Volume 21, Issue 4, Pages 877-886 (April 2013) Generation and Preclinical Characterization of a Fc-optimized GITR-Ig Fusion Protein for Induction of NK Cell Reactivity Against Leukemia  Benjamin Joachim Schmiedel, Antje Werner, Julia Steinbacher, Tina Nuebling, Corina Buechele, Ludger Grosse-Hovest, Helmut Rainer Salih  Molecular Therapy  Volume 21, Issue 4, Pages 877-886 (April 2013) DOI: 10.1038/mt.2013.11 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Schematic illustration of the GITR-Ig fusion proteins. GITR-Ig fusion proteins were generated as dimeric constructs containing the extracellular domain of the GITR receptor (Q26-P162) and a human Fc tail (P217-K447 with C220S) with either the wild-type amino acid sequence or with several modifications (indicated by arrows) to enhance or to knockout its recognition by Fc receptors. Molecular Therapy 2013 21, 877-886DOI: (10.1038/mt.2013.11) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Binding characteristics of the Fc-engineered GITR-Ig fusion proteins. (a–c) Specific binding of the indicated GITR-Ig fusion proteins to surface-expressed GITRL was confirmed by FACS after determination of GITRL positivity using GITRL mAb and isotype control as described in the Materials and Methods section. (a) GITRL transfectants (C1R-GITRL) and control cells (C1R-neo); (b) primary GITRL-positive and -negative AML and (c) primary GITRL-positive and -negative CLL cells. Shaded peaks, specific mAb or fusion proteins (10 µg/ml each); open peaks, respective controls. (d) NK cells were incubated with the different GITR-Ig fusion proteins followed by a secondary PE-conjugate and analysis by FACS to unravel binding of the Fc parts to CD16. Data of one representative experiment each out of at least five with similar results are shown. AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; FACS, fluorescence-activated cell sorting; mAb, monoclonal antibody; NK, natural killer cell. Molecular Therapy 2013 21, 877-886DOI: (10.1038/mt.2013.11) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Modulation of NK cell reactivity by the GITR-Fc constructs. (a–c) Twenty-four well plates were coated with GITR mAb and blocked by addition of 10% FCS-PBS followed by addition of GITR-Ig fusion proteins or Fc controls (10 µg/ml each) and washing. Subsequently, pNK cells were added and cultured for 24 hours in the presence or absence of 100 U/ml IL-2 (+ IL-2) as indicated. (a) Downregulation of CD16 expression and (b) upregulation of the activation marker CD69 was analyzed by FACS; the percentage of positive NK cells is indicated. (c) Supernatants were analyzed for IFN-γ levels by ELISA. (d) Lysis of C1R-GITRL and C1R-neo by pNK cells (E:T ratio, 10:1) upon exposure to the indicated concentrations of GITR-Fc-ADCC was determined by 2-hour europium release assays. (e) C1R-GITRL and pNK cells (E:T ratio, 10:1) were cultured with or without GITR-Fc-ADCC or Fc control (10 µg/ml each) in the presence of the indicated concentrations of sGITRL. Cytotoxicity was determined by 2-hour europium release assay. One representative experiment each of a total of at least three with similar results is shown. FACS, fluorescence-activated cell sorting; E:T, Effector:target; FCS, fetal calf serum; IFN, interferon; IL, interleukin; mAb, monoclonal antibody; NK, natural killer cell; PBS, phosphate-buffered saline. Molecular Therapy 2013 21, 877-886DOI: (10.1038/mt.2013.11) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Induction of NK reactivity against primary AML cells by GITR-Ig fusion proteins. Leukemia cells of AML patients (>80% blast count) were cultured with PBMC or pNK cells as indicated in the presence or absence of GITR-Ig fusion proteins or Fc controls (10 µg/ml each). (a) Cytotoxicity of pNK cells was determined by 2-hour europium release assays. (b) IFN-γ levels released by pNK cells into supernatants were analyzed after 24 hours by ELISA. Exemplary results using GITRL-positive (left) and -negative (middle) AML cells as well as combined results obtained in the indicated number (n) of independent experiments with GITRL-positive patient cells (right) are shown. (c) Granule mobilization on resting (left panel) and briefly activated (12 hours exposure to 50 U/ml IL-2, middle panel) NK cells among PBMC of healthy donors as well as pNK cells (right panel) was analyzed by FACS for CD107a after 3 hours of culture. For combined analysis, results obtained with untreated target cells were set to one (dotted line) in each individual data set to enable statistical analysis. *Statistically significant differences (P < 0.05, Mann–Whitney U-test). AML, acute myeloid leukemia; FACS, fluorescence-activated cell sorting; IFN, interferon; IL, interleukin; NK, natural killer cell; PBMC, peripheral blood mononuclear cell. Molecular Therapy 2013 21, 877-886DOI: (10.1038/mt.2013.11) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Induction of NK reactivity against primary CLL cells by GITR-Ig fusion proteins. Leukemic cells of CLL patients (>80% lymphocyte count) were cultured with PBMC or pNK cells as indicated in the (a–c) absence or (d,e) presence of Rituximab (R) with or without GITR-Ig fusion proteins or Fc controls (10 µg/ml each). (a,d) Cytotoxicity of pNK cells was determined by 2-hour europium release assays. (b,e) IFN-γ levels released by pNK cells into supernatants were analyzed after 24 hours by ELISA. Exemplary results using GITRL-positive (left) and -negative (middle) CLL cells as well as combined results obtained in the indicated number (n) of independent experiments with GITRL-positive patient cells (right) are shown. (c) Granule mobilization on resting (left panel) and briefly activated (12 hours exposure to 50 U/ml IL-2, middle panel) NK cells among PBMC of healthy donors as well as pNK cells (right panel) was analyzed by FACS for CD107a after 3 hours of culture. For combined analysis, results obtained with untreated or Rituximab-treated target cells were set to one (dotted line) in each individual data set to enable statistical analysis. *Statistically significant differences (P < 0.05, Mann–Whitney U-test). CLL, chronic lymphocytic leukemia; E:T, Effector:target; FACS, fluorescence-activated cell sorting; IFN, interferon; IL, interleukin; NK, natural killer cell; PBMC, peripheral blood mononuclear cell. Molecular Therapy 2013 21, 877-886DOI: (10.1038/mt.2013.11) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Reactivity of NK cells against primary AML and CLL cells upon treatment with GITR-Ig in the autologous system. (a,b) Granule mobilization of autologous NK cells within PBMC of (a) AML patients (CD56+CD3−CD33−CD34−) and (b) CLL patients (CD56+CD3−) with 50–90% content of leukemic cells was analyzed by FACS for CD107a after 3 hours of culture in the presence or absence of the indicated GITR-Ig fusion proteins or Fc controls (10 µg/ml each). In case of CLL samples, experiments were performed with and without Rituximab (R) (10 µg/ml) as indicated. Numbers in dot plots represent the percentage of CD107a-positive NK cells. Exemplary results of one experiment are shown in the upper panels. Lower panels represent combined results obtained with 12 different patients of each leukemia entity (percentages of CD107a-positive NK cells in each individual sample as well as medians of results). (c,d) PBMC of (c) AML and (d) CLL patients (both n = 6) were cultured in the presence or absence of the GITR-Ig fusion proteins or Fc controls (10 µg/ml each) for 24 hours. Where indicated, Rituximab (R) (10 µg/ml) was also added to CLL samples. Leukemia cell lysis was determined by a FACS-based assay system as described in the Materials and Methods section; PBMC of leukemia patients that had been left untreated or, in case of CLL were exposed to Rituximab only where indicated served as control. The median within each group is indicated by (:). *Statistically significant differences (P < 0.05, Mann–Whitney U-test). AML, acute myeloid leukemia; CLL, chronic lymphocytic leukemia; FACS, fluorescence-activated cell sorting; NK, natural killer cell; PBMC, peripheral blood mononuclear cell. Molecular Therapy 2013 21, 877-886DOI: (10.1038/mt.2013.11) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions