The use of cytokine-based therapeutics has traditionally been limited by systemic toxicity, but it is now becoming evident that it may be possible to circumvent this problem in a number of ways. Tumor-targeting antibody-cytokine fusion proteins (also called “immunocytokines”) typically allow dose escalation to therapeutically active regimens as a result of their selective localization at the site of disease (Neri 2019; Helguera, Morrison, and Penichet 2002). Despite the enhancement of the therapeutic index, when the concentration of the payload in blood exceeds a certain threshold, patients may experience dose-limiting immune-related adverse events (irAEs). In most cases, irAEs disappear when the immunocytokine is cleared from circulation (Ko et al. 2004; Spitaleri et al. 2013; Rudman et al. 2011). Here, we describe an innovative approach to mask off-target activity of immunocytokines by transient inhibition of their intracellular signaling cascade. Small molecules are particularly attractive for this application since their serum half-life matches the clearance rate of the immunocytokine from circulation. L19-TNF and L19-IL12 are two clinical-stage antibody-cytokine fusion proteins that display potent activity by triggering the receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and Janus Kinase 2 (JAK2) signaling pathways, respectively (Amin et al. 2018; Hu et al. 2021). In a first study, GSK’963, a potent small molecule inhibitor of RIPK1, was tested in tumor-bearing mice with the aim to reduce acute systemic toxicity associated with TNF signaling. Transient inhibition of RIPK1 allowed the administration of L19-murTNF doses which would otherwise be lethal. The combination with GSK’963 did not affect the selective localization of the immunocytokine to tumors. Moreover, L19-murTNF was still able to induce tumor necrosis and to exert potent anti-cancer activity. In a second study, tumor-bearing mice receiving L19-murIL12 were pretreated with Ruxolitinib, a commercially available JAK2 inhibitor. The addition of Ruxolitinib could significantly improve the tolerability profile of L19-murIL12 without affecting the anti-cancer properties of the immunocytokine. In clinical trials, it has been observed that IL12 treatment can induce cytokine release syndrome and liver damage (Atkins et al. 1997). Our data demonstrate that the pre-treatment with Ruxolitinib restricted the pro-inflammatory effects of L19-murIL12 to the tumor site, protecting the mice from body weight loss as well as reducing blood cytokine levels and hepatotoxicity. Overall, this preclinical work is of clinical relevance, as patients treated with targeted cytokines could potentially benefit from judicious combinations treatments using small molecule inhibitors.

Citation Format: Giulia Rotta, Ettore Gilardoni, Domenico Ravazza, Jacqueline Mock, Samuele Cazzamalli, Roberto De Luca, Emanuele Puca, Dario Neri, Sheila Dakhel Plaza. Decreasing toxicity of immunocytokines by transient and selective inhibition of their intracellular signaling activation. [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 5744.