Recombinant canine IgE Fc and an IgE Fc-TRAIL fusion protein bind to neoplastic canine mast cells.

Screening for expression of the high affinity receptor for IgE by reverse transcriptase PCR, revealed that almost all canine mast cell tumors expressed FcɛRIα mRNA, supporting the rationale for developing anti-neoplastic treatments based on molecules that could target this receptor. Use of cytotoxic cytokines to trigger an apoptotic signal is one strategy for inducing cell death in malignant mast cells. The coding sequences for canine IgE and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were identified through genome analyses. Selected regions of the coding sequences for these genes were cloned and compared to the predicted genome sequences. The Fc region of canine IgE, death domain of canine TRAIL and an IgE Fc: TRAIL fusion construct were generated and epitope-tagged proteins expressed, using a eukaryotic expression system. Specific binding of recombinant canine IgE Fc-containing proteins to recombinant human FcɛRIα and to a canine mast cell tumor line expressing FcɛRIα (C2), but not one failing to express FcɛRIα (MCLA), was demonstrated. Specific binding of the IgE: TRAIL fusion protein was not abrogated by the TRAIL moiety. These results are proof of principle that canine IgE targeting to FcɛRIα can be used as a platform for selective delivery of therapies to FcɛRIα-expressing cells, potentially enhancing their therapeutic index and efficacy.

Targeted knockdown of canine KIT (stem cell factor receptor) using RNA interference.

Canine mast cell tumours often express KIT mutations that result in constitutive activation of the c-kit receptor and which are associated with more aggressive disease. The aim of the current study was to determine whether small inhibitory RNA (SiRNA) molecules could specifically target canine KIT mRNA for knock-down. Canine beta-2 microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and KIT sequences were cloned into the psiCHECK™-2 vector. SiRNA molecules, designed to target gene-specific sequences, were co-transfected with plasmid DNA into Chinese hamster ovary (CHO) cells. Renilla and firefly luciferase activity was measured using the Dual-GLO(®) Luciferase Assay (Promega). Using this reporter system, canine housekeeping gene-specific SiRNA molecules demonstrated knockdown of their targets (72.0% knockdown for B2M and 94.5% knockdown for GAPDH). An SiRNA molecule targeting exon 2 of canine KIT successfully knocked-down reporter gene expression of a KIT(26-407) construct (90.8% knockdown). An SiRNA molecule targeting a 48 base-pair in-tandem duplication mutation in KIT exon 11 selectively knocked down expression of the KIT(1569-1966mutant) construct (93.1% knockdown) but had no effect on the KIT(1569-1918wild-type) construct. The results show that RNA interference can be used to inhibit canine KIT mRNA expression and has the potential to selectively target the mutant version of KIT that is expressed by some malignant mast cells.

Susceptibility of the C2 canine mastocytoma cell line to the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which preferentially induces apoptosis in cells that have undergone malignant transformation. In humans, non-neoplastic cells are normally protected from the effects of TRAIL by expressing decoy receptors, lacking death domains. In contrast, neoplastic cells tend to downregulate their decoy receptor expression, increasing their susceptibility to the pro-apoptotic effects of TRAIL, via the functional TRAIL receptors. The aim of the current study was to investigate the effect of TRAIL on the canine C2 mastocytoma cell line to determine whether this agent might be a suitable treatment for mast cell tumors in dogs. C2 and MDCK cells were cultured with recombinant human TRAIL. Apoptosis was assessed using a Caspase 3 and 7 chemiluminescence assay and flow cytometry following Annexin V:FITC labelling. Cell metabolism was assessed using a colorimetric MTT-based assay. C2 cells demonstrated greater sensitivity to TRAIL-induced apoptosis compared to MDCK cells by all assessment methods. The dog genome assembly was searched for orthologs of TRAIL and its receptors using published sequences from other species for reference. Although a canine ortholog for TRAIL was identified, only one TRAIL receptor ortholog (TNFRSF11B) could be found. C2, but not MDCK, cells expressed mRNA for TNFRSF11B, detected by RT-PCR. In other species, TNFRSF11B is a decoy receptor, as even though it has a death domain it is secreted due to its lack of a transmembrane domain. The effect of TRAIL on the C2 cell line suggests that this cytokine might be suitable for treatment of mast cell tumors in dogs.