IL-2 is inactivated by the acidic pH environment of tumors enabling engineering of a pH-selective mutein.

Cytokines interact with their receptors in the extracellular space to control immune responses. How the physicochemical properties of the extracellular space influence cytokine signaling is incompletely elucidated. Here, we show that the activity of interleukin-2 (IL-2), a cytokine critical to T cell immunity, is profoundly affected by pH, limiting IL-2 signaling within the acidic environment of tumors. Generation of lactic acid by tumors limits STAT5 activation, effector differentiation, and antitumor immunity by CD8⁺ T cells and renders high-dose IL-2 therapy poorly effective. Directed evolution enabled selection of a pH-selective IL-2 mutein (Switch-2). Switch-2 binds the IL-2 receptor subunit IL-2Rα with higher affinity, triggers STAT5 activation, and drives CD8⁺ T cell effector function more potently at acidic pH than at neutral pH. Consequently, high-dose Switch-2 therapy induces potent immune activation and tumor rejection with reduced on-target toxicity in normal tissues. Last, we show that sensitivity to pH is a generalizable property of a diverse range of cytokines with broad relevance to immunity and immunotherapy in healthy and diseased tissues. Acid-tolerant IL-2 targets tumors: Hypoxic and acidic conditions prevalent in the tumor microenvironment present a formidable challenge for host lymphocytes trying to eradicate cancer cells. IL-2, a cytokine supporting CD8 T cell and NK cell functions, exhibits markedly compromised binding to the IL-2Rα receptor chain at pH 6 and below. Gaggero et al. used directed evolution to screen for IL-2 variants with mutations at the receptor interface that enhanced receptor binding at low pH. A mutated "Switch-2" variant of IL-2 with strong receptor binding at pH 6 but minimal interaction at neutral pH exhibited enhanced in vivo antitumor activity in mice. Recognition of the pH sensitivity of IL-2 and other cytokines will spur engineering of cytokine variants capable of enhanced performance in the acidic tumor microenvironment. [ABSTRACT FROM AUTHOR]