LB120

Construction and characterisation of novel fusion toxin targeted to cancer (DT-SCF)

Sirisha Potala, Rama Verma

Indian Institute of Technology Madras, Tamil Nadu, India

Background
Fusion toxins, an emerging class of targeted therapies, exhibit remarkable tumour specificity thus promising a better alternative to current therapies in treatment of cancers. The aim of this study was to evaluate the cytotoxicity and specificity of a novel fusion toxin, diphtheria toxin-stem cell factor (DT-SCF). In the present study, a novel recombinant fusion toxin DT-SCF has been designed for selectively targeting malignancies expressing c-kit. C-kit appears to be a reasonably good target as its over-expression has been found on many types of cancers such as ovarian, pancreatic, stomach, liver, small cell lung carcinoma and hematological malignancies.

Method
DT-SCF gene coding for 1-387 amino acids of diphtheria toxin, His-Ala linker, 2-141 amino acids of SCF was cloned into expression vector with C- terminal His tag. The induced DT-SCF protein was exclusively expressed in insoluble fraction. Purification of DT-SCF was achieved by inclusion body isolation and metal affinity chromatography under denaturing and reducing conditions. Purified DT-SCF was renatured partially on-column by gradually reduction of denaturant and was followed by complete refolding through rapid dilution technique.

Results
Plasmid coding for DT-SCF was successfully constructed and expressed. Protein encoding fusion toxin DT-SCF was purified and characterised by SDS-PAGE, western blotting, circular dichroism spectroscopy, fluorescence spectroscopy and cytotoxicity assays on various cell lines. Cell viability assay provided the first evidence that DT-SCF is a potent cytotoxic agent selectively targeted to cells expressing c-kit.

Conclusion
DT-SCF fusion toxin appears to be a promising agent to target malignancies expressing c-kit receptor. The encouraging results of potency and specificity suggest the necessity for further development.