High-dose IL-2 is approved for patients with metastatic melanoma and renal cell cancer, but is associated with significant toxicity. We have described tumor-targeted CEA-IL2v and FAP-IL2v immunocytokines that are based on an engineered IL2v moiety with abolished binding to IL-2Ra (CD25) to avoid undesired CD25-mediated toxicities and Treg expansion. Their antibodies bind with high affinity to either CEA or FAP, which are broadly expressed in various tumors, and mediate retention/accumulation in malignant lesions. In an effort to further maximize the potency of IL-2R activation of T cells (aka signal 3) without the side effects of wildtype IL-2, we have generated PD1-IL2v that binds to the checkpoint inhibitor PD-1 and delivers IL2v preferentially in cis to PD-1+ T cells substituting binding to CD25. This enables high affinity IL2R signaling selectively in recently primed antigen specific TILs and stem-cell like subsets. Binding/competition experiments demonstrated that PD1-IL2v is 50-fold more potent than FAP-IL2v in inducing p-STAT5 in PD-1 positive T cells, whereas it has equivalent potency on PD-1 negative T cells. Notably, although Tregs can express low constitutive levels of PD-1, PD1-IL2v shows preferential binding to Teff vs. Tregs. Furthermore, PD1-IL2v can overcome Treg suppression of Tconv effector cells to a greater extent than PD-1, FAP-IL2v or their combination. For use in syngeneic mouse models muPD1-IL2v was generated. In an orthotopic pancreatic Panc02-Luciferase model administration of muPD1-IL2v (1 mg/kg IV, qw) resulted in rapid and complete elimination of tumor cells (loss of luciferase signal) and in long term survival (> 140 days) in 7/7 treated animals with protection from tumor cell re-challenge, whereas only 1/7 animals in the muPD-1 + muFAP-IL2v combination group (10 mg/kg + 2.5 mg/kg IV, qw) and none of the animals treated with the respective monotherapies showed long term survival. IHC showed that the improved outcome with muPD1-IL2v was related to a strong intra-tumoral increase of CD3+ CD8+ T cells expressing PD-1 and GrzB. In an immuno-PD study with s.c. Panc02 tumors muPD1-IL2v strongly increased the number of IFNg+ TNFa+ multifunctional and cytotoxic GrzB+ PD-1+ T cells in the tumor accompanied with a 20-fold increase in CD8/CD4 ratio and a strong increase in the CD8+ TEM population resulting in subsequent tumor control or remission in 50% of the animals, respectively. In summary, our data demonstrate that preferential cis-targeting of IL2v to PD-1+ antigen specific T cells with PD1-IL2v results in a strong potentiation of T cell response and anti-tumor efficacy as compared to the combination of PD-1 checkpoint inhibition with FAP-IL2v. These preclinical data establish PD1-IL2v as a promising next generation IL-2 for cancer immunotherapy.

Citation Format: Christian Klein, Laura Codarri-Deak, Valeria Nicolini, Stefan Seeber, Laura Lauener, Marine Richard, Esther Bommer, Maria Karagianni, Johannes Sam, Ramona Schlenker, Marisa Mariani, Petra Petra Schwalie, Sylvia Herter, Marina Bacac, Inja Waldhauer, Anne Freimoser-Grundschober, Volker Teichgraeber, Pablo Umana. A novel PD1-IL2v immunocytokine for preferential cis-activation of IL-2R signaling on PD-1 expressing T cell subsets strongly potentiates anti-tumor T cell activity of PD-1 checkpoint inhibition and IL-2R-beta-gamma agonism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1552.