ALBERT

Description: Immunotherapy targeting individual antigens in acute myeloid leukemia (AML) has shown promise. However, in view of leukemia heterogeneity and the loss of tumor antigen expression by AML, it is unlikely that any single antigen will be consistently expressed by all leukemia cells. This highlights the need to identify additional antigens in AML that can be targeted. Azurophil granule proteases have been shown to be effective immunotherapeutic targets. Proteinase 3 and neutrophil elastase, the parent proteins for the HLA-A2 restricted peptide PR1, have been targeted successfully in myeloid leukemia using immunotherapy. We recently discovered the HLA-A2 restricted peptide CG1 (FLLPTGAEA), which is derived from the azurophil granule protease cathepsin G (CG). We showed that CG is highly expressed by AML blasts and leukemia stem cells in a limited number of AML samples and showed that CG1 can be targeted in vitro using CG-specific cytotoxic T lymphocytes (CTL). We sought to determine whether CG1 can be targeted in vivo and to characterize the expression of CG in a large cohort of AML patients. To assess the efficacy of targeting CG1 in vivo, we used a NOD scid gamma (NSG) mouse model engrafted with the human HLA-A2+ AML cell line U937 (U937-A2), which is known to express CG. Mice were injected with U937-A2 (0.5 x 106) cells and on the following day were treated with either CG1-CTL (0.25 x 106), negative control HIV-CTL expanded from the same donor or were left untreated. Mice treated with CG1-CTL demonstrated a significantly greater reduction in U937-A2 in the bone marrow (BM) (8% residual AML; P

Description: Abstract 4291 Natural Killer (NK) cells have important and potent innate immunoregulatory and immune surveillance functions against tumor. The paradoxical coexistence of tumors and anti-tumor immune cells (“Hellstrom Paradox”) may in part be explained by the pathophysiology of the “hostile” tumor microenvironment which suppress immune-cell function, such as hypoxia, low pH, low tissue glucose, and the presence of immunosuppressive cytokines and metabolites. However, the effect of the malignant environment on the ability of NK cells to infiltrate tumor and exhibit effector function is largely unknown. Therefore, we investigated the ability of NK cells to operate under conditions of hypoxia. Importantly, NK cells showed a 1,000-fold reduction in proliferative capacity when grown under chronic hypoxia (4 weeks of 1% O2). In addition, there was a corresponding decrease in cytotoxicity as revealed by chromium release assay. This was in contrast to autologous T cells which could numerically expand under corresponding growth conditions. Expression profiling uncovers profound upregulation of hypoxia-inducible genes such as EGLN1(9.9x), EGLN3(52x), LDHA(11.5x), SLC2A1(30.5x), PDK1(16.8x), VEGFA(286x) and BNIP3(138x) in hypoxic NK cells. Protein expression confirmed these changes, as NK cells under normoxic culture produced 520 nmoles/million cells of ATP, while those under hypoxic culture managed only 100 nmoles/million cells. This is consistent with a bioenergetic switch from oxidative phosphorylation to glycosis resulting from PDK1 upregulation. NK cells in hypoxia produced 61 pg/mL of VEGF compared to 1480 pg/mL for NK cells in normoxia (20% O2), as determined by ELISA. The inability of NK cells to propagate under conditions of hypoxia may be due to a drop in mitochondrial content we observed when cells were exposed to chronic hypoxia, a potential mitophagic effects of BNIP3 upregulation. In addition to the poor proliferative capacity of NK cells under hypoxia, we also noted the loss of CD56 expression on hypoxic NK cells which is associated with loss of cytotoxicity. Sequence analysis reveals that miR-210 can bind to the 3′UTR of CD56 mRNA, targeting it for degradation. Therefore, we investigated whether miR-210 levels are upregulated in hypoxic NK cells and found that increased presence correlated with loss of CD56 expression. This leads to the conclusion that NK-cell immunotherapy may be improved by downregulating miR-210 levels. Indeed, our findings help shape strategies for obtaining robust and sustained NK-cell effector function for adoptive immunotherapy in the hypoxic tumor microenvironment. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3766 Cell therapy by infusion of T cells can reconstitute immunity to combat pathogens and malignancies. However, the time required to manufacture T cells with the desired properties and in sufficient numbers ex vivo is often incompatible with the treatment window for patients. Furthermore, autologous T cells from patients with advanced disease may have compromised function and be tolerant to desired antigens. A potential solution would be an approach to infuse allogeneic T cells that avoids immune-mediated rejection caused by host T cells recognizing disparate major or minor histocompatibility antigens on the infused cells. To broaden the application of T cell therapy, we investigated whether HLA gene expression can be disrupted by designer zinc-finger nucleases (ZFNs). ZFNs comprise a zinc finger DNA binding domain designed to bind a specific DNA sequence fused to the cleavage domain of Fok I endonuclease. Since FokI dimerization is required to introduce a double strand break (DSB), we generated ZFN pairs that flank the intended DNA target sequences in the required spatial conformation. Cellular repair of the DSB by error-prone non-homologous end joining allows disruption of HLA gene expression. As an initial proof of concept experiment, transfection of ZFN pairs designed to target exon 3 of the HLA-A locus into the human kidney cell line HEK293 resulted in 10% genetic modification of the HLA-A loci. We generated clones of HEK293 cells that showed deletion or insertion mutations within the ZFN binding site of one or both HLA-A alleles leading to early termination of translation. These HLA-Anull HEK293 clones evaded HLA-A-restricted lysis by T cell clones, even after interferon-γ and TNF-α treatment was used to upregulate HLA expression. Since only transient expression of ZFNs is needed to disrupt a target gene, we tested the ability to disrupt HLA-A gene expression by electro-transfer of in vitro-transcribed ZFN mRNA into primary T cells. We show that a single administration of the mRNA encoding the ZFNs targeting HLA-A could render over 40% of primary T cells HLA-A negative. We enriched the HLA-Anull population by paramagnetic bead separation to obtain a pool of T cells 〉90% of which lack HLA-A expression. An attractive potential clinical application for HLAnull allogeneic T cells is to redirect their specificity independent of HLA via expression of a chimeric antigen receptor (CAR) targeting CD19. Thus, we eliminated HLA-A expression from CD19-specific CAR+ T cells and demonstrated that they (i) evade HLA-A-restricted lysis by T cell clones, and (ii) specifically lysed CD19+ tumor targets. Finally, to further improve this T cell product and eliminate potential deleterious immune mediated recognition by the endogenous T cell receptor (TCR) on allogeneic CAR+ T cells, we used ZFN pairs targeting the TCR α or the TCR β locus. Transient expression of these ZFNs resulted in permanent disruption of endogenous TCR expression and a highly enriched αβ TCRnull cell population could be generated by paramagnetic bead selection. These data support our plans to develop allogeneic T cells as “off-the-shelf” biologics that can be infused on demand as “drugs”. Disclosures: Reik: Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment.

Description: Abstract 489 Relapse remains the major cause of treatment failure after allogeneic hematopoietic transplantation for AML and MDS. Alloreactive NK cells mediate a potent antileukemic effect and may also enhance engraftment and reduce GVHD. We performed a phase I study infusing “third party” alloreactive NK cells from a haploidentical related donor as a component of the preparative regimen for allotransplantation from a separate HLA identical donor. The goal was to augment the antileukemia cytotoxicity of the preparative regimen by infusion of alloreactive NK cells and improve the overall outcome of hematopoietic transplantation. Patients with advanced AML or high risk MDS in relapse or beyond first remission were eligible. They received the busulfan-fludarabine preparative regimen, followed by infusion of NK cells predicted to be alloreactive by KIR:KIR ligand incompatibility. The NK cell enriched product was produced from a steady state apheresis product by depleting CD3+ cells using the CliniMACS device (Miltenyi Corp). A second step positively selecting CD56+ cells was performed for the first dose level, but discontinued thereafter in order to increase the cell yield. The NK cell product was then cultured in complete media containing rIL-2 for 16 hours and infused intravenously following completion of the busulfan-fludarabine chemotherapy. After 5 days, ATG was administered followed by PBSC infusion from the HLA identical sibling or unrelated donor. Patients received tacrolimus and methotrexate for GVHD prophylaxis. Patients were treated in 4 dose levels of the NK cell enriched product; 1) 106 cells/kg, 2) 5 × 106/kg, 3) 3 × 107/kg, and 4) 3 × 107/kg followed by systemic interleukin-2 0.5 million units/m2 SQ daily for 5 days. CD3+ cells in the NK cell product were required to be

Description: Abstract 2100 Background: Multiple myeloma (MM) is the second most common hematologic malignancy in adults and, to date, is incurable. Allogeneic natural killer (NK) cells are active in various hematologic malignancies and may have a role against MM. Umbilical cord blood is a potential source for allogeneic NK cells and ex vivo expanded umbilical cord blood-derived NK (UCB-NK) cells demonstrate activity comparable to that of peripheral blood-derived NK cells. Here we demonstrate the anti-myeloma activity of UCB-NK cells expanded to clinical grade by a novel technique using artificial antigen presenting feeder cells (“K562 Clone 9” cells) modified to express IL-21 (“K562-cl9-mIL21”). Methods: Cord blood mononuclear cells (CBMCs) were cultured in 10% human serum albumin media with IL-2 (500 IU/ml) and K562-cl9-mIL21 feeder cells (2:1 feeder: CBMC ratio) for 21 days. Thereafter, cells were subjected to CD3-immunomagnetic depletion. CD3-negative cells were then used as effector cells in functional assays. Flow cytometry was used to confirm NK cell purity (C56+/CD3- cells) and a standard chromium-51 assay was performed to determine NK cell cytotoxicity. Targets included K562 cells, MM cell lines RPMI 8226, ARP-1 and U266, autologous non-neoplastic UCB cells (negative control) and bone marrow-derived CD138+ plasma cells from myeloma patients. Results: Expansion of CBMCs with K562-cl9-mIL21 yielded a 〉2000 fold expansion of NK cells, compared with 47 fold expansion of CD56-selected cells cultured with IL-2 alone (p

Description: Abstract 1479 Despite a functioning immune system, malignant tumors develop strategies to avoid immune recognition and elimination by T cells. Gene therapy can be used to render T cells capable of targeting tumors in an antigen-specific manner. The ability to genetically manipulate a T cell ex vivo provides new opportunities to enhance their biological activity in vivo. To explore new treatment approaches we propose a novel paradigm in which T cells are developed as biological delivery vehicles for desired genes and nanoparticles. We have combined gene therapy with nanotechnology to generate T cells that express a chimeric antigen receptor (CAR) selectively expressed on T cells operating within the tumor microenvironment and to introduce radiolabeled gold nanoparticles for tracking T cells in vivo. T cells can home to tumor microenvironments to exert their immunoreceptor-dependent cytolytic activity. However, despite the ability of T-cell receptors (TCRs) and CAR to recognize tumor cells, the anti-tumor effect can be incomplete. The ability to transfer a wide range of materials into T cells via electroporation can be exploited to adapt T cells as biological delivery agents capable of targeting specific tissues. This method may also circumvent the limited biodistribution of “raw” nanomaterials due to rapid clearance by the liver and other reticuloendothelial system (RES) organs. Gold is an attractive biocompatible nanomaterial which can be synthesized in compliance with current good manufacturing practice (cGMP) guidelines required for clinical-grade production and can be chemically functionalized for specific imaging or therapeutic functions. We first tested whether commercially available nanoparticles can be electro-transferred into cultured and primary human T cells. 43 nm diameter latex nanoparticles and 7 nm gold nanoparticles (GNPs) were electro-transferred into T cells and visualized by TEM and confocal imaging (Figure 1). We have modified GNPs on the surface with the chelator, diethylenetriaminepentaacetic acid (DTPA), for stable coordination with 111In followed by GNP PEGylation. We have shown that these radiolabeled GNPs can be readily electro-transferred into T cells suitable for combined single-photon emission computed tomography (SPECT) and computed tomography (CT) (Figure 2). We used a clinical SPECT/CT scanner to detect 111In-GNP in T cells (∼2.1 × 104 nanoparticles/cell) at a sensitivity of ∼760 cells/mL. After developing electroporation protocols of nanoparticles and in vivo imaging of 111In-GNPs, additional sensitivity was achieved by modifying the chelating chemistry using the macrocyclic chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), to bind 64Cu to GNPs, increasing the number of gold particles/cell, and using 64Cu-labeled GNPs for imaging by positron emission tomography (PET). Before tail vein injection to a mouse, 11.4 mCi was detected from 10 million T cells (suspended in 300 μL PBS) electroporated using a BTX ECM830 device with the following settings: 1 kV/cm, 4 ms duration, single square pulse (Figure 3). The estimated concentration of nanoparticles transferred into T cells was ∼2.3 × 105 nanoparticles/cell as determined by a gamma counter (2470 Wizard, PerkinElmer) and nanoparticle titration. While 20 nm GNPs were used for 111In labeling, 7 nm GNPs were chosen for 64Cu labeling because of the improved (10-fold) electroporation efficiency. We are currently investigating whether multi-functional GNPs encapsulating chemotherapy drugs can add both in vivo T-cell imaging capability and enhanced cytotoxicity to CAR-redirected T cells. In aggregate, this may improve the potency of clinical-grade genetically modified T cells as a vehicle with the imaging capability for targeted delivery of drug-loaded GNPs to the tumor microenvironment. Disclosures: No relevant conflicts of interest to declare.

Description: Key Points Fc-engineered mAb promotes NK cell ADCC via better activation, serial killing, and kinetic boosting at higher target cell densities. Enhanced target killing also increased frequency of NK cell apoptosis, but this effect is donor-dependent.

Description: Introduction Signal transducer and activator of transcription 3 (STAT3) is an important regulator of cellular immunity, and in some animal models inhibition of STAT3 has been effective in improving natural killer (NK) cell antitumor efficacy. However, there has been little direct assessment of the STAT3 signaling role in human NK cells. In our previous work we found that (i) human NK cells stimulated with K562-based artificial antigen presenting cells (aAPC) genetically modified to express membrane bound IL-21 (mbIL21), which predominantly activates STAT3, resulted in greater proliferation, longer telomere length, and less senescence than NK cells expanded with mbIL15, which predominantly activates STAT5 (ii) NK cell proliferation, cytotoxicity and expression of the activating receptor NKG2D were enhanced by STAT3 activating cytokines and diminished by STAT3 inhibitors. Based on these results we hypothesized that activation of STAT3 plays a critical role in NK cell expansion and anti-tumor activity and tested our hypothesis by studying NK cells from Job syndrome patients. Hyper-immunoglobulin E syndrome (HIES / Job syndrome) is an immunodeficiency characterized by highly elevated serum IgE and recurrent skin and lung abscesses primarily caused by staphylococcal infection. Both, sporadic and autosomal dominant Job syndromes are caused by mutations in the gene coding for STAT3, which results in impaired cytokine signaling in immune cells. The STAT3 signaling deficiency is responsible for many lymphocyte abnormalities observed in Job syndrome patients such as impaired TH17 differentiation, defective development and maintenance of central memory T cells and reduced memory B cells. However, the effect of STAT3 deficiency on NK cell development, proliferation and function in Job syndrome patients has not been studied yet. Methods We obtained peripheral blood from healthy donors and from HIES patients with documented STAT3 mutations. NK cell proliferation was induced by stimulating peripheral blood mononuclear cells (PBMCs) with K562-based artificial antigen presenting cells genetically modified to express membrane bound cytokines. IL10 and IL21 were applied to induce STAT3 activation. Receptor expression was measured by flow-cytometry. NK cells from normal, healthy donors were used as control. Statistical comparison was performed by Student’s t test using GraphPad Prism. Results Compared to normal donors (1) Flow-cytometric analysis of PBMCs showed a significantly lower percentage of NK cells in Job syndrome patients (2) We observed impaired proliferation of Job syndrome patients’ NK cells upon stimulation of PBMCs not only with mbIL21 but also with mbIL15 in complex with its receptor α (mbIL15Rα), a physiologically relevant presentation of a physiologically relevant cytokine involved in NK cell survival and proliferation (3) Lower percentage of mature, CD56+CD16+ NK cells in Expanded NK cells from Job syndrome patients and (4) Reduced basal expression of NKG2D receptor as well as lower induction of NKG2D receptor expression upon stimulation with STAT3 activating cytokines IL10 and IL21, on Job syndrome patients’ NK cells. Conclusions Our finding of a deficient NK cell number in Job syndrome patients carrying dominant negative STAT3 mutations is the first to show an NK cell defect in this immunodeficiency. Lower percentage of NK cells in the peripheral blood and impaired ex vivo expansion of Job syndrome patient’s NK cells suggest deficient development and/or deficient proliferation and survival of NK cells in Job syndrome patients with dominant negative STAT3 mutations. Along with recurrent bacterial infections, Job syndrome patients are also more prone to viral infections and lymphoma. As NK cells perform surveillance of virally infected and tumorigenic cells through NKG2D receptor, low NK cell number with reduced NKG2D expression may explain the proclivity of Job syndrome patients to viral infections and lymphoma. Work is in progress to assess cytokine generation and anti-tumor activity of Job syndrome patients’ NK cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3293 Background: Hyper-immunoglobulin E syndrome (Job's syndrome) is an immunodeficiency characterized by highly elevated serum IgE and recurrent skin and lung abscesses from inappropriately mild immune responses to staphylococcal infection. Both sporadic and autosomal dominant Job's syndrome patients are caused by mutations in the gene coding for STAT3, which results in impaired cytokine signaling in immune cells. The STAT3 signaling deficiency is responsible for many lymphocyte abnormalities observed in Job's syndrome patients such as impaired TH17 differentiation, defective development and maintenance of central memory T cells and reduced memory B cells. However, the effect of STAT3 deficiency on NK cell development and function in Job's syndrome patients has not been studied yet. Previous work performed in our lab employing STAT3 activating cytokines and STAT3 inhibitors showed that STAT3 activation plays a role in NK cell proliferation, survival, cytolytic activity, and regulation of expression of the activating receptor NKG2D. This finding prompted us to test whether aberrant STAT3 activation affects NK cell proliferation, survival and cytolytic activity, in NK cells of Job's syndrome patients. Objective: To assess proliferation, cytolytic activity and receptor expression on NK cells from Job's syndrome patients with dominant negative STAT3 mutations. Methods: NK cell proliferation was induced by stimulating peripheral blood mononuclear cells (PBMCs) with K562-based artificial antigen presenting cells genetically modified to express membrane bound cytokines. IL10 and IL21 were applied to induce STAT3 activation. Receptor expression was measured by flow-cytometry. NK cytolytic activity was evaluated by Calcein release assay. NK cells from normal, healthy donors were used as control. Statistical comparison was performed by paired Student's t test using GraphPad Prism. Results: Flow-cytometric analysis of PBMCs showed significantly lower percentage of NK cells, identified as CD3− CD56+, in Job's syndrome patients compared to normal donors. The expansion in response to membrane bound IL21 stimulation was found to be impaired in NK cells obtained from Job's syndrome patients compared to those from normal donors. Expanded NK cells from Job's syndrome patients had lower percentage of mature, CD56+CD16+NK cells compared to those from normal donors. We also found significantly reduced cytolytic activity in NK cells from Job's syndrome patients. STAT3 activating cytokines IL10 and IL21 were also found to be impaired in their ability to induce NKG2D expression on NK cells obtained from Job's syndrome patients. Conclusions: Our finding of deficient NK cell number and cytolytic activity in Job's syndrome patients is the first to show an NK cell defect in this immunodeficiency. Lower percentage of NK cells in the peripheral blood and impaired ex vivo expansion of Job's syndrome patient's NK cells suggest deficient development and/or deficient proliferation and survival of NK cells in Job's syndrome patients with dominant negative STAT3 mutations. Along with recurrent bacterial infections, Job's syndrome patients are also more prone to viral infections and lymphoma. Given the role of NK cells in the immune surveillance of virally infected and tumorigenic cells, low NK cell function may explain the proclivity of Job's syndrome patients to viral infections and lymphoma. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1744 Natural Killer (NK) cells are able to extract fragments of cell membrane from antigen presenting cells through the immunological synapse, functionally incorporating the membrane receptors contained therein by a process called trogocytosis. Recently it was demonstrated that NK cells can acquire functional CCR7 from dendritic cells through this process and migrate in response to chemokines (CCL19 and CCL21). We investigated whether this process could be used to transiently modify NK cells ex vivo without genetic intervention. In previous work, we developed a K562-based artificial antigen presenting cell (aAPC) expressing membrane-bound IL-21 (K562-cl9-mIL21) which enables robust NK-cell expansion, and determined that NK cells did not express detectable CCR7 during this process. To investigate trogocytosis in this system, we genetically modified K562-cl9-mIL21 to express membrane-bound CCR7 (K562 cl9 mIL21CCR7) using the Sleeping Beauty transposon/transposase system. After 24 hours of co-culture the NK cells cultured with K562-cl9-mIL21CCR7 demonstrated marked surface expression of CCR7 compared to NK cells cultured on K562-cl9-mIL21 (Figure 1a). In kinetic experiments using three independent donors, CCR7 peak uptake occured at 24 hours (Figure 1b), followed by a decline that corresponded with the loss of aAPCs in the cultures due to lysis by NK cells. This demonstrates that NK cells can be transiently modified during in vitro expansion to bear receptors that are otherwise not a normal part of their transcriptional repertoire. We are currently establishing the functionality of trogocytosed receptors in vitro and in vivo, and investigating the kinetics of CCR7 persistence after removal of the aAPCs. However, even transient expression as demonstrated might be sufficient for bestowing novel NK-cell migration ability in vivo in response to chemokine signaling, giving the engineered NK cells ability to reach desired tissue targets. Disclosures: Lee: Altor BioScience Corp: Research Funding; Celgene: Research Funding.