title: Development of an attenuated interleukin-2 fusion protein that can be activated by tumour-expressed proteases.
abstract: The ability to alter the cytokine microenvironment has the potential to shape immune responses in many physiological settings, including the immunotherapy of tumours. We set out to develop a general approach in which cytokines could be functionally attenuated until activated. We report the development and initial characterization of fusion proteins in which human or mouse interleukin-2 (IL-2), a potent growth factor for immune cells, is joined to a specific IL-2 inhibitory binding component separated by a protease site. The rationale is that upon cleavage by a protease the cytokine is free to dissociate from the inhibitory component and becomes biologically more available. We describe the successful development of two attenuation strategies using specific binding: the first uses the mouse IL-2 receptor alpha chain as the inhibitory binding component whereas the second employs a human antibody fragment (scFv) reactive with human IL-2. We demonstrated that the fusion proteins containing a prostate-specific antigen or a matrix metalloproteinase (MMP) protease cleavage site are markedly attenuated in the intact fusion protein but had enhanced bioactivity of IL-2 in vitro when cleaved. Further, we showed that a fusion protein composed of the IL-2/IL-2 receptor alpha chain with an MMP cleavage site reduced tumour growth in vivo in a peritoneal mouse tumour model. This general strategy should be applicable to other proteases and immune modulators allowing site-specific activation of immunomodulators while reducing unwanted side-effects.
authors.affiliation: Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA. John_Puskas@urmc.rochester.edu
authors.forename: John
authors.initials: J
authors.lastname: Puskas
authors.forename: Denise
authors.initials: D
authors.lastname: Skrombolas
authors.forename: Abigail
authors.initials: A
authors.lastname: Sedlacek
authors.forename: Edith
authors.initials: E
authors.lastname: Lord
authors.forename: Mark
authors.initials: M
authors.lastname: Sullivan
authors.forename: John
authors.initials: J
authors.lastname: Frelinger
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D007376:Interleukin-2 / Q000235:genetics* / Q000378:metabolism*; D053645:Interleukin-2 Receptor alpha Subunit / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009369:Neoplasms / Q000503:physiopathology; D017430:Prostate-Specific Antigen / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000970:Antineoplastic Agents; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D011994:Recombinant Proteins; D017430:Prostate-Specific Antigen