Retargeted cytokine-immunoglobulin fusion proteins to deliver radioisotopes for diagnosis, radioimmunotherapy and TRAIL sensitization of high grade astrocytoma and metastatic breast cancer

Abstract

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Objectives: Interleukin 13 Receptor alpha 2 (IL13Ra2) is a cancer-associated receptor that is present in greater then 80% of High Grade Astrocytomas (HGA) and has recently been recognized as a “virulent” gene that predisposes breast cancer cells to metastasize. This receptor is not present in significant quantities in normal tissue, with the exception of the testes and is distinct from the physiological IL13 receptor, IL13Ra1, which is present in numerous healthy tissues. IL13 binds to its receptors, IL13Ra1 and IL13Ra2, through different binding sites. Glutamic acid at position 13 of human IL13 has been shown to be necessary for its interaction with its physiological receptor, IL13Ra1. Mutations at this position abrogate IL13’s physiological actions, but importantly, do not alter its activity towards the high-affinity cancer receptor, IL13Ra2. This ability to retarget IL13 to preferentially bind to IL13Ra2 have prompted numerous groups to develop strategies to target IL13Ra2(+) HGAs. Some of these strategies that are currently in pre-clinical or clinical trials include IL13/cytotoxin fusion proteins, IL13Ra2 targeted T-cells, IL13Ra2 targeted viruses and IL13Ra2 based genetic and peptide vaccines. Although preliminary data suggests that IL13Ra2-targeting strategies have great clinical potential, no diagnostic agent is available to ascertain the real-time IL13Ra2 status of these devastating tumors. Our objective is to synthesize retargeted cytokine-immunoglobulin fusion proteins to Deliver Radioisotopes for the diagnosis and treatment of IL13Ra2(+) tumors.

Methods: We therefore fused a retargeted IL13 mutant, IL13.E13K, to either the full IgG1 constant region (hinge-CH2-CH3) or only the CH3 region and expressed them in CHO cells as a secreted protein.

Results: We demonstrate that radio-iodination of retargeted IL13 does not alter its ability to bind IL13Ra2(+) cells. Immunoglobulin regions enhance the in vivo stability of fusion proteins and therefore render our retargeted IL13 constructs suitable for diagnostic and therapeutic radioisotope delivery. Of significance, we also demonstrate that the delivery of radiation sensitizes HGA cells to TRAIL, a potent pro-apoptotic ligand that is currently in phase I clinical trials. We are currently examining the in vivo biodistribution and anti-tumor potential of our IL13-based “cytoglobulin” constructs alone and in combination with Trail.

Conclusions: Based on our preliminary data, we conclude that our novel targeted fusion proteins are promising tools for molecular diagnoses and therapy of high grade astrocytoma and metastatic breast cancer.