Diphtheria toxin fused to variant interleukin-2 exhibits potent cytotoxicity and specific binding to interleukin-2α receptor
Authors: Sirisha Potala, Rama Verma
Journal: Clinical Cancer Research (2008)

A27  Immunotoxins, a class of biologic therapeutics provide efficacious and specific treatment for cancer as they target surface specific growth factor receptors or antigens over expressed on cancer cells. Immunotoxins have shown promising results in vitro and in vivo. One of the immunotoxin approved for use by FDA is denileukin diftitox; major toxicities with this drug are acute hypersensitivity, vascular leak syndrome and impaired immune function. The latter two toxicities are due to nonspecific binding of immunotoxin to macrophages and activated lymphocytes. We hereby attempted to design an immunotoxin with unaltered efficacy and decreased toxicity. In these immunotoxins, modified diphtheria toxin (DT) acts as cytotoxic moiety and IL-2 as the targeting moiety for cancer cells that over express IL-2 receptor on their surface. IL-2 receptor has three subunits alpha, beta and gamma. Biological effect of IL2 is mediated through IL2 alpha beta gamma receptor and IL2 beta gamma receptor complexes expressed on T cells, B cells, macrophages and natural killer cells whereas IL2 alpha receptor is uniquely expressed on cancer cells and activated T cells. Targeting specifically alpha receptor eliminates toxicity. Our novel immunotoxin DT (1-390) IL-2 (3-68) is designed with the above strategy. In addition, because of its reduced size compared to denileukin diftitox we also assume better uptake by tumor and improved pharmacokinetics.  We have cloned DT (1-387) his IL-2 (2-133), DT (1-390) IL-2 (3-68) and DT (1-390) into pET28b (+) vector with C terminal histag. The fragment encoding DT was obtained by PCR amplification from genomic DNA of Corynebacterium diphtheria and the fragment encoding IL-2 was obtained by RTPCR from PBMC isolated from blood. The two amplified fragments were simultaneous ligated into vector in correct reading frame. DT (1-387) his IL-2 (2-133) which is similar to denileukin diftitox and DT (1-387) in which receptor binding domain was deleted to avoid normal-tissue binding were used as positive and negative controls respectively. Over expression of these recombinant proteins was carried out by induction with 0.4mM IPTG at 37°C for two hours. Purification of these proteins was carried out by inclusion body isolation and affinity chromatography under denaturing conditions. Purity and molecular weight of the proteins were determined by SDS-PAGE analysis. The purified proteins were immunologically characterized by western blotting. Cytotoxicity and specificity of this protein to different IL-2 receptor subunits, was demonstrated by MTT assay and immunofluorescence in different leukemic cell lines.  Cytotoxicity of this recombinant protein was observed to be more than denileukin diftitox for cells with higher alpha receptor expression whereas in cells expressing both alpha and beta receptor cytotoxicity varies from cell line to cell line. Cytotoxic activity was observed to be same as denileukin diftitox in case of K562 cell line and less than denileukin diftitox in case of KG1 and HUT78 cell lines. From this data we conclude that the novel immunotoxin has comparable efficacy to denileukin diftitox and is more specifically targeted to IL2 alpha receptor. Its probable decreased toxicity and increased uptake by tumor should be validated further.