IL2-caspase 3 chimeric protein may provide a novel approach to the therapy of human IBD, and a possible suggested treatment for other pathological conditions that involve uncontrolled expansion of activated T cells.

One of the main problems of conventional anticancer therapy is multidrug resistance (MDR), whereby cells acquire resistance to structurally and functionally unrelated drugs following chemotherapeutic treatment. One of the main causes of MDR is overexpression of the P‐glycoprotein transporter. In addition to extruding the chemotherapeutic drugs, it also inhibits apoptosis through the inhibition of caspases. To overcome MDR, we constructed a novel chimeric protein, interleukin (IL)‐2 granzyme A (IGA), using IL‐2 as a targeting moiety and granzyme A as a killing moiety, fused at the cDNA level. IL‐2 binds to the high‐affinity IL‐2 receptor that is expressed in an array of abnormal cells, including malignant cells. Granzyme A is known to cause caspase 3‐independent cell death. We show here that the IGA chimeric protein enters the target sensitive and MDR cancer cells overexpressing IL‐2 receptor and induces caspase 3‐independent cell death. Specifically, after its entry, IGA causes a decrease in the mitochondrial potential, triggers translocation of nm23‐H1, a granzyme A‐dependent DNase, from the cytoplasm to the nucleus, where it causes single‐strand DNA nicks, thus causing cell death. Moreover, IGA is able to overcome MDR and kill cells resistant to chemotherapeutic drugs. We believe that overcoming MDR with targeted molecules such as IGA chimeric protein that causes caspase‐independent apoptotic cell death could be applied to many other resistant types of tumors using the appropriate targeting moiety. Thus, this novel class of targeted molecules could open up new vistas in the fight against human cancer.

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Designing a chimeric protein and developing a procedure for its stable production as a biologically active protein, are key steps in its potential application to clinical trails. IL2-Caspase3 chimeric protein designed to target activated T lymphocytes was found to be a promising molecule for targeted treatment, however was found to be difficult to produce as a biological active molecule. Thus, we designed a new version of the molecule, IL2-Caspase3s, in which six amino acids (aa 29-34) from the N-terminus of the large subunit of caspase 3 were excluded. Repeated expressions, productions, and partial purifications of the IL2-Caspase3s yielded reproducible batches with consistent results. We found that IL2-Caspase3s causes cell death in a specific, dose-, and time-dependent manner. Cell death due to IL2-Caspase3s is caused by apoptosis. This improved and biologically stable IL2-Caspase3s chimeric protein may be developed in the future for clinical trails as a promising therapy for several pathologies involving activated T-cells. Moreover, this truncated caspase 3 sequence, lacking the N-terminal six amino acids of its large subunit, may be used in other caspase 3-based chimeric proteins targeted against various human diseases, using the appropriate targeting moiety.

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