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Background Therapeutic delivery of Interleukin 21 (IL-21) has been reported to reduce T cell exhaustion and prolong effector responses in chronic viral infection and cancer. 1 2 However, dysregulated IL-21 activity is also linked to adverse reactions and autoimmune disease.3 Strategies to safely control and direct IL-21 activity in vivo are limited by the poor stability and pharmacokinetics of this cytokine. We deployed computational tools to develop a stabilized murine IL-21 (cs21) that is amenable to modification. Intratumoral administration of cs21 conjugated to antibodies targeting CD45 results in sustained immune cell activation without systemic toxicity.Methods We simulated the interaction between murine IL-21 and its receptors using AlphaFold 3. 4 Using this computational prediction and homology with the known human structure, we identified key contact residues to preserve while permitting modification of non-contact residues. IL-21 variants were generated using Protein MPNN and screened for expression by mammalian cells, along with their ability to stimulate IL-21 reporter cell lines.5 Further in vitro validation was performed with pSTAT staining on primary murine CD8+ T cells. Activity in vivo was confirmed with pSTAT staining of immune cells in the tumor draining lymph nodes of mice bearing syngeneic B16F10 melanoma tumors following intratumoral administration.Results The IL-21 variant cs21 was selected for its ~25-fold improvement in mammalian expression and ~3-fold improvement in activity on IL-21 reporter cells. On primary murine T cells, an IgG conjugate of cs21 exhibited a ~100-fold decrease in the EC 50 of pSTAT3 upregulation compared with murine IL-21. To potentiate locally retained intratumoral therapy, we conjugated cs21 to a CD45 targeting antibody. In B16F10 melanoma, αCD45-cs21 induced robust pSTAT3 in CD45+ cells in the tumor draining lymph node. Monotherapy αCD45-cs21 induced enhanced median survival while the combination of αCD45-cs21 with other cytokines such as IL-12 and IL-15 exhibited potent synergy without apparent toxicity.Conclusions Computational stabilization of IL-21 results in a sequence with enhanced immunostimulatory activity, and therapeutic utility. Conjugation of cs21 to CD45 targeting antibodies permits safe and efficacious intratumoral therapy.References Elsaesser H, Sauer K, Brooks DG. IL-21 is required to control chronic viral infection. Science. 2009;324:1569–1572.Zander R, Schauder D, Xin G, Nguyen C, Wu X, Zajac A, Cui W. CD4+ T cell help is required for the formation of a cytolytic CD8+ T cell subset that protects against chronic infection and cancer. Immunity. 2019;51:1028–1042.Ren HM, Lukacher AE, Rahman ZSM, Olsen NJ. New developments implicating IL-21 in autoimmune disease. J Autoimmun. 2021;122.Abramson J, Adler J, Dunger J, et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024;630:493–500.Dauparas J, Anishchenko I, Bennett N, et al. Robust deep learning-based protein sequence design using ProteinMPNN. Science. 2022;378:49–56.Ethics Approval All animal experiments were approved by the Massachusetts Institute of Technology institutional animal care and use committee and conducted in compliance with federal, state, and local regulations.