TransCon IL-2 ß/γ: a novel long-acting prodrug with sustained release of an IL-2Rß/γ-selective IL-2 variant with improved pharmacokinetics and potent activation of cytotoxic immune cells for the treatment of cancer.

BACKGROUND: Recombinant interleukin-2 (IL-2, aldesleukin) is an approved cancer immunotherapy but causes severe toxicities including cytokine storm and vascular leak syndrome (VLS). IL-2 promotes antitumor function of IL-2Rß/γ+ natural killer (NK) cells and CD8+, CD4+ and gamma delta (γδ) T cells. However, IL-2 also potently activates immunosuppressive IL-2Rα+ regulatory T cells (Tregs) and IL-2Rα+ eosinophils and endothelial cells, which may promote VLS. Aldesleukin is rapidly cleared requiring frequent dosing, resulting in high Cmax likely potentiating toxicity. Thus, IL-2 cancer immunotherapy has two critical drawbacks: potent activation of undesired IL-2Rα+ cells and suboptimal pharmacokinetics with high Cmax and short half-life. METHODS: TransCon IL-2 ß/γ was designed to optimally address these drawbacks. To abolish IL-2Rα binding yet retain strong IL-2Rß/γ activity, IL-2 ß/γ was created by permanently attaching a small methoxy polyethylene glycol (mPEG) moiety in the IL-2Rα binding site. To improve pharmacokinetics, IL-2 ß/γ was transiently attached to a 40 kDa mPEG carrier via a TransCon (transient conjugation) linker creating a prodrug, TransCon IL-2 ß/γ, with sustained release of IL-2 ß/γ. IL-2 ß/γ was characterized in binding and primary cell assays while TransCon IL-2 ß/γ was studied in tumor-bearing mice and cynomolgus monkeys. RESULTS: IL-2 ß/γ demonstrated selective and potent human IL-2Rß/γ binding and activation without IL-2Rα interactions. TransCon IL-2 ß/γ showed slow-release pharmacokinetics with a low Cmax and a long (>30 hours) effective half-life for IL-2 ß/γ in monkeys. In mouse tumor models, TransCon IL-2 ß/γ promoted CD8+ T cell and NK cell activation and antitumor activity. In monkeys, TransCon IL-2 ß/γ induced robust activation and expansion of CD8+ T cells, NK cells and γδ T cells, relative to CD4+ T cells, Tregs and eosinophils, with no evidence of cytokine storm or VLS. Similarly, IL-2 ß/γ enhanced proliferation and cytotoxicity of primary human CD8+ T cells, NK cells and γδ T cells. SUMMARY: TransCon IL-2 ß/γ is a novel long-acting prodrug with sustained release of an IL-2Rß/γ-selective IL-2. It has remarkable and durable pharmacodynamic effects in monkeys and potential for improved clinical efficacy and tolerability compared with aldesleukin. TransCon IL-2 ß/γ is currently being evaluated in a Phase 1/2 clinical trial (NCT05081609).

Increasing MinD's Membrane Affinity Yields Standing Wave Oscillations and Functional Gradients on Flat Membranes.

The formation of large-scale patterns through molecular self-organization is a basic principle of life. Accordingly, the engineering of protein patterns and gradients is of prime relevance for synthetic biology. As a paradigm for such pattern formation, the bacterial MinDE protein system is based on self-organization of the ATPase MinD and ATPase-activating protein MinE on lipid membranes. Min patterns can be tightly regulated by tuning physical or biochemical parameters. Among the biochemically engineerable modules, MinD's membrane targeting sequence, despite being a key regulating element, has received little attention. Here we attempt to engineer patterns by modulating the membrane affinity of MinD. Unlike the traveling waves or stationary patterns commonly observed in vitro on flat supported membranes, standing-wave oscillations emerge upon elongating MinD's membrane targeting sequence via rationally guided mutagenesis. These patterns are capable of forming gradients and thereby spatially target co-reconstituted downstream proteins, highlighting their functional potential in designing new life-like systems.

Probing Biomolecular Interactions by a Pattern-Forming Peptide-Conjugate Sensor.

As a key mechanism underpinning many biological processes, protein self-organization has been extensively studied. However, the potential to apply the distinctive, nonlinear biochemical properties of such self-organizing systems to biotechnological problems such as the facile detection and characterization of biomolecular interactions has not yet been explored. Here, we describe an in vitro assay in a 96-well plate format that harnesses the emergent behavior of the Escherichia coli Min system to provide a readout of biomolecular interactions. Crucial for the development of our approach is a minimal MinE-derived peptide that stimulates MinD ATPase activity only when dimerized. We found that this behavior could be induced via any pair of foreign, mutually binding molecular entities fused to the minimal MinE peptide. The resulting MinD ATPase activity and the spatiotemporal nature of the produced protein patterns quantitatively correlate with the affinity of the fused binding partners, thereby enabling a highly sensitive assay for biomolecular interactions. Our assay thus provides a unique means of quantitatively visualizing biomolecular interactions and may prove useful for the assessment of domain interactions within protein libraries and for the facile investigation of potential inhibitors of protein-protein interactions.

Design of biochemical pattern forming systems from minimal motifs.

Although molecular self-organization and pattern formation are key features of life, only very few pattern-forming biochemical systems have been identified that can be reconstituted and studied in vitro under defined conditions. A systematic understanding of the underlying mechanisms is often hampered by multiple interactions, conformational flexibility and other complex features of the pattern forming proteins. Because of its compositional simplicity of only two proteins and a membrane, the MinDE system from Escherichia coli has in the past years been invaluable for deciphering the mechanisms of spatiotemporal self-organization in cells. Here, we explored the potential of reducing the complexity of this system even further, by identifying key functional motifs in the effector MinE that could be used to design pattern formation from scratch. In a combined approach of experiment and quantitative modeling, we show that starting from a minimal MinE-MinD interaction motif, pattern formation can be obtained by adding either dimerization or membrane-binding motifs. Moreover, we show that the pathways underlying pattern formation are recruitment-driven cytosolic cycling of MinE and recombination of membrane-bound MinE, and that these differ in their in vivo phenomenology.

The MinDE system is a generic spatial cue for membrane protein distribution in vitro.

The E. coli MinCDE system has become a paradigmatic reaction-diffusion system in biology. The membrane-bound ATPase MinD and ATPase-activating protein MinE oscillate between the cell poles followed by MinC, thus positioning the main division protein FtsZ at midcell. Here we report that these energy-consuming MinDE oscillations may play a role beyond constraining MinC/FtsZ localization. Using an in vitro reconstitution assay, we show that MinDE self-organization can spatially regulate a variety of functionally completely unrelated membrane proteins into patterns and gradients. By concentration waves sweeping over the membrane, they induce a direct net transport of tightly membrane-attached molecules. That the MinDE system can spatiotemporally control a much larger set of proteins than previously known, may constitute a MinC-independent pathway to division site selection and chromosome segregation. Moreover, the here described phenomenon of active transport through a traveling diffusion barrier may point to a general mechanism of spatiotemporal regulation in cells.