IL-15:IL-15 receptor alpha superagonist complex: high-level co-expression in recombinant mammalian cells, purification and characterization.

IL-15, a promising cytokine for treating cancer and viral diseases, is presented in trans by the IL-15 receptor (IL-15R) alpha-chain to the IL-15Rßγc complex displayed on the surface of T cells and natural killer (NK) cells. We previously reported that an asparagine to aspartic acid substitution at amino acid 72 (N72D) of IL-15 provides a 4-5-fold increase in biological activity compared to the native molecule. In this report, we describe Chinese hamster ovary (CHO) cell expression of a soluble complex (IL-15 N72D:IL-15RαSu/Fc) consisting of the IL-15 N72D superagonist and a dimeric IL-15Rα sushi domain-IgG1 Fc fusion protein. A simple but readily scalable affinity and ion exchange chromatography method was developed to highly purify the complex having both IL-15 binding sites fully occupied. The immunostimulatory effects of this complex were confirmed using cell proliferation assays. Treatment of mice with a single intravenous dose of IL-15N72D:IL-15RαSu/Fc resulted in a significant increase in CD8+ T cells and NK cells that was not observed following IL-15 treatment. Pharmacokinetic analysis indicated that the complex has a 25-h half-life in mice which is considerably longer than <40-min half-life of IL-15. Thus, the enhanced activity of the IL-15N72D:IL-15RαSu/Fc complex is likely the result of the increased binding activity of IL-15N72D to IL-15Rßγc, optimized cytokine trans-presentation by the IL-15RαSu domain, the dimeric nature of the cytokine domain and its increased in vivo half-life compared to IL-15. These findings indicate that this IL-15 superagonist complex could serve as a superior immunostimulatory therapeutic agent.

Interleukin-15:Interleukin-15 receptor α scaffold for creation of multivalent targeted immune molecules.

Human interleukin-15 (hIL-15) and its receptor α (hIL-15Rα) are co-expressed in antigen presenting cells allowing trans-presentation of the cytokine to immune effector cells. We exploited the high-affinity interactions between hIL-15 and the extracellular hIL-15Rα sushi domain (hIL-15RαSu) to create a functional scaffold for the design of multispecific fusion protein complexes. Using single-chain T cell receptors (scTCRs) as recognition domains linked to the IL-15:IL-15Rα scaffold, we generated both bivalent and bispecific complexes. In these fusions, the scTCR domains retain the antigen-binding activity and the hIL-15 domain exhibits receptor binding and biological activity. As expected, bivalent scTCR fusions exhibited improved antigen binding due to increased avidity, whereas fusions comprising two different scTCR domains were capable of binding two cognate peptide/MHC complexes. Bispecific molecules containing scTCR and scCD8αß domains also exhibit enhanced binding to peptide/MHC complexes, demonstrating that the IL-15:IL-15Rα scaffold displays flexibility necessary to support multi-domain interactions with a given target. Surprisingly, functional heterodimeric molecules could be formed by co-expressing the TCR α and ß chains separately as fusions to the hIL-15 and hIL-15RαSu domains. Together, these properties indicate that the hIL-15 and hIL-15RαSu domains can be used as versatile, functional scaffold for generating novel targeted immune molecules.

Targeting activity of a TCR/IL-2 fusion protein against established tumors.

We have previously reported that a single-chain T cell receptor/IL-2 fusion protein (scTCR-IL2) exhibits potent targeted antitumor activity in nude mice bearing human tumor xenografts that display cognate peptide/HLA complexes. In this study, we further explore the mechanism of action of this molecule. We compared the biological activities of c264scTCR-IL2, a scTCR-IL2 protein recognizing the aa264-272 peptide of human p53, with that of MART-1scTCR-IL2, which recognizes the MART-1 melanoma antigen (aa27-35). In vitro studies showed that c264scTCR-IL2 and MART-1scTCR-IL2 were equivalent in their ability to bind cell-surface IL-2 receptors and stimulate NK cell responses. In mice, MART-1scTCR-IL2 was found to have a twofold longer serum half-life than c264scTCR-IL2. However, despite its shorter serum half-life, c264scTCR-IL2 showed significantly better antitumor activity than MART-1scTCR-IL2 against p53(+)/HLA-A2(+) tumor xenografts. The more potent antitumor activity of c264scTCR-IL2 correlated with an enhanced capacity to promote NK cell infiltration into tumors. Similar differences in antigen-dependent tumor infiltration were observed with activated splenocytes pre-treated in vitro with c264scTCR-IL2 or MART-1scTCR-IL2 and then transferred into p53(+)/HLA-A2(+) tumor bearing recipients. The data support a model where c264scTCR-IL2 activates immune cells to express IL-2 receptors. Following stable interactions with cell-surface IL-2 receptors, c264scTCR-IL2 fusion molecule enhances the trafficking of immune cells to tumors displaying target peptide/HLA complexes where the immune cells mediate antitumor effects. Thus, this type of fusion molecule could be used directly as a targeted immunotherapeutic or in adoptive cell transfer approaches to activate and improve the anti-cancer activities of immune cells by providing them with pre-selected antigen recognition capability.

Potent antitumor activity of a tumor-specific soluble TCR/IL-2 fusion protein.

We previously have generated a single-chain T cell receptor-cytokine fusion protein (264scTCR/IL-2) comprising interleukin-2 genetically linked to a soluble HLA-A2.1-restricted TCR recognizing a peptide of human p53 protein. In this report, we show that 264scTCR/IL-2 inhibits the growth of primary tumors derived from the A375 (p53+/HLA-A2.1+) human melanoma and exhibits significantly better antitumor activity than recombinant human IL-2 alone. Moreover, treatment with 264scTCR/IL-2 results in tumor growth retardation in mice bearing large A375 tumors and other p53+/HLA-A2.1+ human tumors but does not affect tumor outgrowth of HLA-A2.1-negative tumors. This suggests that antigen targeting plays a substantial role in the efficacy of 264scTCR/IL-2 against p53+/HLA-A2+ tumors. Further, the antitumor activity of 264scTCR/IL-2 was found to be likely mediated by NK cell activation and tumor infiltration. A biologically active chimeric version of the molecule (c264scTCR/IL-2) also exhibits favorable pharmacokinetic properties required of a clinical candidate for this novel class of potent antitumor activities and targeted anticancer immunotherapeutics.

Amphitrite ornata dehaloperoxidase: enhanced activity for the catalytically active globin using MCPBA.

Dehaloperoxidase (DHP) from Amphitrite ornata is the only heme-containing, hydrogen peroxide-dependent globin capable of oxidatively dehalogenating halophenols to yield the corresponding quinones. To ascertain that this enzymatic activity is intrinsic to DHP, we have cloned and expressed the enzyme in Escherichia coli. We also find that an alternate oxygen atom donor, meta-chloroperbenzoic acid, gives appreciably higher activity than hydrogen peroxide. Under optimal turnover conditions (large peroxide/peracid excess), after an initial burst of activity, DHP appears to become trapped in a non-catalytic state (possibly Compound II) and is unable to fully convert all halophenol to product. However, full substrate conversion can be achieved under more physiological conditions involving a much smaller excess of oxygen atom donor. Parallel studies have been carried out using horseradish peroxidase and myoglobin to calibrate the activity of DHP versus typical peroxidase and globin proteins, respectively.