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Introduction IL-2 therapy can lead to durable responses in cancer patients, but is associated with significant toxicity. None of the known IL-2-based immunocytokines has yet progressed to pivotal clinical trials due to various constraints in their design; in particular, the fusion of two wild-type IL-2 moieties to the antibody and retained FcgR binding of IgG-based immunocytokines. This design results in 1) high affinity binding with pM affinity to IL-2Raβγ on immune cells compromising tumor targeting and inducing rapid systemic clearance and short half-life; 2) high affinity for CD25 (IL-2Ra) expressed on pulmonary vascular endothelium contributing to pulmonary toxicity; and 3) preferential activation of Tregs over immune effectors. Here we describe a novel class of monomeric tumor-targeted immunocytokines where a single, engineered IL-2 variant (IL2v) with abolished CD25 binding is fused to the C-terminus of an antibody with a heterodimeric Fc-part. FcγR and C1q binding is completely abolished by a novel Fc mutation. For tumor targeting, human(-ized) high affinity antibodies against CEA (GA504, CEA-IL2v) or FAP (GA501, FAP-IL2v) were selected. CEA-IL2v recognizes a membrane proximal epitope of human carcinoembryonic antigen (CEA) and binds preferentially to membrane-bound CEA, but not shed CEA. Methods CEA- and FAP-IL2v were produced as recombinant proteins and their activity tested oneffector cells by assessing the activation of P-STAT5, cell proliferation, sensitivity to Fas-induced apoptosis, expression of activation markers and cytokine release upon treatment. Safety, pharmacokinetics (PK), pharmacodynamics (PD) and anti-tumor efficacy were analyzed in SCID and fully immunocompetent C57Bl/6 mice as single agent and in combination with trastuzumab and cetuximab. Tumor targeting was investigated in the orthotopic syngeneic Renca renal cell cancer tumor model in Balb/c mice by SPECT imaging. Results FAP- and CEA-IL2v completely lack binding to CD25, but retain IL-Rβγ binding, and show pM binding affinity to respective antigens, FAP on fibroblasts and CEA on tumor cells. As consequence of abolished binding to CD25 these molecules do not preferentially activate Tregs. The treatment of effector cells with IL2v reduces their sensitivity for Fas-mediated apoptosis (also known as activation induced cell death) as compared to wild-type IL-2 based immunocytokine. IL-2Rβγ bioactivity was retained and FAP- and CEA-IL2v activate NK, CD4+ and CD8+ T cells as shown by induction of activation markers, cell proliferation and cytokine release. Furthermore, CEA-IL2v and FAP-IL2v enhanced the cytotoxic activity of NK cells when combined with ADCC-competent antibodies. Mechanism of action studies in fully immunocompetent mice showed that the molecules strongly expand and activate NK, CD8+ T cells and gd T cells (up to 100-fold) and skew the CD4:CD8 ratio strongly towards CD8+ T cells in the peripheral blood, lymphoid tissues, and in the tumor. In C57Bl/6 mice, CEA- and FAP-IL2v demonstrate improved safety despite a higher exposure and circulatory half-life than the analogous IL-2 based immunocytokine. MicroSPECT/CT imaging with radioactively labeled FAP-IL2v revealed good FAP-mediated tumor targeting in the orthotopic syngeneic Renca model with low normal tissue uptake and low accumulation in lymphoid tissues, contrary to analogous IL-2 based immunocytokine that showed preferential targeting to lymphoid tissue. Studies in tumor-bearing mice showed dose-dependent anti-tumor efficacy of FAP-IL2v and CEA-IL2v in syngeneic models. Additional studies in xenograft models in SCID mice transgenic for human CD16A showed that CEA-IL2v strongly enhances the antitumor efficacy and/or survival mediated by ADCC-competentantibodies, including trastuzumab and cetuximab. Conclusion CEA- and FAP-IL2v demonstrate superior safety, PK and tumor targeting, while lacking preferential induction of Tregs due to abolished CD25 binding, monovalency and high-affinity tumor-targeting as compared to classical IL-2-based immunocytokines. They retain capacity to activate and expand NK and CD8+ effector T cells through IL-2Rβγ in the periphery and the tumor microenvironment. These data support their further nonclinical and clinical investigation for immunotherapy of cancer. Clinical trials with CEA-IL2v are foreseen in 2014. Disclosures: Klein: Roche Glycart AG: Employment. Waldhauer:Roche: Employment. Nicolini:Roche: Employment. Dunn:Roche: Employment. Freimoser-Grundschober:Roche: Employment. Danny:Roche: Research Funding. Boerman:Roche: Research Funding. Nayak:Roche: Employment. Herter:Roche: Employment. Van Puijenbroek:Roche: Employment. Ast:Roche: Employment. Hofer:Roche: Employment. Hosse:Roche: Employment. Lang:Roche: Employment. Neumann:Roche: Employment. Kettenberger:Roche: Employment. Neubauer:Roche: Employment. Gorr:Roche: Employment. Tuerck:Roche: Employment. Evers:Roche: Employment. Gerdes:Roche: Employment. Levitsky:Roche: Employment. Bacac:Roche: Employment. Moessner:Roche: Employment. Umana:Roche: Employment, Equity Ownership.