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

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 EC 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 EC 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.

GPCRomics: GPCR Expression in Cancer Cells and Tumors Identifies New, Potential Biomarkers and Therapeutic Targets

“…GPCRs are commonly targeted by small molecules but other approaches may be possible, for example, techniques to blunt GPCR expression, GPCR-directed antibodies, antibody-drug conjugates, and bio-conjugates. ( Junttila et al, 2015 ; Lelle et al, 2015 ; Gong et al, 2016 ; Gilabert-Oriol et al, 2017 ).…”

Expression and activity of the calcitonin receptor family in a sample of primary human high-grade gliomas

“…We therefore performed a preliminary screen by western blot for CALCR in our 4 HGG cell lines, which supported expressed protein of molecular weight and reactivity consistent with CTR in all 4 cell lines (see below). From this preliminary experiment we chose to further characterise these 4 cell lines, which appeared to have detectable CTR and were previously used in an anti-CTR immunotoxin study [25]. These cells were grown as adherent cultures in a monolayer on matrigel with EGF and FGFb growth factors that, in the absence of serum, showed a consistent morphology to previous publications (Fig.…”

“…In spite of this we could not detect a functional CTR response as assessed by cAMP accumulation (PB1, JK2, WK1) and i Ca 2+ assay (PB1 and WK1). However, we have previously shown that both JK2 and WK1 cell lines are susceptible to anti-CTR-immunotoxin (N-terminally directed) mediated cell killing [25]. In other systems, CTR has been reported to internalise extremely rapid in a ligand independent receptor manner [53, 55], suggesting that perhaps the CTR may not be present at the plasma membrane for sufficient time to generate detectable functional response in these GBM lines.…”

“…This expression correlated with GBM stem cell morphology and co-expression of GBM stem cell markers, glial fibrillary acidic protein and CD133 [24]. Additionally, toxin conjugates of monoclonal anti-CTR antibody mAb2C4 promote cell death in JK2, SB2b and WK1 high-grade glioma and U87MG glioblastoma-derived cell lines with effective concentrations in the picomolar range, supportive of CTR expression [25]. Other recent reports show variable expression of CALCR mRNA with expression in only a subset 12/42 [26] or 115/152 [27] of primary tumours.…”

Dianthin and Its Potential in Targeted Tumor Therapies

“…In another study, Gilabert-Oriol et al determined the IC 50 value of recombinant His-tagged dianthin-30 chemically coupled to a monoclonal anti-calcitonin receptor antibody to 10–20 nM on high-grade glioma SB2b cells. A conjugate of the same antibody with the small molecule toxin monomethyl auristatin E revealed a similar cytotoxicity (IC 50 = 25.1 nM) [85] (Table 5).…”