Conditionally Activated IL-12 or IFNα Indukine™ Molecules Inhibit Syngeneic Lymphoma Tumor Growth in Mice, Induce Anti-Tumor Immune Responses and Are Tolerated in Non-Human Primates
Authors: Steiner Philipp , Brodkin Heather , Canales Josue , Hicklin Daniel , Isaacs Randi , Ismail Nesreen , Morris Kristin , Nirschl Christopher , Salmeron Andres , Seidel-Dugan Cindy , Steuert Zoe , Sullivan Jenna , Tyagi Ethika , Winston William 
Published: November 2021

Abstract
Systemic therapy with proinflammatory immune modulators to activate anti-tumor immunity is a promising approach to treat cancer. However, poor pharmacokinetic properties and dose-limiting toxicities such as inflammation, cytokine release syndrome, and tissue damage have prevented or limited the clinical use of cytokines such as interleukin 12 (IL-12) and interferon α (IFNα). Previous clinical development of rhIL-12 was terminated due to toxicity, and although rIFNα is approved for the treatment of melanoma, lymphoma, and leukemia, its use has been limited by systemic toxicity and modest efficacy.
INDUKINE™ molecules, engineered using Werewolf Therapeutics' Predator™ discovery platform, are novel, systemically delivered cytokine pro-drugs which prevent systemic toxicity and deliver cytokines to the tumor microenvironment (TME) where they are activated. Several INDUKINE™ molecules using Werewolf's proprietary linkers have been previously shown to be cleaved by a wide range of human solid tumors but have yet to be tested in primary human lymphoma samples or syngeneic mouse lymphoma models.
IL-12 is a potent, pleiotropic cytokine for immune-mediated killing of cancer cells, whose mechanism of action (MOA) includes activation of both cytotoxic T and NK cells. The WTX-330 INDUKINE™ molecule, a wild-type IL-12 pro-drug, contains a half-life extension (HLE) domain to support infrequent dosing and a high affinity anti-IL-12 neutralizing antibody domain to maintain the molecule in its inactive state in the periphery. Both the HLE and blocking domains are tethered to IL-12 via two identical tumor protease-sensitive linkers. Linker cleavage in the TME removes the HLE domain and the blocker, resulting in active IL-12 within the TME with wild-type IL-12 biologic and PK profiles.
IFNα is a member of the type-I IFN family and activates innate immune responses either directly by engaging IFNα receptors (IFNAR) ubiquitously expressed on immune cells or indirectly by inducing chemokines that attract myeloid and lymphoid cells to the tumor site. The WTX-613 INDUKINE™ molecule is an inactive IFNα2b pro-drug with two identical HLE domains tethered to IFNα2b via two identical tumor protease-sensitive linkers. The HLE domains sterically block binding of WTX-613 to IFNAR until cleavage of the linkers in the TME releases active IFNα.
Since human IL-12 and IFNα2b are not cross reactive in mice, surrogate WTX-330 and WTX-613 INDUKINE™ molecules were created, consisting of a mouse/human chimeric IL-12 or a mouse IFNα1 to explore anti-tumor responses in syngeneic hematologic cancer models. In the subcutaneously (s.c.) A20 B cell lymphoma model, the WTX-330 surrogate showed dose-dependent anti-tumor activity with 4 out of 10 tumor-free mice at the top dose. The WTX-613 surrogate demonstrated tumor stasis lasting beyond the treatment phase. Utilizing the s.c. EG7.OVA T lymphoblast line, tumor growth was efficiently blocked by the WTX-613 surrogate while the WTX-330 surrogate inhibited tumor growth during the dosing period. Both treatments were well tolerated by the mice at active dose levels.
The WTX-330 and WTX-613 surrogates strongly activated NK and CD8+ cell responses and induced APC and effector cell markers in the MC38 syngeneic tumor model supporting a MOA as described for wild-type IL-12 and IFNα. Similar studies are on-going in lymphoma models. PK analysis in mice revealed extended half-life (T1/2) for both WTX-330 and WTX-613 surrogates compared to the short T1/2 of native IL-12 or IFNα1. Finally, WTX-330 and WTX-613 were well tolerated in non-human primates (NHP), resulting in plasma exposure levels for INDUKINE™ molecules that exceeded those needed for anti-tumor activity in mice. In addition, plasma levels of free IL-12 after dosing with WTX-330 were very low compared to tolerated levels of wild-type IL-12. Similar studies for WTX-613 are on-going.
Preclinical data obtained so far for both programs support the continued development and future evaluation of these innovative and differentiated therapies in hematologic malignancies, both as monotherapies and in multiple combinations with standard of care.

Disclosures
Isaacs: Werewolf Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Seidel-Dugan: Werewolf Therapeutics: Current Employment, Other: current shareholder .