Novel design of interleukin-2 derivatives with reversed pH and potent antitumor activity

Interleukin-2 (IL-2) displays high affinity for IL-2Rα at physiological pH, but low affinity in the acidic tumor microenvironment (TME), which brings side effects and reduces anti-tumor activity. This pH-dependent affinity modulation is attributed to protonation/deprotonation states of key acidic and basic amino acid residues within their binding interface, significantly impacting complex formation. Therefore, we designed three classes of IL-2 derivatives via a single-step process combining protein oxidative refolding and site-specific cysteine modification using the four prepared redox pairs based on IL-2(K64C). Surface plasmon resonance screening identified ZAP-IL-2(K64C) and Ac-TSAP-IL-2(K64C) derivatives with lower affinity for IL-2Rα at physiological pH and higher affinity under acidic TME conditions. Furthermore, toxicity assessment demonstrated that the pH-reversed IL-2 derivatives significantly mitigated the severe side effects associated with wild-type (wt) IL-2. In anti-tumor efficacy studies, the pH-reversed IL-2 derivatives demonstrated superior efficacy compared to wt IL-2, achieving a 50% tumor inhibition rate (TIR) versus 35% for wt IL-2 monotherapy. Strikingly, combination therapy with the pH-reversed IL-2 derivatives and the anti-PD-1 elicited a significant synergistic effect, further elevating the TIR to 85%. Our study provides a promising strategy for developing next-generation IL-2 therapeutics characterized by enhanced efficacy and reduced toxicity.