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Cancer Immunology, Immunotherapy

, Volume 66, Issue 9, pp 1217–1228 | Cite as

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

  • Roger Gilabert-Oriol
  • Sebastian G. B. Furness
  • Brett W. Stringer
  • Alexander Weng
  • Hendrik Fuchs
  • Bryan W. Day
  • Angela Kourakis
  • Andrew W. Boyd
  • David L. Hare
  • Mayank Thakur
  • Terrance G. Johns
  • Peter J. WookeyEmail author
Original Article

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.

Keywords

Calcitonin receptor Immunotoxins Targeting High-grade glioma cell lines Glioblastoma 

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

Notes

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.

Compliance with ethical standards

Conflict of interest

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.

Supplementary material

262_2017_2013_MOESM1_ESM.mp4 (3 mb)
Supplementary material 1 (MP4 3119 kb)
262_2017_2013_MOESM2_ESM.pdf (627 kb)
Supplementary material 2 (PDF 626 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Roger Gilabert-Oriol
    • 1
    • 7
  • Sebastian G. B. Furness
    • 2
  • Brett W. Stringer
    • 3
  • Alexander Weng
    • 4
    • 5
  • Hendrik Fuchs
    • 4
  • Bryan W. Day
    • 3
  • Angela Kourakis
    • 1
  • Andrew W. Boyd
    • 3
  • David L. Hare
    • 1
  • Mayank Thakur
    • 4
  • Terrance G. Johns
    • 6
  • Peter J. Wookey
    • 1
    Email author
  1. 1.Department of Medicine/Cardiology (Austin Health, Heidelberg)University of MelbourneHeidelbergAustralia
  2. 2.Drug Discovery Biology Laboratory, Monash Institute of Pharmaceutical Sciences and Department of PharmacologyMonash University (Parkville)ParkvilleAustralia
  3. 3.QIMR-Berghofer Medical Research InstituteBrisbaneAustralia
  4. 4.Institut für Laboratoriumsmedizin, Klinische Chemie und PathobiochemieCharité-Universitätsmedizin Berlin, Campus Virchow-KlinikumBerlinGermany
  5. 5.Institute of PharmacyBerlinGermany
  6. 6.Hudson Institute of Medical ResearchMonash University (Clayton)ClaytonAustralia
  7. 7.Department of Experimental TherapeuticsBC Cancer Research CentreVancouverCanada

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