker DG, Lackmann M, Osborne G, Khanna KK, Reynolds BA, Lickliter JD, Boyd AW (2013) EphA3 maintains tumorigenicity and is a therapeutic target in glioblastoma multiforme. Cancer Cell 23:238–248

    Article  CAS  PubMed  Google Scholar 

  37. Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, Russell R, Bayani J, Head R, Lee M, Bernstein M, Squire JA, Smith A, Dirks P (2009) Glioma stem cell lines expanded in adherent culture have tumor-specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell 4:568–580

    Article  CAS  PubMed  Google Scholar 

  38. Francisco JA, Cerveny CG, Meyer DL, Mixan BJ, Klussman K, Chace DF, Rejniak SX, Gordon KA, DeBlanc R, Toki BE, Law CL, Doronina SO, Siegall CB, Senter PD, Wahl AF (2003) cAC10-vcMMAE, an anti-CD30-monomethyl auristatin E conjugate with potent and selective antitumor activity. Blood 102:1458–1465

    Article  CAS  PubMed  Google Scholar 

  39. Grundy TJ, De Leon E, Griffin KR, Stringer BW, Day BW, Fabry B, Cooper-White J, O’Neill GM (2016) Differential response of patient-derived primary glioblastoma cells to environmental stiffness. Sci Rep 6:23353

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Tivnan A, Zhao J, Johns TG, Day BW, Stringer BW, Boyd AW, Tiwari S, Giles KM, Teo C, McDonald KL (2014) The tumor suppressor microRNA, miR-124a, is regulated by epigenetic silencing and by the transcriptional factor, REST in glioblastoma. Tumour Biol 35:1459–1465

    Article  CAS  PubMed  Google Scholar 

  41. Hosein AN, Lim YC, Day B, Stringer B, Rose S, Head R, Cosgrove L, Sminia P, Fay M, Martin JH (2015) The effect of valproic acid in combination with irradiation and temozolomide on primary human glioblastoma cells. J Neuro Oncol 122:263–271

    Article  CAS  Google Scholar 

  42. Dickinson A, Yeung KY, Donoghue J, Baker MJ, Kelly RD, McKenzie M, Johns TG, St John JC (2013) The regulation of mitochondrial DNA copy number in glioblastoma cells. Cell Death Differ 20:1644–1653

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Gilabert-Oriol R, Weng A, Trautner A, Weise C, Schmid D, Bhargava C, Niesler N, Wookey PJ, Fuchs H, Thakur M (2015) Combinatorial approach to increase efficacy of Cetuximab, Panitumumab and Trastuzumab by dianthin conjugation and co-application of SO1861. Biochem Pharmacol 97:247–255

    Article  CAS  PubMed  Google Scholar 

  44. Rosenblum MG, Kohr WA, Beattie KL, Beattie WG, Marks W, Toman PD, Cheung L (1995) Amino acid sequence analysis, gene construction, cloning, and expression of gelonin, a toxin derived from Gelonium multiflorum. J Interf Cytokine Res 15:547–555

    Article  CAS  Google Scholar 

  45. Cumber AJ, Henry RV, Parnell GD, Wawrzynczak EJ (1990) Purification of immunotoxins containing the ribosome-inactivating proteins gelonin and momordin using high performance liquid immunoaffinity chromatography compared with blue sepharose CL-6B affinity chromatography. J Immunol Methods 135:15–24

    Article  CAS  PubMed  Google Scholar 

  46. Weng A, Thakur M, von Mallinckrodt B, Beceren-Braun F, Gilabert-Oriol R, Wiesner B, Eichhorst J, Böttger S, Melzig MF, Fuch H (2012) Saponins modulate the intracellular trafficking of protein toxins. J Control Release 164:74–86. doi:10.1016/j.jconrel.2012.10.002

    Article  CAS  PubMed  Google Scholar 

  47. Seck T, Baron R, Horne WC (2003) Binding of filamin to the C-terminal tail of the calcitonin receptor controls recycling. J Biol Chem 278:10408–10416

    Article  CAS  PubMed  Google Scholar 

  48. Ross JL, Ali MY, Warshaw DM (2008) Cargo transport: molecular motors navigate a complex cytoskeleton. Curr Op Cell Biol 20:41–47

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Cizeau J, Grenkow DM, Brown JG, Entwistle J, MacDonald GC (2009) Engineering and biological characterization of VB6-845, an anti-EpCAM immunotoxin containing a T-cell epitope-depleted variant of the plant toxin bouganin. J Immunother 32:574–584

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

Dr Furness was supported by the National Health and Medical Research Council of Australia (Program Grant 1055134 and Project Grant 1061044). Dr Gilabert-Oriol was supported by an Endeavour Research Fellowship (Australian Government). This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors are grateful for the gift of rGelonin from Professor Rosenblum of the University of Texas MD Anderson Cancer Center, Texas, USA. Confocal microscopy was performed at the Biological Optical Microscopy Platform, The University of Melbourne (http://www.microscopy.unimelb.edu.au) and the Leibniz-Institut für Molekulare Pharmakologie (FMP), Campus Berlin-Buch, Berlin, Germany. Thanks go to the team at the Antibody Facility (Walter and Eliza Hall Institute, Bundoora) for production of the anti-CTR antibodies.

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Author notes

  1. Roger Gilabert-Oriol

    Present address: Department of Experimental Therapeutics, BC Cancer Research Centre, 675 W 10th Ave, Vancouver, BC, V5Z IL3, Canada

  2. Roger Gilabert-Oriol and Sebastian G. B. Furness contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine/Cardiology (Austin Health, Heidelberg), University of Melbourne, Lance Townsend Building, Level 10, Austin Campus, Studley Road, Heidelberg, VIC, 3084, Australia

    Roger Gilabert-Oriol, Angela Kourakis, David L. Hare & Peter J. Wookey

  2. Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University (Parkville), Parkville, VIC, Australia

    Sebastian G. B. Furness

  3. QIMR-Berghofer Medical Research Institute, Brisbane, QLD, Australia

    Brett W. Stringer, Bryan W. Day & Andrew W. Boyd

  4. Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353, Berlin, Germany

    Alexander Weng, Hendrik Fuchs & Mayank Thakur

  5. Institute of Pharmacy, Königin-Luise-Str. 2+4, 14195, Berlin, Germany

    Alexander Weng

  6. Hudson Institute of Medical Research, Monash University (Clayton), Clayton, VIC, Australia

    Terrance G. Johns

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  1. Roger Gilabert-Oriol
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Correspondence to Peter J. Wookey.

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Dr Wookey is a Director of Welcome Receptor Antibodies Pty Ltd (Australia) which developed the anti-CTR antibodies. All other authors declare no conflict of interest.

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Gilabert-Oriol, R., Furness, S.G.B., Stringer, B.W. et al. Dianthin-30 or gelonin versus monomethyl auristatin E, each configured with an anti-calcitonin receptor antibody, are differentially potent in vitro in high-grade glioma cell lines derived from glioblastoma. Cancer Immunol Immunother 66, 1217–1228 (2017). https://doi.org/10.1007/s00262-017-2013-z

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