NKTR-214, an Engineered Cytokine with Biased IL2 Receptor Binding, Increased Tumor Exposure, and Marked Efficacy in Mouse Tumor Models.

PURPOSE: Aldesleukin, recombinant human IL2, is an effective immunotherapy for metastatic melanoma and renal cancer, with durable responses in approximately 10% of patients; however, severe side effects limit maximal dosing and thus the number of patients able to receive treatment and potential cure. NKTR-214 is a prodrug of conjugated IL2, retaining the same amino acid sequence as aldesleukin. The IL2 core is conjugated to 6 releasable polyethylene glycol (PEG) chains. In vivo, the PEG chains slowly release to generate active IL2 conjugates. EXPERIMENTAL DESIGN: We evaluated the bioactivity and receptor binding of NKTR-214 and its active IL2 conjugates in vitro; the tumor immunology, tumor pharmacokinetics, and efficacy of NKTR-214 as a single agent and in combination with anti-CTLA-4 antibody in murine tumor models. Tolerability was evaluated in non-human primates. RESULTS: In a murine melanoma tumor model, the ratio of tumor-killing CD8(+) T cells to Foxp3(+) regulatory T cells was greater than 400 for NKTR-214 compared with 18 for aldesleukin, supporting preferential activation of the IL2 receptor beta over IL2 receptor alpha, due to the location of PEG molecules. NKTR-214 provides a 500-fold greater exposure of the tumor to conjugated IL2 compared with aldesleukin. NKTR-214 showed efficacy as a single agent and provided durable immunity that was resistant to tumor rechallenge in combination with anti-CTLA-4 antibody. NKTR-214 was well tolerated in non-human primates. CONCLUSIONS: These data support further evaluation of NKTR-214 in humans for a variety of tumor types, adding to the repertoire of potent and potentially curative cancer immunotherapies.

Dystonia-associated forms of torsinA are deficient in ATPase activity.

Early-onset dystonia is caused by mutations in the torsinA protein, a putative member of the AAA+ class of ATPases. In this study we have evaluated the ATPase activity of bacterially expressed wild-type torsinA and its disease-associated mutant forms. Upon overexpression in Escherichia coli, recombinant torsinA proteins were accumulated as insoluble inclusion bodies and required refolding to become soluble and catalytically active. The refolded wild-type and mutant torsinA proteins were capable of hydrolyzing ATP, but their specific ATPase activities differed significantly. Deletions of the amino acid residues E302/303 and F323-Y328 resulted in a decrease of ATPase activity to approximately 35% and approximately 75% of the wild-type level, respectively. ATPase activity of wild-type and mutant torsinA proteins was influenced by factors that varied with cell stress, such as temperature, pH, and ionic strength, and was inhibited by sodium vanadate. Our results provide the first direct evidence for a role of torsinA as an active ATPase and suggest that the mutations in torsinA might affect normal functions of the protein by reducing its enzymatic activity.

Development and anatomic localization of torsinA.

We have mapped the distribution of torsinA, the protein that is mutated in dystonia type 1 (DYT1), during postnatal development in rat brain. TorsinA was expressed in most brain regions at postnatal day 7, and its expression became more intense and widespread with age. The distribution of torsinA, however, showed marked age-dependent differences among regions of the cerebral cortex and hippocampus. Notably, large cholinergic interneurons of the striatum displayed intense torsin labeling between postnatal days 14 and 21, a period of intense synaptogenesis in this region.