Purpose The effectiveness of NK cell infusions to induce leukemic remission is limited by lack of both antigen specificity and in vivo expansion. To address the first issue we previously generated a bispecific killer engager (BiKE) containing single chain scFV against CD16 and CD33 to create an immunologic synapse between NK cells and CD33+ myeloid targets. We have now incorporated a novel modified human IL-15 crosslinker, producing a 161533 trispecific killer engager (TriKE) to induce expansion, priming, and survival, which we hypothesize will enhance clinical efficacy. Methods Reagents were tested in proliferation and functional assays and in an in vivo xenograft model of AML. Results When compared to the 1633 BiKE, the 161533 TriKE induced superior NK cell cytotoxicity, degranulation, and cytokine production against CD33+ HL-60 targets and increased NK survival and proliferation. Specificity was shown by the ability of a 1615EpCAM TriKE to kill CD33-EpCAM+ targets. Using NK cells from patients after allogeneic stem cell transplantation when NK cell function is defective, the 161533 TriKE restored potent NK function against primary AML targets and induced specific NK cell proliferation. These results were confirmed in an immunodeficient mouse HL-60-Luc tumor model where the 161533 TriKE exhibited superior anti-tumor activity and induced in vivo persistence and survival of human NK cells for at least 3 weeks. Conclusions Off-the-shelf 161533 TriKE imparts antigen specificity and promotes in vivo persistence, activation, and survival of NK cells. These qualities are ideal for NK cell therapy of myeloid malignancies or targeting antigens of solid tumors.

Natural killer (NK) cells play an important role in the immunosurveillance of hematopoietic malignancies. Their reactivity is influenced by activating and inhibitory signals mediated by tumor-expressed ligands for NK receptors. Many members of the tumor necrosis factor (TNF) family modulate differentiation, proliferation, activation and death of both tumor and immune effector cells. The TNF receptor family member glucocorticoid-induced TNFR-related protein (GITR) stimulates anti-tumor immunity in mice, but available data indicate that GITR may mediate different effects in mice and men and impairs the reactivity of human NK cells. Here, we comprehensively studied the expression and function of GITR ligand (GITRL) in leukemia. Among the different leukemia entities, pronounced expression of GITRL on leukemic cells was observed in chronic lymphocytic leukemia (CLL), and the GITR receptor was expressed at significantly higher levels on NK cells of CLL patients compared with healthy controls. Upon GITR --GITRL interaction, signaling via GITRL into the leukemia cells induced the release of interleukin (IL)-6, IL-8 and TNF, which act as growth and survival factors for CLL cells. In addition, GITRL impaired both direct and Rituximab-induced degranulation, cytotoxicity and interferon-g production of NK cells, which could be restored by GITR blocking antibodies. Thus, GITRL may contribute to disease pathophysiology and resistance to direct and Rituximab-induced NK reactivity in CLL.

Invariant natural killer T (iNKT)/natural killer (NK)/cytokine-induced killer (CIK) cells are important for immune surveillance. (I) Novel combinations of antibody 6B11 (targeting the Vα24-Jα18-invariant T-cell receptor) with CD4/CD8/CD1d/Vα24 for iNKT subset detection and "T/NK cell-like"-iNKT subsets were defined. Compared with healthy peripheral blood mononuclear cells (MNC) (significantly) lower proportions of iNKT cells (6B11/6B11CD3/6B11CD161), NK cells (CD3CD56/CD3CD161), and CIK cells (CD3CD56/CD3CD161) were found in peripheral blood MNC from acute myeloid (AML)/acute myeloid, lymphoid (ALL)/chronic lymphoid leukemia (CLL) patients in acute disease stages. Subtyping of iNKT cells revealed (significantly) higher proportions of CD3 T cells and CD161 NK cells in AML/ALL/CLL expressing 6B11 compared with healthy MNC. Prognostic evaluations showed higher proportions of iNKT/NK/CIK cells in favorable AML subgroups (younger age, primary, no extramedullary disease, achievement/maintenance of complete remission) or adult ALL and CLL patients. (II) iNKT/NK/CIK cell frequencies increased after (vs. before) mixed lymphocyte cultures of T-cell-enriched immune reactive cells stimulated with MNC/whole blood with or without pretreatment with "cocktails" (dendritic cells generating methods/kits inducing blasts' conversion to leukemia-derived dendritic cells from AML patients). Individual "cocktails" leading to "highest" iNKT cell frequencies could be defined. Antileukemic blast lytic activity correlated significantly with frequencies of iNKT/NK/CIK cells. In summary healthy MNC show significantly more iNKT/NK/CIK cells compared with AML/ALL/CLL MNC, a shift in the iNKT cell composition is seen in healthy versus leukemic samples and iNKT/NK/CIK cell-proportions in AML/ALL/CLL MNC samples correlate with prognosis. "Cocktail"-treated AML blasts lead to higher iNKT/NK/CIK cell frequencies and samples with antileukemic activity show significantly higher frequencies of iNKT/NK/CIK cells. Proportions of iNKT/NK/CIK cells should regularly be evaluated in AML/ALL/CLL diagnosis panels for quantitative/prognostic estimation of individual patients' antileukemic potential and their role in dendritic cells/leukemia-derived dendritic cells triggered immune surveillance.

Myelofibrosis (MF) is a rare disease responsible for an increasing ineffective hematopoesis by a progressive fibrosing process in the bone marrow. The only curative treatment option is allogeneic hematopoietic cell transplantation (HCT). In this single-center analysis, we evaluated retrospectively 54 consecutive patients suffering from primary or secondary MF which underwent HCT from 1997 to 2014 after either myeloablative (MAC, n = 19) or reduced-intensity conditioning (RIC, n = 35). Overall survival (OS) and disease-free survival (DFS) after 3 years was 54/53 % for RIC versus 63/58 % for MAC (p = 0.8/0.97). Cumulative incidence of relapse was 34 % after RIC and 8 % after MAC (p = 0.16). Three-year non-relapse mortality (NRM) was 15 % after RIC and 34 % after MAC (p = 0.29). We found that RIC was associated with a lower incidence of acute graft versus host disease (GvHD; II-IV 26 vs. 0 %, p = 0.004). Evaluation of prognostic relevance of the Dynamic International Prognostic Scoring System (DIPSS) score showed a significant better OS in patient with risk score ≤3 versus >3 (after 3 years, 71 vs. 39 %, p = 0.008). While similar OS and DFS were observed with MAC or RIC, the use of RIC resulted in lower incidence of acute GvHD. RIC regimens may be therefore the preferred conditioning approach for allogeneic HCT in patients with MF.

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Our study revealed that RANK-L is a promising marker to forecast pts' prognosis in AML. Immune checkpoint receptor PD-1/L as well as RANK and HLA-ABC did not show an impact on pts' survival.

Costimulatory ligands (COLs) and their receptors (COR) regulate immune reactions and cellular survival and might be relevant in acute myeloid leukemia (AML). This study evaluated the clinical relevance of 4-1BBL, glucocorticoid-induced TNFR-related protein (GITR) and ligand (GITRL), CD80, and CD86 in case of expression on AML blasts. 98 patients were evaluated at initial diagnosis. Immunophenotypically evaluated specific fluorescence index (SFI) levels of COR and COL on blasts were correlated with morphological, cytogenetic, and several prognostic parameters. Significantly higher COR expression was seen in monocytic versus non-monocytic AML subtypes; GITR, p=0.05; GITRL, p=0.005; CD86, p=0.001). Cut-off values for two COR and their ligands were evaluated: cases presenting with 4-1BB values above cut-off 1.2 SFI levels correlated (tendentially) significantly with a higher probability for disease-free survival (DFS, p=0.06) and a favorable HR of 0.2; p=0.04 for relapse. HR for death was also significantly lower in this group (0.12; p=0.04). In contrast, a lower probability for DFS and overall survival was seen in cases with 4-1BBL expression above 2.2 SFI levels (p=0.08 and p=0.09). In addition, multivariate analysis showed a significantly higher probability of death in this group (HR 10.3, p=0.04). Expression of CD80 and CD86 did not show significant prognostic relevance. On initial diagnosis, 4-1BB and 4-1BBL qualify as markers for prediction of patients' course and represent a valuable screening target for patients with AML at initial diagnosis.

Natural killer (NK) cells are capable of immune surveillance mediated by a balance of activating and inhibitory receptors. We have shown that adoptive transfer of NK cells can induce complete remissions in patients with refractory AML when combined with lymphodepleting chemotherapy and IL-2 to stimulate survival and in vivo expansion of the NK cells. Using this approach, 30-50% of patients with refractory attain clinical remissions. We hypothesize that clinical benefit is limited by the lack of antigen specificity and because IL-2 induces regulatory T cells (Treg) that inhibit NK cell proliferation. We have developed trispecific killer engagers (TriKE) to overcome these limitations. We have previously shown that bispecific killer engagers (BiKEs) are capable of creating immunologic synapses between NK cells and CD33 antigens on AML and MDS targets leading to NK cell signaling through the highly potent CD16 (FcγRIII) receptor. We observed that although CD16 engagement leads to enhanced killing and cytokine production by NK cells, there is no effect on proliferation (designated 1633 in Figure, Panel B). Because IL-15 is the homeostatic factor for NK cell proliferation, survival, activation, and development and because unlike IL-2, it does not stimulate CD4+ CD25+ Treg, we developed a TriKE that includes a modified human IL-15 crosslinker sandwiched between single chain Fv against CD16 and CD33 (designated 161533). The final target gene was spliced into pET21d vector expression plasmid and transformed into the Escherichia coli and compared to a 1633 BiKE with the identical Fv regions as above. TriKEs were recovered from inclusion bodies, refolded, and purified. The addition of the IL-15 crosslinker to the molecule reduced its isoelectric point by two pH units creating more favorable conditions for purification and enhancing yield. Despite identical amounts of starting inclusion bodies, the final yield of 161533 TriKE was twice the yield of 1633 BiKE indicating more favorable purification dynamics. To establish the selectivity of the anti-CD33 binding moiety, BiKEs and TriKEs were analyzed using flow cytometry and both bound to CD16 and CD33 selectively without interference from the IL-15 crosslinker. To determine the capacity of BiKE and TriKE reagents to mediate NK cell killing, activity was compared in a 4-hour chromium release assay (representative of 3 separate experiments) against CD33+ HL60 targets (Figure Panel A) or CD33- HT29 (not shown). The highest specific activity is with the 161533 TriKE containing a modified IL-15 linker as control samples of anti-CD16 and anti-CD33 alone did not augment cytotoxicity while 1633 BiKE mediating killing was intermediate. CD33- HT29 was not killed. We next tested BiKE and TriKE in a proliferation assay using CSFE labeled primary NK cells. NCI produced recombinant human IL-15 was used as a positive control. The TriKE exhibited potent proliferation of primary NK cells compared to BiKE (Figure Panel B). As target cell induced NK cell production of cytokines is an important aspect of the anti-leukemia response, we compared the BiKE and TriKE for their ability to mediate cytokine production with primary NK cells and HL60 targets. TriKE induced greater target cell specific TNFα, INFγ, IL-6, MIP1α, GM-CSF and IL-8 compared to BiKE. We then tested the activity of the TriKE in a xenogeneic model of human NK cell adoptive transfer where NSG mice are given 0.75 million HL60-Luc cells followed by 250 cGy XRT ± 1 million NK cells 3 days later from a CD3 and CD19 depleted clinical product and daily IP injection of 50 mcg/kg TriKE. Significant anti-tumor activity without toxicity was seen in the NK and TriKE treated mice (P<0.002). In addition, the 161533 TriKE had potent in vivo IL-15 activity to support the in vivo persistence and expansion of NK cells while BiKE did not (data not shown). In summary, we have shown that a novel TriKE construct can mediate CD16 directed cytotoxicity against CD33 targets and this is specifically maintained when adding a modified IL-15 linker. The IL-15 activity is fully preserved in vitro and in vivo and unexpectedly enhances the purification product. This will allow us to redirect NK cells to malignant targets while providing cytokine stimulation with an easily exportable off-the-shelf non-gene therapy strategy using a drug that also maintains NK cell persistence, survival and in vivo expansion. Drug production is in progress for clinical testing in 2016. Figure 1. Figure 1. Disclosures Miller: Coronado: Speakers Bureau; BioSciences: Speakers Bureau; Celegene: Speakers Bureau.