ALBERT

Description: Natural killer (NK) cells are components of the innate immunity and play an important role in cancer surveillance through their cytolytic and immunomodulatory capabilities. Infusion of NK cells is a promising tool for cell therapy of hematologic malignancies and solid tumors. However, the potent cytotoxicity of NK cells might be hampered by tumor immune escape mechanisms and intrinsic resistance. We and others previously demonstrated intrinsic resistance of leukemia cells to NK cell lysis can be overcome by the transduction of artificial antigen receptor into NK cells. The genetic engineering of primary NK cells with chimeric antigen receptor improved cytotoxic activity and cytokine production, and this enhanced function was target-specific. Thus, a novel method to enhance NK cell activity against a wide range of tumors is also required. Several cytokines are associated with enhanced cytotoxicity, in vivo survival, and proliferation of NK cells. In particular, interleukin (IL)-21, which shares the common cytokine-receptor gamma chain with IL-2, was reported to enhance the cytotoxicity of human NK cells. In the present study, we investigated whether the enforced expression of human IL-21 in primary human NK cells enhanced their cytotoxicity against leukemic cells and allowed prolonged survival. We collected peripheral blood samples from healthy adult donors, and mononuclear cells were isolated by density gradient centrifugation. Primary NK cells were expanded by stimulation with K562-mbIL15-41BBL cell line following standard procedures. After 7 days of expansion, residual T cells were removed with magnetic beads and NK cells were transduced with a retroviral vector containing human IL-21 cDNA and GFP. Fourteen days after transduction, more than 95% of cells were CD56+CD3- NK cells. Median GFP expression in the CD56+CD3- cells was 84.2% (74.5%-97.1%, n=6). We confirmed that NK cells transduced with human IL-21 cDNA (NK-IL21) had intracellular expression of IL-21 as assessed by flow cytometry, while NK cells transduced with a vector containing GFP only (NK-mock) did not. 4-hour cytotoxicity assays revealed significantly enhanced cytotoxicity exerted by NK-IL21 (Fig. 1). Cytotoxic activity of NK-IL21 against K562 cells and Jurkat cells was significantly higher than that of NK-mock. We found that the intracellular expression levels of both perforin and granzyme B were higher in NK-IL21 cells than in NK-mock cells, in accordance with their higher cytotoxicity against target cells. However, NK-IL21 did not show increased expression of the apoptosis-inducing molecule TRAIL, NK cell activating receptor NKG2D, or natural cytotoxicity receptors p30, p44, or p46. The success of NK cell infusions might rely on the in vivo persistence of NK cells. We therefore tested whether the enforced expression of IL-21 in NK cells enhanced their proliferation and survival, and found that IL-21 expression in NK cells did not prevent apoptosis induced by IL-2 withdrawal and therefore did not favorably alter cell proliferation without IL-2. In contrast to the favorable results obtained by short-time cytotoxicity assays, NK-IL21 did not exert effective tumor control in long-term coculture experiments. The residual leukemic cell burden in NK-IL21 cocultures was not decreased and did not differ from that in NK-mock coculture experiments where cocultures were extended to 7 days without IL-2. However, by adding IL-2 (100 U/ml) to the culture, we demonstrated a dramatic suppression of residual leukemia burden exerted by NK-IL21. As shown in Figure 2, the number of residual K562 cells in the NK-IL21 cocultures was much lower than in the NK-mock cocultures (1.9% ± 0.4% vs 61.5% ± 3.8% of control culture without NK cells at a 1:1 E:T ratio, p

Description: Genetic modification of T cells with an artificial tumor-targeting chimeric antigen receptor (CAR) is a new approach for adoptive cell therapy for cancer. Defining cell surface molecules that are both selectively expressed on cancer cells and can be safely targeted with T cells or NK cells is a significant challenge in this research field. NKp44 is a member of the natural cytotoxicity receptor (NCR) families and also known as NCR2. Expression of NKp44 is limited to activating NK cells, which leads to a marked increase in cytotoxicity against tumors. The receptor contains one extracellular immunoglobulin domain, type I transmembrane (TM) domain, and intracellular (IC) domain, and its surface expression seems to require binding of the TM domain to adaptor molecules of DAP12 accessory protein that contains ITAMs. The ligand for NKp44 is considered damage-associated molecular pattern molecules, which have been reported to be expressed by various types of cancer cells but not by healthy cells. Therefore, a wide range of cancer cells may be safely targeted if the ligand-binding domain of this receptor is used in a construction of a chimeric antigen receptor (CAR) as an antigen recognition site, instead of using single chain variable domains derived from monoclonal antibody. We created several NKp44-based CAR constructs, which shares the extracellular NKp44 IG domain as a ligand-binding domain. Surface expression levels and subsequent functional properties can differ among T cells or NK cells transduced with novel CARs with different structural characteristics. We thus tested whether swapping the domains other than the antigen-binding domain affected expression and function. The CAR genes were retrovirally transduced into human primary T cells according to a standard method. We also transduced human primary NK cells with NKp44-based CARs, by a previously reported method (Imai C, et al. Blood 2005), to compare the expression pattern of the CAR in NK cells with that in human T cells. Retroviral transfer of wild type NKp44 gene and a construct harboring IC(p44) both did not induce NKp44 surface expression (Fig 1A,B). By sharp contrast, primary NK cells were able to express the CAR protein on the cell surface after transfer of these two genes. Removal of the IC(p44) [EH(p44)-TM(p44)-IC(CD3z)] allowed slight surface expression in T cells (Fig1C). The replacement of TM(p44) with TM(CD8a) resulted in higher surface expression in T cells (Fig 1D). These observations indicated the presence of IC(p44) as well as TM(p44) in the CAR constructs hampered surface expression in T cells most likely due to the lack of DAP12 expression. In addition to TM replacement, replacement of EH(p44) with EH(CD8a) markedly increased surface expression of the CAR (Fig 1E). Similarly, we tested use of CD28 domains instead of CD8a. Surprisingly, as different from the case of CD8a, the construct EH(p44)-TM(CD28)-IC(CD3z) yielded highest surface expression among the all CAR constructs created in this study in T cells as well as in NK cells (Fig1F), while the replacement of EH(p44) of the abovementioned CAR with EH(CD28) resulted in marked reduction of the CAR expression (Fig 1G). We confirmed surface expression of NKp44 ligand with flow cytometric analysis using recombinant human NKp44 Fc chimera protein (R&D Systems, McKinley Place, Minneapolis, USA) on various tumor cell lines including myeloid leukemia (K562, THP-1, U937, KY821, HL60), T-cell leukemia (PEER, MOLT4, HSB2), Burkitt lymphoma (Raji), BCR-ABL-positive B-ALL (OP-1), osteosarcoma (MG63, NOS1, NOS2, NOS10, U2OS, SaOS2), rhabdomyosarcoma (Rh28, RMS-YM), neuroblastoma (SK-N-SH, NB1, NB16, IMR32), and cervical carcinoma (Hela). Function of the best construct [EH(p44)-TM(CD28)-IC(CD3z)] was further evaluated. Primary T cells transduced with this NKp44-based CAR exerted powerful cytotoxicity against tumor cell lines tested and produced interferon-g and granzyme B, while GFP-transduced T cells and control T cells transduced with truncated NKp44-based CAR did not. In conclusion, we have created a novel CAR based on the antigen-binding property derived from NKp44 receptor immunoglobulin domain. This CAR should be effective to redirect T cells as well as NK cells against various types of cancer including hematological malignancies. Figure 1 Schematic representation of gene constructs and their surface expression of NKp44-based CARs in human T cells and NK cells. Figure 1. Schematic representation of gene constructs and their surface expression of NKp44-based CARs in human T cells and NK cells. Disclosures Imai: Juno Therapeutics: Patents & Royalties.

Description: Leukemic cells could survive after aggressive therapies including mega-dose chemotherapy. Natural killer (NK) cells have great promise for treatment of such refractory disease because many kinds of hematopoietic malignancies and solid tumors are reported to be intrinsically sensitive to NK cell lysis. Indeed, NK cell allo-reactivity has been shown to reduce leukemia relapse after haploidentical stem cell transplantation (SCT) and cord blood transplantation (CBT) in patients with myeloid leukemia (Ruggeri et al. Science 2002; Willemze, et al. EBMT 2008 abstract). However, NK cell allo-reactivity against acute lymphoblastic leukemia (ALL) has been reported to be generally weak regardless of the presence of killer immunoglobulin receptor (KIR) ligand mismatch. We previously showed that genetic modification of primary NK cells with anti-CD19 chimeric receptor could overcome the refractoriness of B-lineage ALL cells to NK cell lysis (Imai et al, Blood 2005). For T-lineage ALL, however, development of antigen-specific artificial receptors against T-ALL cells has been hampered by the similarity in immunophenotype between NK cells and T cells. Novel strategy to augment anti-T-ALL activity of NK cells, such as combined use of a kind of agents which has anti-leukemic activity and does not inhibit NK cell activity, is needed. Valproic acid (VPA) has been widely used as an anticonvulsant and a mood stabilizer for a long time, and its safety has been established. Recently, a number of studies have shown that VPA could act as a histone deacetylase (HDAC) inhibitor and possess anti-cancer activity. We thus studied the effect of VPA on NK cell cytotoxicity against T-ALL cell lines and asked whether VPA could be incorporated into NK cell therapy. NK cells were activated and expanded by coculture of peripheral blood mononuclear cells with K562 cells transfected with 4-1BB ligand and membrane-bound interleukin-15. After 7 days, cells were subjected to CD3-depletion procedure to earn highly pure (〉99%) NK cells, and then cultured in the presence of high dose interleukin (IL)-2 (1000 U/ml). Efficient killing of T-ALL cell lines MOLT4, Jurkat and CEM-C7 by activated NK cells were observed in 4-hour cytotoxicity assay, even if KIR ligand mismatch were not present. VPA suppressed leukemic cell proliferation and induced apoptosis in MOLT4, Jurkat and CEM-C7 at clinically achievable concentrations and in a dose-dependent fashion. Exposure of activated NK cells to VPA at 100 mg/ml for 48 hours in the presence of IL-2 resulted in mild inhibition of NK cell proliferation by approximately 20% and decreased surface expression of activating receptors such as NKG2D, 2B4, and natural cell cytotoxicity receptors p30, p44, p46 (10–40% decrease in mean fluorescence intensity). However, the addition of VPA to coculture of NK cells with MOLT-4, Jurkat and CEM-C7 did not result in interfering the anti-leukemia activity of NK cells. When anti-leukemic activity was evaluated in more prolonged culture experiments at very low effector: target (E/T) ratio, the number of residual leukemic cells was significantly lower in culture with VPA compared to those without VPA (MOLT-4: residual leukemia cells 12.2 ± 1.9% vs 46.0 ± 2.3% of control culture at E/T ratio of 0.13:1; CEM-C7: 21.0 ± 0.4% vs 40.1 ± 1.1% of control culture at E/T ratio of 0.13:1, 72-hour incubation). Similar results were also obtained when VPA was substituted by another HDAC inhibitor Suberoylanilide hydroxamic acid (SAHA). We found that upregulation of NK-activating receptor ligands MICA/B, ULBP1 and ULBP2 occurred in MOLT-4, Jurkat and CEM-C7 after 24–48 hour exposure to VPA at 100 mg/ml. On the contrary, T-ALL cells did not show increased sensitivity to NK cell lysis after pretreatment by VPA for 48 hours. Therefore, we thought that the synergistic effect of VPA observed in prolonged culture experiments could be mainly attributed to growth suppression of continuously growing leukemic cells, not to its immunomodulatory activity. These data suggest that simultaneous use of VPA would not abrogate NK cell cytotoxic activity and VPA might be used in combination with infusion of NK cells for patients bearing VPA-sensitive tumors. In patients after haploidentical SCT and CBT, NK cell allo-reactivity might be potentiated by the use of VPA.