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: Abstract 2094 Haploidentical natural killer (NK) cells can induce and consolidate remission in patients with high-risk acute myeloid leukemia (AML) (Rubnitz et al. J Clin Onc 24: 371, 2010). Recently, significantly reduced relapse rates were observed in AML patients who received killer immunoglobulin-like receptor ligand-mismatched cord blood, suggesting effective alloreactivity of cord blood-derived NK cells (Willemze et al. Leukemia 23: 492, 2009). Cord blood transplantation (CBT) is an effective alternative source for allogeneic hematopoietic cell transplantation in both children and adults. However, its therapeutic efficacy for malignant diseases is limited by the lack of available donor effector cells, such as cytotoxic T lymphocytes, lymphokine-activated killer cells, NK-like T cells and NK cells, for treatment of hematological relapse and posttransplant lymphoproliferative disorder and/or for scheduled posttransplant cellular immunotherapy against refractory diseases. We previously reported a method that induces NK cells to proliferate and reliably allows their genetic modification in healthy individuals and leukemia patients in remission receiving maintenance chemotherapy (Imai et al. Blood 106: 376, 2005). To explore the possibility of using patients’ peripheral blood as a source for posttransplant NK cell therapy, we used our method to expand donor-derived NK cells from peripheral blood of CBT recipients early after engraftment. We also examined whether NK cells can be rendered cytotoxic against original leukemia blasts by transferring an antigen-specific artificial immunoreceptor gene. This study was approved by an institutional ethical committee. Patients received CBT for consolidation of hematological malignancy (n=7), neuroblastoma (n=1) or resolution of refractory EBV-associated hemophagocytic syndrome (n=1) with myeloablative (n=7) or reduced intensity conditioning (RIC) regimens (n=2). The patients were enrolled in the study after engraftment and peripheral blood was obtained after appropriate written consent was obtained. A chimerism study using short tandem repeat assays showed complete donor chimerism in all patients except one who received RIC-CBT. The peripheral blood was obtained at a median of 92 days post-CBT (range: 46–303 days) and subjected to ex vivo activation and expansion using a previously described protocol with slight modifications. Briefly, peripheral blood was coincubated with modified K562 cells expressing membrane-bound IL-15 and 4-1BB ligand (K562-mb15-41BBL) in the presence of low-dose IL-2 (10 U/mL). Most patients were on maintenance immunosuppressive therapy with calcineurin inhibitors with (n=3) or without (n=6) systemic corticosteroids. After 7 days of culture, a median 11.0-fold expansion (range: 5.3–28.9-fold) was observed in all but one patient who had been administered chemotherapy with Mylotarg for relapsed AML a few days before the blood sampling. The expansion rate in the first week was less efficient in CBT recipients than in healthy individuals (〉20-fold), probably because of the immunosuppressants administered. However, an additional 2-week culture in the presence of high-dose IL-2 (1000 U/mL) yielded a median 206-fold expansion (range: 101–1381-fold in 21 days). The expanded NK cells exhibited upregulation of activating receptors including NKG2D, NCRp30 and NCRp44, and vigorous cytotoxicity against K562 cells (86.8–97.7% at an E/T ratio of 1:1). The NK cells were susceptible to retroviral genetic modification with the MSCV-IRES-GFP vector (median GFP-positive cells, 52.7%, n=10). Finally, peripheral NK cells from patients with acute lymphoblastic leukemia were expanded and transduced with the chimeric immunoreceptor gene anti-CD19-BB-ζ. The donor-derived NK cells expressed large amounts of anti-CD19 chimeric receptors on their surface and killed original leukemia blasts that were highly resistant to NK cell lysis (e.g. anti-CD19 vs. non-signaling receptor: 69% vs. 0% at an E/T ratio of 1:1). These results suggest that, in CBT recipients, ex vivo expansion and genetic modification of donor-derived NK cells from the patients’ peripheral blood is feasible. Because peripheral blood can be easily and repeatedly obtained, the method described here will allow multiple scheduled infusions. This preliminary study may lead to a novel strategy for posttransplant donor-NK cell therapy in CBT recipients. Disclosures: No relevant conflicts of interest to declare.

Description: Dose adjustment of busulfan (BU) based on individual pharmacokinetics (PK) has been established as an important clinical tool in conditioning of hematopoietic stem cell transplantation (HSCT). Wide interindividual variety of achievable BU concentration in infants by recommended formula is a problematic issue. Nevertheless, there is no sizable BU PK data in infants. We report here the characteristics of BU PK data in infants with MLL gene-rearranged acute lymphoblastic leukemia (MLL-r ALL) and its relevance to the outcome of HSCT. MLL03, a nationwide study conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG), enrolled 62 infants with MLL-r ALL from 2004 to 2009 and tested whether an early intervention of allogeneic HSCT in first complete remission (1CR) could prevent early relapse and produce improved event-free survival (EFS) (Koh K, et al. Leukemia 2015). Protocol determined conditioning consisted of BU combined with etoposide and cyclophosphamide. Plasma BU concentration was measured by high performance liquid chromatography assay based on one-compartment model. The initial BU dose was determined according to an individual BU PK test measured one week prior to the start of HSCT conditioning, and further adjusted on the PK results. Among the 43 infants who received HSCT in 1CR, complete BU PK data were available in 30 cases (21 cases were transplanted from unrelated cord blood donor and 9 from family donor). Oral (PO) BU was given in 18 cases and intravenous (IV) BU in 12 cases. These 30 cases were analyzed for correlation with the clinical outcome. We set the target range of steady state concentration (Css) of BU calculated from PK within 600-900 ng/ml. As a result of BU PK test, the level of estimated Css in the group receiving PO-BU and IV-BU reached the range of 405-949 ng/ml and 484-1247 ng/ml, respectively; 61% of PO-BU group and 46% of IV-BU group were within the desired range. There was a trend of IV-BU group to attain higher targeted Css level with relatively lower dose of recommended formula, while higher dose of PO BU was necessary to achieve proper Css level. Thus, higher level of Css could be easily reached in the IV-BU group compared to the PO-BU group. After adjustment of BU dose based on the PK test results, no case showed abnormally high Css and one case showed extremely low Css than the targeted range despite twice as higher recommended PO BU dose were given. Despite wide diversity of PK data observed in each individual, permissible Css levels could be attained in most of the cases. All the cases achieved full engraftment. There was no statistical significance in the outcome including overall survival, EFS, relapse rate, and treatment-related mortality according to the level of Css nor BU formula. Sinusoidal obstruction syndrome (SOS) occurred in 6 cases (20%; two grade 3, three grade 2, and one grade 1), although Css levels of all the 6 cases did not exceed the upper limit (median 638 ng/ml, range 540-790 ng/ml). However, SOS occurred in another 5 cases out of 13 patients who did not undergo BU PK adjustment, with trend of more severe disease (38%; one grade 4, three grade 3, and one grade 2). We therefore consider that the BU dose adjustment based on PK test is useful to reduce both severity and incidence of SOS. Besides SOS, one case died of transfusion related lung injury. In conclusion, wide interindividual variety of BU PK was observed in infants with MLL-r ALL and dose adjustment of BU based on PK test would be essential for BU containing conditioning of allogeneic HSCT for infants in terms of both safety and efficacy. Disclosures Off Label Use: Off-label use of CliniMACS purified CD34+ cells.

Description: Background Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, infections, autoimmunity and lymphoma. Gene therapy (GT) using autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplant for patients who lack well matched donors, avoiding graft-versus-host-disease. An initial experience with gene therapy using a γ-retroviral vector showed correction of hematological defects in 9/10 patients, but was aggravated by development of leukemia in 7 of them. We report the outcomes of a phase I/II clinical trial in which 5 WAS patients underwent GT using a self-inactivating lentiviral (SIN-LV) vector expressing the human WAS cDNA under the control of a 1.6kB fragment of the human WAS promoter. Subjects and Methods Five patients with severe WAS (clinical score 3-5) were enrolled at a median age of 1.8 years (1.4 - 8 years) at a single pediatric tertiary care center. WAS protein (WASP) was absent or markedly decreased in 2 and 3 subjects, respectively. Purified CD34+ cells from mobilized peripheral blood (n = 4) or both mobilized peripheral blood and bone marrow (n = 1) were transduced ex-vivo with the SIN-LV vector and re-infused after conditioning with busulfan (target AUC of 70-80 mg*h/L) and fludarabine (120mg/m2). The median dose of CD34+ cells infused was 9.8 x 106 cells/kg (6.3 - 24.9 x 106 cells/kg) with a mean vector copy number (VCN) of 1.7 copies/cell in CD34+ cells (0.54 - 3.37). In addition to eczema, thrombocytopenia and WAS-related infections in all patients, two subjects also had autoimmunity pre-GT, manifested as skin vasculitis and autoimmune cytopenias. Results All 5 subjects were alive and well at median follow-up of 4.8 years (2.5 - 5.9 years). Multi-lineage vector gene marking was sustained over time. All subjects had improvement or resolution of eczema and none have had any intercurrent severe infectious events. WASP expression measured by flow cytometry in T cells was increased over baseline in all patients, but remained below normal levels and correlated with VCN and cell dose received. Proliferation of T cells in response to anti-CD3, which was initially defective in 4/5 patients, improved post-GT. Humoral immune deficiency was also ameliorated, as evidenced by independence from Ig replacement and vaccine responses in those tested. All subjects remained platelet transfusion-free and none have had severe bleeding events. Platelet levels increased to 〉50 x 103 cells/uL in two patients with a VCN ≥2 in transduced stem cells and myeloid VCN ~1 copy/cell in neutrophils; the other 3 subjects sustained platelet counts 20% of the transgene-marked cell population. To date, there have been no malignancies reported, either related to GT or WAS itself. Conclusion In summary, our data confirm and extend the safety and efficacy of GT in correcting disease manifestations associated with WAS, as seen in other studies using SIN-LV. Higher VCN in the drug product and in transduced stem cells correlated with better reconstitution of platelets and myeloid function. In contrast to other groups, we found in our study that patients with poor lymphocyte reconstitution post-GT may be at risk of ongoing autoimmunity despite high-level gene marking. Disclosures London: ArQule, Inc: Consultancy; United Therapeutics: Consultancy. Despotovic:Novartis: Research Funding; Amgen: Research Funding; Dova: Honoraria. Forbes:Takeda: Consultancy. Galy:Genethon: Employment. Williams:Novartis: Membership on an entity's Board of Directors or advisory committees; bluebird bio: Other: License of certain IP relevant to hemoglobinopathies. Potential for future royalty/milestone income. Received payment in past through BCH institutional licensing agreement., Research Funding; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder, potential for future royalty/milestone income, Research Funding; Alerion Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder. OffLabel Disclosure: CliniMACS technology for CD34+ cell selection

Description: Novel therapies are required to circumvent the resistance of leukemic cells to chemotherapy and improve cure rates. Infusions of natural killer (NK) cells might enhance the anti-leukemic effects of chemotherapy and hematopoietic stem cell transplantation. Indeed, haploidentical NK cells were shown to induce remission in patients with high-risk acute myeloid leukemia (AML; Miller at al. Blood105: 3051, 2005). Current NK-cell therapy protocols include administration of interleukin (IL)-2 to sustain the survival and activation of the infused NK cells in vivo. In this study, we tested whether enforcing the expression of the IL-2 gene in NK cells would abrogate their requirement for exogenous IL-2, prolong their survival and augment their anti-leukemic capacity., We previously reported a method that induces NK cells to proliferate and reliably allows their genetic modification (Imai et al, Blood106: 376, 2005). We used this method to expand primary NK cell from healthy individuals for 7 days. After depletion of residual T cells, NK cells were transduced with a retroviral vector containing human IL-2 cDNA and GFP. Fourteen days after transduction, more than 99% of cells were CD3− CD56+ NK cells; median transduction efficiency, assessed by GFP positivity, was 70.0% (40.4%–91.4%, n=9). NK cells expressing IL-2 cDNA (NK-gIL2) showed intracellular expression of IL-2 and secreted IL-2 (1.3 ± 0.5 U/106 cells/24 hours). Karyotyping of NK-gIL2 cells propagated for more than 6 weeks revealed no chromosomal aberration. The cytotoxicity of NK-gIL2 cells against the AML cell line K562 was similar to that of NK cells transduced with a vector containing GFP only when the cytotoxicity assay was performed with 4-hour coculture. However, NK-gIL2 exerted a much more potent cytotoxicity if the cultures were prolonged to 7 days. For example, residual leukemia was 58.4% ± 1.3% versus 97.3% ± 2.1% in control culture at a 0.5:1 E:T ratio. The superiority of NK-gIL2 was particularly evident in experiments where exogenous IL-2 was withdrawn for 72 hours before testing. In 4-hour cytotoxicity assays performed this way, cytotoxicity against K562 cells, as well as the T-ALL cell line MOLT4 and the Burkitt lymphoma cell line Daudi was significantly higher than that exerted by control NK cells (e.g., K562 killing: 82.9% ± 1.1% versus 30.9% ± 4.1% at a 1:1 ratio). NK-gIL2 showed significantly lower apoptotic rates than control NK cells after 72 hours of culture without exogenous IL-2 by Annexin V/7AAD staining. Survival after 7 days of culture in the absence of exogenous cytokines was also much higher in NK-gIL2 than NK cells transduced with GFP only. Cell death of NK cells may occur after killing of K562. We found that this was significantly lower in NK-gIL2 than in control NK cells, thus explaining the more potent anti-leukemic activity of NK-gIL2 in long-term culture. The addition of more than 100 U/ml IL-2 was needed to suppress cell death in control NK cells to a degree similar to that measured for NK-gIL2 cells in cultures without exogenous IL-2, suggesting that secretion of low amounts of IL-2 in an autocrine/paracrine fashion could sustain NK cell survival better than exogenous IL-2. We also found that the expression of the natural cytotoxicity receptor p30 and p44 was higher in NK-gIL2 cells than in control NK cells after 72-hour of IL-2 depletion, in line with their higher cytotoxic activities after IL-2 withdrawal. Our results indicate that endogenous IL-2 production can replace the requirement for exogenous IL-2 in NK cells and significantly improve their survival and cytotoxicity. An in vivo infusion of 1010 NK-gIL2 cells would result in the secretion of 1 × 104 U IL-2, an amount much lower than that administered in most immunotherapy protocols (1–2 × 106 U/sqm/day). These data suggest that infusion of NK-gIL2 could be more effective than that of unmodified NK cells while minimizing adverse effects of IL-2 administration.

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: The X-linked disorder Wiskott-Aldrich Syndrome (WAS), characterized by thrombocytopenia, eczema, recurrent infections, autoimmunity and cancer, is typically treated with allogeneic transplantation. Somatic gene therapy (GT) using autologous CD34+ cells is a promising treatment alternative for high-risk patients lacking matched allogeneic donors, as it avoids graft versus host disease. GT using a γ-retroviral vector with intact viral enhancers was efficacious but had a high rate of leukemia due to insertional oncogenesis. We report here preliminary results of 4 WAS patients who underwent GT using a self-inactivating lentiviral (SIN-LV) vector, in which viral enhancers have been deleted and the human WAS cDNA is controlled by a 1.6kB fragment of the human WAS promoter (w1.6_hWASP_WPRE SIN-LV). WASP expression per cell induced by this vector was lower than in normal cells when examined in murine models and human trials. We hypothesized that the SIN-LV would readily correct immune abnormalities due to selective advantage for WAS protein (WASP) expressing T cells whereas correction of myeloid and platelet abnormalities would require high vector copy number (VCN) in the infused cells. Patients with severe WAS (clinical scores 3-5) were enrolled at a median age of 32 months (17 months-8 years). Patients 1 and 3 had detectable but low WASP expression. Patients 2 and 4 carried mutations that abrogated WASP expression but had evidence of somatic reversion in T and/or NK cells. CliniMACS purified CD34+ mobilized peripheral blood or bone marrow cells were transduced with the vector and infused after busulfan (12-15mg/kg) and fludarabine (120mg/m2) conditioning. CD34+ cell doses ranged from 6.3-24.91 x 106 cells/kg. VCN of the infused cells was variable (3.37, 1.34, 0.54, 1.01 copies/cell). Busulfan exposure was myeloablative or near-myeloablative in patients 1, 3, and 4 (81.2, 77.2, 84.5 mg*h/L) and submyeloablative in patient 2 (48.8 mg*h/L). All patients are alive with median follow-up of 13.5 months (9-24 months). All patients had improvement in eczema, remain platelet transfusion independent and have had no severe bleeding events. WASP expression in T cells was increased post-GT over baseline. Selective advantage for WASP expressing T cells was apparent in patients 1, 2 and 4, who had higher VCN in T cells at 6 months post-GT (0.93-2.21) than in B (0.48-1.7) or myeloid (0.13-0.89) cells. The presence of revertants in patients 2 and 4 did not appear to interfere with T cell reconstitution. In contrast patient 3 who had the highest WASP expression at baseline and the lowest VCN of infused cells (0.5 copies/cell), had the lowest VCN in T cells at 8 months (0.1 copies/cell). Defective T cell proliferation in response to anti-CD3 stimulation, characteristic of WAS, was improved post-GT. Next generation sequencing of T cell receptors in sorted naïve, memory and regulatory T cells revealed profound abnormalities of diversity, decreased entropy and marked clonal expansions pre-GT; most of these improved at 6 months post-GT. Cytoskeletal function in myeloid cells was highly abnormal pre-GT, as shown by absence of podosome formation in monocyte-derived dendritic cells (0-1% vs. 61% in controls). Podosome formation at 6 months post GT was improved but subnormal (4-40%). Only patient 1, who received the highest cell dose, the highest VCN, and myeloablative busulfan exposure had robust platelet reconstitution (pre-GT 24 versus 110 x 109/L 6 months post-GT) and high level gene marking in myeloid cells 0.89 copies/cell. At the same timepoint, patients 2, 3 and 4 had platelet counts of 20-30 x 109/L and correspondingly lower VCN in myeloid cells (0.13-0.27 copies/cell). No severe adverse events related to GT have occurred to date, with relatively short follow-up. Integration site analysis of sorted cells showed highly polyclonal reconstitution, with distributions of integration acceptor sites as expected for the lentiviral vector backbone. In summary, GT using a SIN-LV that induces expression of WASP at levels below wild type improved the clinical and laboratory manifestations of WAS, with better reconstitution in patients receiving cells with high VCN. Disclosures Off Label Use: Off-label use of CliniMACS purified CD34+ cells.

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.