title: Design and characterization of a protein superagonist of IL-15 fused with IL-15Rα and a high-affinity T cell receptor.
abstract: To avoid high systemic doses, strategies involving antigen-specific delivery of cytokine via linked antibodies or antibody fragments have been used. Targeting cancer-associated peptides presented by major histocompatibility complex (MHC) molecules (pepMHC) increases the number of potential target antigens and takes advantage of cross-presentation on tumor stroma and in draining lymph nodes. Here, we use a soluble, high-affinity single-chain T cell receptor Vα-Vβ (scTv), to deliver cytokines to intracellular tumor-associated antigens presented as pepMHC. As typical wild-type T cell receptors (TCRs) exhibit low affinity (K(d) = 1-100 μM or more), we used an engineered TCR, m33, that binds its antigenic peptide SIYRYYGL (SIY) bound to the murine class I major histocompatability complex protein H2-K(b) (SIY/K(b) ) with nanomolar affinity (K(d) = 30 nM). We generated constructs consisting of m33 scTv fused to murine interleukin 2 (IL-2), interleukin 15 (IL-15), or IL-15/IL-15Rα (IL-15 linked to IL-15Rα sushi domain, called "superfusion"). The fusions were purified with good yields and bound specifically to SIY/K(b) with high affinity. Proper cytokine folding and binding were confirmed, and the fusions were capable of stimulating proliferation of cytokine-dependent cells, both when added directly and when presented in trans, bound to cells with the target pepMHC. The m33 superfusion was particularly potent and stable and represents a promising design for targeted antitumor immunomodulation.
authors.affiliation: Dept. of Biochemistry, University of Illinois, Urbana, IL 61801, USA. jstone@illinois.edu
authors.forename: Jennifer D
authors.initials: JD
authors.lastname: Stone
authors.forename: Adam S
authors.initials: AS
authors.lastname: Chervin
authors.forename: Hans
authors.initials: H
authors.lastname: Schreiber
authors.forename: David M
authors.initials: DM
authors.lastname: Kranz
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D016503:Drug Delivery Systems; D006801:Humans; D019409:Interleukin-15 / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053720:Interleukin-15 Receptor alpha Subunit / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D051381:Rats; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D019409:Interleukin-15; D053720:Interleukin-15 Receptor alpha Subunit; D009842:Oligopeptides; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C406862:superagonist SIYR