Natural killer (NK) cells are cytotoxic lymphocytes that largely contribute to the efficacy of therapeutic strategies like allogenic stem cell transplantation in acute myeloid leukemia (AML) and application of Rituximab in chronic lymphocytic leukemia (CLL). The tumor necrosis factor (TNF) family member GITR ligand (GITRL) is frequently expressed on leukemia cells in AML and CLL and impairs the reactivity of NK cells which express GITR and upregulate its expression following activation. We developed a strategy to reinforce NK anti-leukemia reactivity by combining disruption of GITR-GITRL interaction with targeting leukemia cells for NK antibody-dependent cellular cytotoxicity (ADCC) using GITR-Ig fusion proteins with modified Fc moieties. Neutralization of leukemia-expressed GITRL by the GITR domain enhanced cytotoxicity and cytokine production of NK cells depending on activation state with NK reactivity being further largely dependent on the engineered affinity of the fusion proteins to the Fc receptor. Compared with wild-type GITR-Ig, treatment of primary AML and CLL cells with mutants containing a S239D/I332E modification potently increased cytotoxicity, degranulation, and cytokine production of NK cells in a target-antigen-dependent manner with additive effects being observed with CLL cells upon parallel exposure to Rituximab. Fc-optimized GITR-Ig may thus constitute an attractive means for immunotherapy of leukemia that warrants clinical evaluation.

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2143 NK cells play an important role in anti-tumor immunity and largely contribute to the efficacy of therapeutic strategies like allogenic stem cell transplantation in AML and application of Rituximab that induces antibody-dependent cellular cytotoxicity (ADCC) in CLL. Recently, we demonstrated that the TNF family member GITR ligand (GITRL) is expressed on leukemia cells in a high proportion of AML and CLL patients and impairs direct and Rituximab-induced reactivity of NK cells which constitutively express its counterpart GITR (e.g., Buechele et al., Leukemia 2012). Here we developed a strategy to reinforce NK anti-leukemia reactivity by combining disruption of NK-inhibitory GITR-GITRL interaction with induction of ADCC against the GITRL-expressing leukemia cells using GITR-Ig fusion proteins with modified Fc moieties. Fc parts were engineered by amino acid exchange as previously described (Lazar et al., PNAS 2006; Armour et al., Eur. J. Immunol. 1999). Compared to wild type GITR-Ig (GITR-Fc-WT), our mutants (S239D/I332E and E233P/L234V/L235A/deltaG236/A327G/A330S) displayed highly enhanced (GITR-Fc-ADCC) and abrogated (GITR-Fc-KO) affinity to the Fc(gamma)RIIIa receptor (CD16) expressed on NK cells, respectively. In functional analyses of NK cells and primary leukemia cells, GITR-Fc-KO, which does not induce ADCC, already increased NK reactivity due to disruption of GITR-GITRL interaction. Treatment with GITR-Fc-WT further enhanced NK reactivity due to modest induction of ADCC, while GITR-Fc-ADCC induced highly increased NK-mediated target cell lysis, degranulation and cytokine production in a target-antigen dependent manner. With CLL cells, combined treatment with GITR-Fc-ADCC fusion protein and Rituximab caused additive effects, resulting in significantly enhanced NK cell ADCC. Notably, the effects of our fusion proteins were observed both in an allogenic setting and when employing NK cells of patients with autologous leukemia cells as targets. Our results demonstrate that Fc-engineered GITR-Fc-ADCC fusion protein may combine both neutralization of the NK-inhibitoryeffects of GITR-GITRL interaction and targeting GITRL-expressing malignant cells for NK anti-tumor reactivity and thus constitute an attractive immunotherapeutic means for the treatment of AML and CLL. Disclosures: No relevant conflicts of interest to declare.