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Prepublished online as a Blood First Edition Paper on February 27, 2003; DOI 10.1182/blood-2002-10-3089.
Submitted October 18, 2002
Accepted January 8, 2003
The generation of low toxicity interleukin-2 fusion proteins devoid of vasopermeability activity
Peisheng Hu, Myra Mizokami, Gina Ruoff, Leslie A Khawli, and Alan L Epstein*
Department of Pathology, USC Keck School of Medicine, Los Angeles, CA, USA
* Corresponding author; email: aepstein{at}usc.edu.
Because of its key role in immunity, interleukin-2 (IL-2) has been studied extensively for the adoptive immunotherapy of cancer. While systemic administration of this biologic response modifier has been shown to stimulate anti-tumor responses in vivo, its efficacy in the clinic has been limited by the development of serious side effects including the induction of vascular leak syndrome. Previously, we have identified a small peptide fragment of IL-2 which was found to contain the entire vasopermeability activity of the cytokine. Designated Permeability Enhancing Peptide (PEP), this fragment of IL-2 spans from amino acid 22-58 and partly overlaps the receptor binding domains of the molecule. The identification of the location of this potentially undesirable property of IL-2 enabled us to focus on the generation of mutant derivatives which might be lacking vasopermeability activity but which retain cytokine functionality. In addition to this discovery, our laboratory has constructed monoclonal antibody/IL-2 fusion proteins which can target this potent cytokine directly to tumor for the immunotherapy of both solid and lymphoid malignancies. Using this fusion protein technology, we have constructed a series of point mutations in the newly identified vasopermeability region of IL-2 for the purpose of deleting this activity. Using the monoclonal antibody chTNT-3, which binds to necrotic regions in solid tumors, a panel of fusion proteins consisting of wild-type (WT) and mutant IL-2 constructs were generated and tested for vasopermeability activity using an in vivo assay developed in our laboratory. Fusion proteins showing reduced or deleted vasopermeability activity were then tested for their cytokine potency by several methods including their binding to IL-2 receptors, T-cell proliferation assays, the induction of secondary cytokines, dose-escalating toxicity, and finally their ability to treat established solid tumors in syngeneic immunocompetent mice. The results of these studies clearly show that the vasopermeability activity of IL-2 can be substantially deleted by single point mutations without grossly affecting the immune function of the cytokine. Mutants showing good cytokine functionality and no vasopermeability activity do appear to be less toxic in mice and can retain their immunotherapeutic potential for the treatment of solid tumors. It is suggested that a particular mutant, R38W, is a promising candidate for the generation of an improved IL-2 reagent useful for the treatment of cancer and related diseases such as AIDS.
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