Design and characterization of a protein superagonist of IL-15 fused with IL-15Rα and a high-affinity T cell receptor

• Published in: Biotechnology progress 2012-11, Vol.28 (6), p.1588-1597
• Main Authors: Stone, Jennifer D., Chervin, Adam S., Schreiber, Hans, Kranz, David M.
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Biotechnology; Cell Line, Tumor; Cell Proliferation; Drug Delivery Systems; Fundamental and applied biological sciences. Psychology; high-affinity TCR; Humans; IL-15/IL-15Rα; IL-2; immunokine; Interleukin-15 - agonists; Interleukin-15 - chemistry; Interleukin-15 - genetics; Interleukin-15 - metabolism; Interleukin-15 Receptor alpha Subunit - agonists; Interleukin-15 Receptor alpha Subunit - chemistry; Interleukin-15 Receptor alpha Subunit - genetics; Interleukin-15 Receptor alpha Subunit - metabolism; Mice; Oligopeptides - chemistry; Oligopeptides - metabolism; Protein Binding; Protein Engineering - methods; Rats; Receptors, Antigen, T-Cell - chemistry; Receptors, Antigen, T-Cell - genetics; Receptors, Antigen, T-Cell - metabolism; Recombinant Fusion Proteins - agonists; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; TCR-cytokine fusion
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1002/btpr.1631

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 (Kd = 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‐Kb (SIY/Kb) with nanomolar affinity (Kd = 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/Kb 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. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012