In the past decade, great strides have been made in the development of novel immunotherapies, such as immune checkpoint inhibitors (ICI) to treat cancer. However, despite these improvements, subsets of patients do not respond to ICI, highlighting an unmet need for alternative immunotherapies. As potent immunomodulators, cytokines have been explored as treatments for cancer but have been limited due to toxicity and poor pharmacokinetics (PK). One of these key cytokines, interleukin 21 (IL-21), is a pluripotent cytokine that activates anti-tumor T cell responses, induces B cell activation, and promotes generation and maintenance of germinal centers and tertiary lymphoid structures. A member of the common γ-chain family of cytokines, IL-21 acts on a broader range of cells than IL-2 and does not induce vascular leak syndrome. Despite being a potent inducer of immune activation, clinical activity of IL-21 has been hampered by poor PK and adverse events at dose levels associated with efficacy. To explore the potential therapeutic benefits of IL-21 as a treatment for cancer, Werewolf Therapeutics has developed different formats of IL-21 INDUKINETM polypeptides containing native human IL-21, an inactivation domain and a half-life extension domain tethered together by protease sensitive linkers. IL-21 INDUKINETM polypeptides are peripherally inactive, but due to dysregulation of the protease milieu in the tumor microenvironment, upon dosing and distribution to the tumor, the linkers are cleaved and IL-21 is released intratumorally. We will present data demonstrating that IL-21 INDUKINETM polypeptides showed in vitro and in vivo inducibility and activity. In mouse syngeneic tumor models, the IL-21 INDUKINETM polypeptides showed improved tolerability and therapeutic window compared to equal molar dosing of a half-life extended IL-21 protein (IL-21-HLE). Mechanistically, IL-21 triggered a pronounced activation of the adaptive immunity in the tumor. Efficacy in these tumor models was linked to expansion and activation of tumor infiltrating T cells, increased polyfunctionality in CD8+ T cells, as well as signs of increased activation of B cells. Together, these data support continued exploration of an IL-21 INDUKINETM polypeptide as a therapy for cancer. Citation Format: Jenna M. Sullivan, Pamela A. Aderhold, Heather R. Brodkin, Kyriakos Economides, Daniel J. Hicklin, Nesreen Ismail, Yuka Lewis, Cynthia Seidel-Dugan, Cierra Spencer, William M. Winston, Andres Salmeron. Generation of IL-21 INDUKINETM molecules for the treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1829.

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A member of the type-I IFN family, IFNα was also one of the first cytokines tested in the clinic as a therapy for cancer patients, leading to the approval of its use in chronic myelogenous leukemia, lymphoma, and malignant melanoma. Mechanistically, IFNα directly inhibits tumor cell proliferation and induces apoptosis, while simultaneously activating various innate and adaptive immune cell populations. Unfortunately, clinical use of IFNα has been hindered by the adverse events associated with its systemic administration, demonstrating the need for novel approaches to utilize this cytokine in the clinic. WTX-613 is a conditionally activated IFNα prodrug, or INDUKINE™ molecule, which has two identical half-life extension (HLE) domains tethered to IFNα2b via a proprietary tumor protease-sensitive linker. Following systemic administration, WTX-613 circulates as an inactive prodrug that is unable to bind to IFNα receptors in normal tissues due to the steric hinderance of the HLE domains complexed with albumin. However, when WTX-613 enters the tumor, proteases found in the tumor microenvironment (TME) cleave the protease-sensitive linkers, releasing fully active IFNα2b selectively in the tumor. Since human IFNα is not active in mice, a surrogate INDUKINE™ molecule containing mouse IFNα1 (mWTX-613/WW0610) was created. mWTX-613 treatment generated robust anti-tumor immunity in several murine syngeneic tumor models. In the MC38 model, mWTX-613 treatment was more efficacious than equimolar amounts of the recombinant cytokine (mIFNα1) and was accompanied by an increase in the therapeutic window. Mechanistically, systemic treatment with mWTX-613 resulted in prolonged peripheral exposure extending over a week beyond the time of the final dose. mWTX-613 treatment also resulted in a significant increase in the CD8/Treg ratio, and a robust increase in MHC I and MHC II expression by various professional antigen presenting cell populations. In addition, mWTX-613 treatment resulted in a clear and prolonged activation of both tumor infiltrating CD8+ T cells and NK cells. Transcriptionally, systemic treatment with mWTX-613 resulted in prolonged IFNα signaling within the TME, as demonstrated by significant enrichment in multiple Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with an anti-viral immune response. Finally, the combination of mWTX-613 treatment with various checkpoint inhibitor molecules resulted in improved anti-tumor efficacy in syngeneic models. Together, these data support the advancement of this innovative IFNα therapy into clinical testing. Citation Format: Christopher J. Nirschl, Heather R. Brodkin, Celesztina Domonkos, Kyriakos Economides, Daniel J. Hicklin, Nesreen Ismail, Cynthia Seidel-Dugan, Philipp Steiner, Zoe Steuert, William M. Winston, Andres Salmeron. WTX-613, (JZP898) a selectively activated IFNα INDUKINE™ molecule, reprograms the tumor microenvironment and generates robust anti-tumor immunity as a monotherapy and in combination with checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1817.