Expanded Natural Killer Research Articles

Specific Impaired CD56+ NK Cell Development in GATA2 Deficiency As Revealed By Ex Vivo NK Expansion

Patients with GATA2 deficiency specifically lack CD56bright/CD16- natural killer (NK) cells, while the CD56dim/CD16+ (DP) subset is preserved. CD56bright/CD16- cells have been postulated to be the precursor population for more mature and cytotoxic CD16+NK cells. The mechanism by which an immature population disappears while a putatively more mature population is preserved is not understood. Our group has recently shown that CD16+/CD56dim/- NK cells have a distinct ontogeny, showing little overlap with CD56+/CD16- NK cells, which share clonal derivation with myeloid, T and B lineages (Wu et al, Cell Stem Cell 2014). We hypothesize that the missing CD56bright NK population in GATA2 deficiency is due to a maturation defect in a specific hematopoietic stem/progenitor population, separate from those relatively preserved progenitors producing CD16+ NK cells.We performed ex vivo expansion of NK cells from 4 healthy volunteers and 5 patients with GATA2 mutations and compared phenotypes at baseline and upon expansion. Sorted peripheral blood total NK (TNK) cells (CD3, CD20, CD14 depleted and expressing CD16 and/or CD56) and double negative (DN) cells (CD3-/CD20-/CD14- and CD16-/CD56-) were cultured in the presence of IL2 and irradiated Epstein-Barr virus-transformed lymphoblastoid cells. After 14 days of culture, each cell culture was characterized and re-sorted into TNK cells and DN cells, and cultured for an additional 14 days with restimulation. Subject 1 had GATA2 c.229+1 G>A mutation and presented with pancytopenia, recurrent warts, classic B/NK lymphopenia/monocytopenia and MDS with trisomy 8. Subjects 2 and 5, and subjects 3 and 4 were from the same families, respectively, and all shared the same intron 5 GATA2 c.1017+572C>T mutation. The proband, subject 2 had low grade MDS, recurrent warts and classic B/NK lymphopenia and monocytopenia. However, subjects 3, 4 and 5 had normal peripheral blood counts and bone marrow morphology, with only mild B or NK lymphopenia.TNK cells from subject 1 had profound lack of CD56bright cells at baseline, and showed decreased ex vivo expansion efficiency; 18.5-fold increase at day 14 compared to a 39.4-fold increase for control TNK cells. Even after expansion, the CD56bright population was still absent. When DN cells from subject 1 were placed into culture, they did not give rise to a CD56brightpopulation after 14 or 28 days, while control DN cells did give rise to a clear CD56bright population, also expressing additional NK-defining markers such as NKG2D and NKp46. TNK cells from patients with GATA2 intron 5 mutations had a less striking phenotype and were more variable. Subject 2 had greatly decreased CD56bright NK cells at baseline, in contrast to her asymptomatic father, subject 5, and to subjects 3 and 4 from another family. Ex vivo expansion kinetics for the TNK cells from all 4 subjects were similar to controls. By day 14 in culture, their TNK cells showed preserved generation of CD56bright/+ cells and DP cells. DN cells from subject 2 showed no CD56 expression upon expansion at day 14 and only a minor CD56dim population at day 28. Subjects 3, 4 and 5 showed comparable phenotypes at baseline and upon expansion until day 14 (Figure 1). GATA2 DN cells sorted and expanded twice from DN cells at days 1 and 14, at day 28 had much lower CD56bright NK cells (1.1±0.6% vs. 10.1±3.9%; p<0.05) and higher expression of NKG2D, an activating receptor on NK cells, compared to controls (68.23±9.91% vs. 12.3±4.63%; p=0.0036), suggesting a differentiation block in precursor NK cells.CD107a functional analysis showed impaired NK degranulation capacity upon K562 co-incubation in expanded GATA2 TNK cells and DN cells compared to their controls at day 14 (12.4±0.6 vs. 3.5±0.1 vs. 6.7±0.1 (p<0.001); 8.43±0.38 vs. 4.53±0.09 vs. 1.23±0.03 (p<0.001) fold increase from baseline in control 3, subjects 3 and 4 respectively).Our findings suggest that NK differentiation defects in GATA2 deficiency may be due to a differentiation block from a precursor present in the DN subset which is impaired in giving rise to CD56bright/CD16- cells upon prolonged culture, and also potentially in vivo. The differences between the GATA2c.229+1 G>A and GATA2 c.1017+572C>T (intron 5) samples in both phenotype and expansion characteristics correlate with the clinical severity of patients, and suggest a mutation-specific, dose-dependent role of GATA2 in NK development, as well as other modifying factors. [Display omitted] DisclosuresTownsley:GSK: Research Funding.

Abstract CT304: Anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 plus low-dose interleukin-2 (IL-2) in patients with recurrent prostate cancer (PC)

Abstract Purpose J591 is a deimmunized monoclonal antibody that recognizes the extracellular domain of prostate specific membrane antigen and was engineered to induce antibody-dependent cellular cytotoxicity (ADCC). Low dose IL-2 may enhance ADCC by the clonal expansion of natural killer (NK) cells. Immunotherapy may result in survival benefits independent of immediate response. This study was designed to evaluate the efficacy of J591 plus low-dose IL-2 in men with recurrent PC. Experimental Design Men with non-castrate PSA relapse or progressive metastatic castration-resistant PC (mCRPC) received low-dose subcutaneous IL-2 (1.2 x 106 IU/m2/day) daily for 8 weeks with weekly intravenous infusions of J591 (25 mg/m2) on weeks 4-6 (considered 1 cycle). A maximum of 3 cycles of therapy was delivered. Flow cytometry was used to assess NK cell fraction and PSA changes and survival were assessed. Results Seventeen men were treated between 2001-03 with at least 1 cycle (11 PSA relapse, 6 mCRPC) with 1 mCRPC patient invaluable for PSA response due to a protocol violation (9 received 2 cycles, 2 received 3 cycles). Toxicity was mild, mainly limited to fatigue and injection site reactions. PSA declined following therapy in 7 of 16 evaluable men (2 of 5 mCRPC). Flow cytometry revealed an average increase in absolute NK cell count of 107% at week 4 and 117% at the end of cycle 1. NK cell percent change had an inverse relationship with PSA (2-tailed Pearson correlation coefficient r = -0.46; P= 0.13). The actual vs. predicted survival for 5 of the 6 men with mCRPC disease was greater than predicted by the Halabi nomogram (Spearman correlation analysis r=0.29; P=0.57, i.e. nomogram did not predict survival). Conclusions The combination of J591 with low-dose IL-2 was well tolerated in patients with biochemical relapse and mCRPC. In addition, combination therapy expanded NK cell fraction which correlated with PSA decline. Interestingly, long term follow up reveals that 83% of the mCRPC group lived significantly longer than predicted, mostly in an era prior to the approval of life-prolonging therapy. These data support further testing of the hypothesis that PSMA-directed immunotherapy influences outcome in men with advanced PC. Citation Format: Yulian Khagi, Gurveen Kaur, Paul Christos, Naveed H. Akhtar, David M. Nanus, Neil H. Bander, Scott T. Tagawa. Anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 plus low-dose interleukin-2 (IL-2) in patients with recurrent prostate cancer (PC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT304. doi:10.1158/1538-7445.AM2014-CT304

Efficacy of ex vivo activated and expanded natural killer cells and T lymphocytes for colorectal cancer patients.

Immune cell-based therapies using natural killer (NK) cells and cytotoxic T cells are under constant scrutiny, with the aim to design an effective and reduced-toxicity therapy, which will benefit patients via improved quality of life and improved prognosis. Four patients with stage IV colon cancer were administered 1, 3, 5 and 6 effector cell intravenous infusions, respectively. Peripheral blood was collected from the patients and the ex vivo activation and expansion of NK and T cells was performed in Good Manufacturing Practice-certified clean rooms for ~12-15 days. Immunophenotypic analysis of the peripheral blood mononuclear cells (PBMCs) and expanded NK and T cells was conducted using flow cytometry and the patients were followed up. On average, 4.8×107 initial PBMCs and 2.7×109 total expanded cells were obtained. The intravenous infusions of the expanded cells were not accompanied by adverse reactions. Improved prognosis, reflected by a considerable decrease in the cancer markers, accompanied by an improved quality of life in the patients were observed. In conclusion, potential strategies are currently under development for the large-scale production of effectors cells; therefore, autologous immune enhancement therapy (AIET) may be considered as a viable approach to cancer treatment.

Anti-CD20 Chimeric Antigen Receptor (CAR) Modified Expanded Natural Killer (NK) Cells Significantly Mediate Rituximab Sensitive and Resistant Burkitt Lymphoma (BL) Regression and Improve Survival in Human BL Xenografted NSG Mice

Epithelialemesenchymal transition (EMT) plays a central role in cancer progression and metastatic dissemination, and may be induced by local inflammation. Cell lines of lung adenocarcinoma (a549), breast cancer (MCF7) and hepatocellular carcinoma (HepG2) were co-cultured with T, B and NK cells before and after EMT induced by either inflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) or transforming growth factor-beta. The onset of EMT triggered multiple immunosuppressive mechanisms in cancer cells resulting in NK and T cell apoptosis, inhibition of lymphocyte proliferation and stimulation of regulatory T and B cells. Indoleamine 2,3-dioxygenase (IDO) pathway was the main responsible for these effects, as shown by the use of specific inhibitors. Thus, EMT induced by inflammatory stimuli confers to cancer cells immunomodulatory properties resembling those shown in mesenchymal stromal cells, which could be a cue for cancer progression and metastatic dissemination.

Association Of Large Granular Lymphocytic Leukemia (LGL) With B-Cell Lymphoproliferative Disorders

IntroductionLarge granular lymphocytic (LGL) leukemia is an uncommon disease, characterized by a clonal proliferation of mature, post-thymic T-cells, typically CD3+, CD4-, CD8+, CD16+, CD57+ phenotype, representing constitutively active T-cells Less commonly, LGL leukemia is derived from CD3-, CD56+ natural killer (NK) cells. Clonal T-LGLs escape apoptosis by failure to respond to the Fas/Fas ligand (FasL) pathway. Activating mutations in the STAT3gene occur frequently in LGL leukemia, and may play a role in pathogenesis. Autoimmune disorders are frequently associated with LGL leukemia (∼1/3 present with rheumatoid arthritis).The association between LGL leukemia and B-cell lymphoproliferative disorders has been reported, often with low-grade histologies, but is deemed uncommon and the pathogenesis is not well established. We have analyzed a series of patients (pts) diagnosed with both LGL leukemia or expansion and clonal B-cell disorders. Patients and methodsPts with NK or T-LGL leukemia or expansion who were evaluated at Fox Chase Cancer Center or the Cleveland Clinic Taussig Cancer Institute were reviewed, after Institutional Review Board approval. Inclusion criteria were age ≥ 18 yrs and diagnosis of both LGL and B-cell lymphoproliferative disorder. ResultsOne hundred and twenty six pts with a diagnosis of T-LGL leukemia, NK-LGL leukemia or T-LGL expansion were identified. Of these, 44 (34.9%) pts were diagnosed with a clonal B-cell disorder. Twenty-six pts (20.6%) were diagnosed with a clonal B-cell disorder concomitantly with or shortly after the LGL diagnosis, 15 of whom presented with monoclonal gammopathy of unknown significance (MGUS) as their B-cell disorder, 9 with monoclonal B-cell lymphocytosis (MBL), 5 of whom also had monoclonal gammopathy. Eighteen pts (14.2%) had a previous diagnosis of clonal B-cell disorder, including diffuse large B cell lymphoma (DLBCL) (N= 6), CLL (N = 3), mantle cell lymphoma (N=3), multiple myeloma (N = 2), Hodgkin lymphoma (N = 2), Burkitt lymphoma (N = 1) and hairy cell leukemia (N = 1). Fifteen pts (11.9%) received treatment prior to the diagnosis of LGL, 10 of them (7.9%) with regimens including rituximab. The median time from completion of last treatment with rituximab to diagnosis of LGL disorder was 33 months. An additional patient with prior DLBCL was diagnosed with LGL shortly after receiving an oral SYK inhibitor.Two illustrative patients had unexpectedly prolonged remissions of their B cell disorder. A 66 years old man with multiple myeloma who achieved complete remission (CR) after 8 months of bortezomib therapy was then diagnosed with T-LGL, and his myeloma is in ongoing remission now 5 years after T-LGL diagnosis without further therapy. A 66 years old woman with relapsed DLBCL treated with 2ndline immunochemotherapy (R-ICE) for 3 cycles developed lymphocytosis and was diagnosed with T-LGL. With no further therapy, DLBCL is in ongoing remission now 5 years after diagnosis of T-LGL. DiscussionWe report a large series of patients with both clonal B-cell disorders and LGL. Where diagnosis of B-cell disorder and LGL are concurrent, we hypothesize an underlying immune dysregulation leading to both B-cell and T-cell proliferations. Where B-cell disorder precedes LGL, we hypothesize that the underlying disease and/or its treatment creates the environment for LGL, either directly allowing LGL expansion or permitting persistence of antigens that drive LGL expansion. Most pts with antecedent B lymphoma received rituximab (R), with a median time from R-treatment to LGL diagnosis of 33 months. Late onset neutropenia (LON) has been linked to bone marrow expansion of LGL in patients treated with rituximab, suggesting a possible pathogenetic role in our cases as well. Further, in some pts primary B-cell malignancies unexpectedly entered prolonged remission after T-LGL developed, suggesting a possible anti-B cell immune component of LGL. Further studies are warranted. Disclosures:No relevant conflicts of interest to declare.

Anti-CD20 Chimeric Antigen Receptor (CAR) Modified Expanded Natural Killer (NK) Cells Significantly Mediate Burkitt Lymphoma (BL) Regression and Improve Survival In Human BL Xenografted NSG Mice

BackgroundBurkitt lymphoma (BL) is the most common form of non-Hodgkin lymphoma that occurs in children and adolescents (Miles/Cairo, BJH, 2012). The 5-year event-free survival (EFS) of children and adolescents with newly diagnosed BL has been significantly improved but for patients with relapsed/refractory disease, the prognosis is dismal due to chemotherapy resistance (Cairo, et al, J Clin Oncol, 2012; Cairo, et al Blood 2007). Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. We have previously demonstrated that CD20 is expressing in ≥98% of pediatric BL (Perkins/Cairo, Clin Adv Hematol Oncol. 2003) and also reported that expanded Peripheral Blood Natural Killer (exPBNK) Cells electroporated with anti-CD20 Chimeric Antigen Receptor (CAR) mRNA have significant cytotoxicity against CD20+ BL in vitro (Chu/Cairo, et al, ASH, 2012). ObjectiveTo examine the anti-tumor effect of anti-CD20 chimeric antigen receptor (CAR+) modified expanded PBNK cells against disseminated CD20+ Burkitt Lymphoma in vivo using human CD20+ BL xenografted NSG mice. MethodsPBMC were expanded with lethally irradiated K562-mbIL15-41BBL cells for 14 days as we previously reported. CD56+CD3- expanded PBNK (exPBNK) cells were isolated using Miltenyi NK cell isolation kit. Anti-CD20-4-1BB-CD3ζ mRNA (CAR mRNA) was produced using the mMESSAGE mMACHINE T7 Ultra kit. CAR mRNA was nucleofected and CAR expression was detected using anti-mouse IgG, F(ab')2 fragment-specific antibody (Chu & Cairo, et al, ASH, 2012).5x105 Raji cells expressing luciferase (Raji-Luc) were intraperitoneally (i.p.) injected into the NSG mice (6 weeks, Jackson Labs). Raji engraftment and progression was evaluated using the Xenogen IVIS-200 system (Caliper Life Sciences) after i.p. injection of 150mg D-luciferin/kg/mouse. Photons emitted from luciferase-expression cells were quantified using the Living Image software. After Raji-Luc engraftment was verified in mice at day 7, 5x106 anti-CD20 CAR exPBNK or MOCK exPBNK (no anti-CD20 CAR mRNA electroporation) were i.p. injected to each mouse once a week (day 9,16, 23). Control mice received culture medium instead of NK cells. The cumulative luciferase signals were measured weekly to indicate the tumor growth, dissemination and progression.Statistical probability of survival and comparison of survival curves were determined by Kaplan Meier Method and the log-rank test. The results with a P value < 0.05 were deemed statistically significant. ResultsWe found that luciferase signals from Raji-luc were not significantly different between the untreated, mock exPBNK treated, and CAR exPBNK treated groups (p=0.0538) after the first NK injection. However, the luciferase signals measured in the CAR exPBNK treated group were significantly different than that in the control mice (two tailed P= 0.0013) and the mock exPBNK treated mice (two tailed P= 0.0229) after the second NK injection. After the third NK injection, the luciferase signals measured in the CAR exPBNK treated group were also significantly different than that in the control mice (two tailed P= 0.0173) and the mock exPBNK treated mice (two tailed P= 0.0256) (Fig. 1A). But luciferase signals were not significantly different between the untreated mice and the mock exPBNK treated mice (two tailed P= 0.0729).Consistent with the reduced luciferase signals, the CAR exPBNK treated mice had significantly extended survival time with median 40 days compared to the untreated mice (29 days, P<0.001) and the mock exPBNK treated mice (30 days, P<0.001) (Fig. 1B). There was no significant difference of survival between the untreated mice and the mock exPBNK treated mice. ConclusionMultiple injections of anti-CD20 CAR mRNA electroporated exPBNK cells can significantly mediate disseminated Raji tumor regression in Raji xenografted NSG mice compared to untreated and mock exPBNK treated mice. Furthermore, anti-CD20 CAR exPBNK significantly increased the survival of Raji xenografted mice. These results indicate therapeutic potential of multiple injections of anti-CD20 CAR mRNA modified exPBNK cells against BL in patients with chemo-immunotherapy resistance. Future directions include examining the anti-tumor activity of CAR+ exPBNK against rituximab/chemotherapy resistant BL in xenografted NSG mice. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Both Intermittent and Continuous Dosing Regimens Of Pomalidomide Mediate Broad Activation Of Innate and Adaptive Immunity In Relapsed/Refractory Myeloma: Results Of A Phase II Randomized Trial

Pomalidomide (POM) is a novel IMiD® immunomodulatory agent with clinical activity in several settings including in relapsed / resistant myeloma (RRMM). Several preclinical studies have documented the immunologic effects of IMiD® immunomodulatory drugs. IMiD® now form the backbone of several emerging combination therapies in hematologic malignancies. In prior studies, the clinical activity of POM in relapsed myeloma has been demonstrated using both continuous and intermittent dosing regimens with and without steroids. However the impact of the specific POM dosing regimen and the effect of concurrent steroids (as commonly utilized in most combination regimens) on POM-induced immune activation in vivo is unknown. In order to evaluate these issues more directly, we analyzed samples from patients enrolled in a randomized phase II clinical trial comparing two POM dosing schedules. Comparison of drug-induced immune activation between the two dosing schedules was one of the pre-specified endpoints in this study. Patients (n=39) with RRMM documented to be refractory to lenalidomide were randomized to therapy with POM 2 mg/day for 28/28 days (Arm A, n=19) or POM 4 mg/day for 21/28 days (Arm B, n=20) of a 28 day cycle. All patients (pts) received POM alone for cycle 1, followed by the addition of dexamethasone (DEX) at 40 mg weekly in subsequent cycles in both arms. In recent studies, we have shown that immune effects of lenalidomide are manifest early, within a week of initiation of therapy (Richter et al, Blood 121:423, 2013). Therefore each patient was analyzed 1 week after initiation of POM alone at 2 or 4 mg (cycle 1) or POM + DEX (cycle 2). POM therapy led to an increase in T cells at day 7 after initiation of therapy (mean percent increase compared to baseline 47%, p <0.01). This POM-induced T cell expansion consisted predominantly of CD8+ T cells (mean increase compared to baseline 40%, p<0.01). POM therapy also led to an increase in circulating natural killer (NK) cells (mean increase compared to baseline 92%, p<0.01) which peaked at 7 days post-initiation of therapy. POM-induced NK expansion was associated with an increase in the expression of NKG2D as well as CD16 and CD56, consistent with NK activation in vivo. Changes in T and NK cells following cycle 2 (POM+DEX) were compared with the data on cycle 1 (POM alone) in the same patient to evaluate the impact of DEX. Addition of DEX led to dampening of drug-induced T cell activation in vivo, but surprisingly did not dampen drug induced NK activation. Comparison of data from the two cohorts demonstrated that both 2 mg and 4 mg dose of POM led to comparable degree of immune activation in vivo, particularly for NK cells. In selected patients, post-treatment bone marrow samples were available after completion of at least 2 cycles of therapy. Comparison of the transcriptome of the CD138-depleted fraction from these marrows (representing bulk tumor microenvironment) identified nearly 300 genes upregulated in the bone marrow microenvironment post therapy compared to paired baseline samples. Interestingly, these genes were highly enriched in genes associated with T and NK activation. As POM led to activation of both innate and adaptive immunity, we also analyzed drug-induced changes in the myeloid compartment. Comparison of gene expression profiles (GEP) of purified CD14+ monocytes before and after POM indicated that POM therapy leads to a distinct GEP signature in myeloid cells in vivo. Finally, correlation of drug-induced changes in immune cells with clinical response demonstrated that an increase in post-therapy T cells correlated with clinical response, but post-treatment increase in NK cells did not. Together these data demonstrate that both 2 mg and 4 mg dose of POM can mediate broad activation of both innate and adaptive immunity in vivo, even in the setting of immune paresis associated with advanced, heavily pre-treated disease. Both continuous and intermittent dosing schedules of POM have comparable pharmacodynamic effects on immune activation. POM-induced immune activation is detected even with concurrent steroids and immune-related genes constitute a dominant component of drug induced changes in tumor microenvironment. POM-mediated immune activation may thus be exploited in the context of combination with immune therapies as well as monoclonal antibodies. Disclosures:No relevant conflicts of interest to declare.

Natural killer expansion, human leukocyte antigens‐E expression and CD14+CD56+ monocytes in a myelodysplastic syndrome patient

Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and possible evolution to acute leukemia. Occurrence of stem cell defects and of immune-mediated mechanisms was evidenced as relevant for pathophysiology of MDS. Here, we described one case of MDS patient carrying CD14(+) CD56(+) monocytes in bone marrow (BM), in the presence of a defective human leukocyte antigen (HLA)-E expression on peripheral blood (PB) cells and of natural killer (NK) cell expansion in PB and BM. The defective HLA-E expression and the NK expansion are proposed to be relevant for the pathogenesis of myelodysplasia in those patients showing CD14(+) CD56(+) monocytes in BM.

IL-18-MyD88 signaling axis is important in the attainment of a robust virus-specific effector NK cell response but dispensable for memory formation (P1248)

Abstract Although natural killer (NK) cells are considered part of the innate immune system, studies have demonstrated the ability of NK cells to contribute to potent recall responses similar to T and B cells. The precise signals that promote the generation of a long-lived NK cell response are largely undefined. Using cytokine and cytokine receptor deficient mice we investigated the role of the pro-inflammatory cytokine IL-18 on NK homeostasis, activation, expansion, and memory cell formation in response to mouse cytomegalovirus (MCMV) infection. We demonstrate that IL-18 receptor-deficient (IL-18RKO) NK cells exhibit a defect in NK cell expansion following MCMV infection; however, the generation of IL-18RKO memory NK cells was unaffected, as IL-18RKO and wildtype NK cells were detected greater than 5 weeks following MCMV challenge. NK cells deficient in MyD88, the adapter protein which mediates signaling downstream of the IL-1 and IL-18 receptors, also showed a similar defect in expansion. No defect was observed in IL-1RKO NK cells. Interestingly, IL-18RKO NK cells exhibit comparable homeostatic proliferation as wildtype. Thus, these data together highlight the importance of an IL-18-MyD88 signaling axis in NK cell priming, and in the attainment of a robust virus-specific effector NK cell response. Understanding the full contribution of the signals necessary for a protective anti-viral NK cell response will be of interest in the development of vaccines and therapeutics.

Ex Vivo Expansion of Highly Cytotoxic Human NK Cells by Cocultivation with Irradiated Tumor Cells for Adoptive Immunotherapy

Adoptive natural killer (NK) cell therapy may offer an effective treatment regimen for cancer patients whose disease is refractory to conventional therapy. NK cells can kill a wide range of tumor cells by patterned recognition of target ligands. We hypothesized that tumor targets sensitive to NK lysis would drive vigorous expansion of NK cells from human peripheral blood mononuclear cells (PBMC). Here, we provide the basis for developing a novel ex vivo expansion process. By screening class I-negative or -mismatched tumor cell lines we identified a Jurkat T-lymphoblast subline termed KL-1, which was highly effective in specifically expanding NK cells. KL-1 addition to PBMC cultures achieved approximately 100-fold expansion of NK cells with nearly 90% purity, accompanied by reciprocal inhibition of T-cell growth. Marked elevations in expression of activation receptors, natural cytotoxicity receptors (NKp30, NKp44), and adhesion molecules (CD11a, ICAM-1) were associated with high tumor-lytic capacity, in both in vitro and in vivo models. KL-1-mediated expansion of NK cells was contact dependent and required interactions with CD16, the Fcγ receptor on NK cells, with ligands that are expressed on B cells. Indeed, B-cell depletion during culture abrogated selective NK cell expansion, while addition of EBV-transformed B cells further augmented NK expansion to approximately 740-fold. Together, our studies define a novel method for efficient activation of human NK cells that employs KL-1-lysed tumor cells and cocultured B cells, which drive a robust expansion of potent antitumor effector cells that will be useful for clinical evaluation.

Autologous immune enhancement therapy against an advanced epithelioid sarcoma: A case report

Rare types of cancer are often not effectively treated by approaches such as chemotherapy and radio-therapy, although their side-effects persist. Immunotherapy has been gaining attention worldwide with growing examples of its anticancer activity demonstrated in vivo. This case report describes a 35-year-old male who suffered from advanced epithelioid sarcoma and underwent 18 cycles of chemotherapy without any significant response, who suffered adverse effects that caused lung collapse. A notable response was observed following the administration of autologous immune enhancement therapy (AIET), which involves a process of isolation, activation and expansion of natural killer (NK) and T cells, which were obtained from the patient’s own (autologous) peripheral blood. With the present data and the response of the patient to AIET, it may be proposed that AIET is beneficial for patients suffering from advanced epithelioid sarcoma without producing adverse effects.

Enhanced Targeting B Cell Lymphoma/Leukemia (B-L/L) by Anti-CD20 Chimeric Antigen Receptor (CAR) Modified Expanded Natural Killer (NK) Cells Combined with a Histone Deacetylase Inhibitor, Romidepsin

Enhanced Targeting B Cell Lymphoma/Leukemia (B-L/L) by Anti-CD20 Chimeric Antigen Receptor (CAR) Modified Expanded Natural Killer (NK) Cells Combined with a Histone Deacetylase Inhibitor, Romidepsin

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCRα chain rearrangement: Vα14Jα18 in mice and Vα24Jα18 in humans, which is associated with Vβ chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with α-Galactosylceramide (α-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCRα chain rearrangement: Vα14Jα18 in mice and Vα24Jα18 in humans, which is associated with Vβ chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with α-Galactosylceramide (α-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Expanded Natural Killer (NK) Cells for Immunotherapy: Fresh and Made to Order

AbstractAbstract 1912 IntroductionRemarkable increases in the dose and activity of NK cells can be achieved by co-culture with the HLA class I deficient cell line K562 that has been genetically modified to express membrane-bound IL15 and the co-stimulatory molecule 41BB-ligand (K562-mb15-41BBL; Fujisaki et al. Cancer Res. 2009;69:4010–4017). We are conducting a clinical trial utilizing these ex-vivo expanded NK cells (ENK) which are produced at the Center for Cell and Gene Therapy (CAGT) at Baylor and then shipped to the University of Arkansas for Medical Sciences (UAMS) for infusion to high-risk relapsed multiple myeloma (MM) patients using the NHLBI-PACT mechanism. Here we report on the characteristics of the ENK cell products sent fresh versus frozen. MethodsApheresis products were collected from MM patients or healthy donors (HD), cryopreserved, and then shipped to CAGT for GMP grade production, as described (Lapteva et al. Cytotherapy 2012; in press). Briefly, mononuclear cells from thawed and ficolled apheresis products were cultured in Stem Cell Growth Medium (CellGenix) supplemented with 10% fetal bovine serum and 10 U/mL IL2 with stimulator cells at a ratio of 1 NK cell to 10 irradiated K562-mb15-41BBL cells (developed at St. Jude Children’s Research Hospital, Memphis, TN). Cells were harvested on day 8–9; products from HD were CD3-depleted. Clinical-grade products were shipped to UAMS overnight either cryopreserved in a dry shipper (n=7) or fresh in 5% human serum albumin on cold packs at 1–11°C (n=4). Cell purity, expression of activating molecules, and viability by 7AAD exclusion was assessed by flow cytometry. Standard 4h chromium-release assays were used to assess potency against K562 cells at a 20:1 ENK: K562 ratio. Student’s t-Test was used to determine significance. ResultsFrom 0.9–1.5×107 starting NK cells, the total number of ENK cells produced was 5.4×109 (range 1.8–24×109). The fold NK-cell expansion was significantly lower for MM patients (n=5, median 22, 12–70 fold) than for HD (n=6, median 95, 31–160 fold; p<0.05). At harvest, median CD3+/CD56+ NK cell purity was 70% (52–88); CD3 depletion of HD products increased CD3+/CD56+ purity to 93% (86–95) resulting in a median CD3+/CD56- T cell content of 0.02% (0.04–1.02). Overall, median viability was 93% (67–98) and potency (defined as lysis of K562 cells at a 20:1 E:T ratio) was 74% (26–92). One product derived from a patient with 21% CD138+ MM cells in the apheresis collection had low expansion (12-fold), viability (66.7%) and potency (26%). For cryopreserved products, viability immediately after thawing was acceptable (median 94%, 75–99) but recovery of viable cells varied from 61% to 100% and thawed ENK failed to lyse K562 cells unless rested overnight. Further, recovery was extremely poor after overnight incubation (median 16%, 10–21). We therefore validated shipment of fresh ENK products. In contrast with frozen NK cells, the median recovery for fresh clinical products post-shipping was 101% (87–151). We confirmed that NK purity, viability, potency and expression of the key activating molecules NKG2D, NKp30, NKp44 and CD226 were retained up to 48h after transfer. ENK further increased by 34% after 72h in vitro incubation in the presence of IL2. Significant in vivo expansion of ENK was observed after infusion of fresh ENK cell products (n=3) but not after infusion of thawed products (n=3, see separate abstract). An additional advantage was that the fresh cells arrived ready to infuse and changes in release criteria relying on rapid and in process testing significantly reduced the time from apheresis collection to ENK infusion, an important consideration when treating high-risk MM patients who can experience rapid disease progression. ConclusionWe conclude that large numbers of clinical grade ENK cells can be generated from both MM patient and HD derived apheresis products by co-culture with IL2 and K562-mb15-41BBL although less vigorous expansion was observed with patient-derived cells. Upon thawing, cryopreserved ENK cells exhibited inferior recovery and potency, and survived poorly during further in vitro culture. In contrast, freshly formulated and shipped ENK cells have excellent recovery and retain cytolytic ability. Robust in vivo expansion was only seen after infusion of fresh ENK cells. Production assistance by CAGT allowed for the rapid implementation of a novel therapy utilizing fresh ENK cells for poor prognosis MM patients. Disclosures:No relevant conflicts of interest to declare.

Expanded Natural Killer (NK) Cells Transfected with Anti-CD20 Chimeric Antigen Receptor (CAR) mRNA Have Significant Cytotoxicity Against Poor Risk B-Cell (CD20+) Leukemia/Lymphoma (B-L/L).

AbstractAbstract 3007 Background:The outcome for children and adolescents with B-L/L has improved significantly but for patients who relapse or progress, the prognosis is dismal due to chemo-immuno-radiotherapy resistance (Cairo, et al, J Clin Oncol, 2012). Novel, non-chemotherapy-based therapies are desperately needed for this specific poor risk population. Natural Killer (NK) cells play an important role in tumor surveillance post allogeneic stem cell transplantation (Ruggeri L et al., Science 2002) but cell number and tumor specific recognition limit adoptive NK cell therapy (Shereck/Cairo, PBC 2007). Genetically engineered and expanded NK cells with an anti-CD19 CAR have been previously reported by Campana et al (Li L et al, Cancer Gene Ther. 2010). Anti-CD20 CAR transduced primary NK cells by retrovirus were reported by our group (Chu & Cairo, et al, ASH, 2011). Objective:To generate large-scale, efficiently modified NK cells with low cost, clinical applicable and a non-virus method, we investigated the functional activities of anti-CD20 chimeric antigen receptor (CAR+) modified PBNK cells following mRNA nucleofection against CD20+ B-L/L. Methods:PBMC were expanded with mitomycin C treated K562-mbIL15–41BBL cells for 7 or 14 days. CD56+CD3− expanded PBNK (exPBNK) cells were isolated using Miltenyi NK cell isolation kit. CD56, CD3 and receptor expression were evaluated by flow cytometry. Anti-CD20-4-1BB-CD3ζ was subcloned into a pcDNA3 vector. Anti-CD20-4-1BB-CD3ζ mRNA (CAR mRNA) was produced using the mMESSAGE mMACHINE T7 Ultra kit from T7 promoter. CAR mRNA was nucleofected into exPBNK using Amaxa nucleofector. CAR expression was detected using FITC-conjugated goat anti-mouse IgG, F(ab’)2 fragment-specific antibody. exPBNK cytotoxicity was assessed by europium release assay at different E:T ratios against CD20+ B-L/L. CD107a degranulation and intracellular IFNgƒnproduction in exPBNK were measured by flow cytometry after stimulation with medium, K562-mbIL15–41BBL, CD20+ Ramos, CD20+Daudi, or CD20− RS4;11 for 4–6 hrs. Results:CD56+CD3− exPBNK cells were significantly expanded by mitomycin C treated K562-mbIL15–41BBL cells at day7. exPBNK cells were selected with more than 96% purity of CD56+CD3−. 50 to 95% exPBNK cells were detected to express CAR at 16 hrs after CAR mRNA nucleofection. CAR mRNA nucleofection did not affect the expression of exPBNK activating receptors (CD16, CD69, NKG2D, CD244, NKp30, NKp44, NKp46) or inhibitory receptors (NKG2A, KIR2DS4, CD94, CD158a, CD158b, CD158e). exPBNK in vitro cytotoxicity was significantly enhanced by CAR+ exPBNK compared to CAR− exPBNK against CD20+ B-L/L at 10:1 (n>3): Ramos (97.25+ 2.61% vs 82.5+ 4.058%, p<0.05), Daudi (71.5+ 3.26% vs 36.34+ 6.31%, p<0.001), Raji (21.45+ 1.98% vs 6.94+ 5.64%, p<0.05), Raji-2R (a Rituximab resistant cell line) (96.39+ 1.03% vs 86.3+ 1.52%, p<0.01), and U-698-M (82.84+ 1.17% vs 26.2+ 0.776%, p<0.001). However, there was no significant difference against CD20− RS4;11 or Jurkat cells. Consistently, CD107a degranulation was enhanced in CAR+ exPBNK compared to CAR− exPBNK in response to CD20+ Ramos (31.47+ 1.74% vs 15.2+ 0.26%, p<0.001, n=3) and Daudi (38.9+ 2.7% vs 19.73+ 0.58%, p<0.001, n=3) stimulation, however, there was no significant difference in response to RS4;11 or medium. Intracellular IFNγ production was also enhanced in CAR+ exPBNK compared to CAR− exPBNK in response to CD20+ Ramos and Daudi specific stimulation. We also observed that the expression of exPBNK activating receptors (CD69, NKp44 and NKG2D) were enhanced similarly and inhibitory receptors (CD94 and CD158b) were unchanged in mock exPBNK and CAR+ exPBNK cells after incubation with U-698-M compared to medium, implying CAR+PBNK directed enhanced cytotoxicity is mainly mediated by engineered CAR but not by endogenous NK receptors. Conclusion:Anti-CD20 CAR expression in exPBNK cells by mRNA nucleofection was associated with a significant increase in CD107 degradulation and INF-g production after stimulated with CD20+ BL/L compared to mock exPBNK cells. Consequently, Anti-CD20 CAR expression in exPBNK cells results in significant and specific exPBNK in vitro cytotoxicity against CD20+ B-L/L. Future directions include examining CAR+ exPBNK cytotoxic activity against CD20+ primary B-L/L tumor cells isolated from patients and testing the anti-tumor activity of CAR+ exPBNK against B-L/L and animal survival in xenograft mice. Disclosures:No relevant conflicts of interest to declare.

Myeloma Can Modulate Expanded Natural Killer Cell Function Through Multiple Mechanisms

Abstract 4020We previously showed that highly activated natural killer cells can be generated in large numbers from the peripheral blood mononuclear cells (PBMC) of healthy donors and multiple myeloma (MM) patients by co-culturing with K562 stimulator cells modified to express 41BBL and membrane bound IL15 (Garg et al. Haematologica, 2012; in press). We are utilizing these expanded NK (ENK) cells in a clinical trial to treat genomically-defined high-risk relapsed MM. In this study we investigated possible mechanisms of MM escape from ENK cell control. First we determined whether it is feasible to induce ENK cell resistance by co-culturing 11 MM cell lines with ENK cells from the same donor in 6 well-plates at E:T ratios of 10:1. Fresh ENK cells were added every 3–4 weeks. The MM cell lines were tested for emergence of resistance at regular intervals using standard chromium release cytotoxicity assays. In seven of 11 cell lines tested all MM cells died upon adding ENK cells. However, from 4 MM cell lines (OPM2, JJN3, ANBL-6, INA-6), subpopulations emerged which were resistant to ENK cell-mediated lysis (Figure 1A). These cell lines retained resistance even when passaged for ≥3 months in the absence of ENK cells. Flow cytometry showed down regulation of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand-Receptors (TRAIL-R) –R1 and -R2 on all ENK resistant cell lines. Further experiments with OPM2 showed that the resistant cell line was less sensitive to recombinant TRAIL-induced apoptosis. Bortezomib treatment of both ENK-resistant and-sensitive OPM2 cell lines increased TRAIL-R1 and –R2 expression and susceptibility to recombinant TRAIL. Interestingly, gene expression profiling of CD138 positive MM patient cells failed to demonstrate a statistically significant difference in the level of TRAIL-R1 and-R2 expression among molecular subgroups, high- versus low-risk 70 gene score, diagnosis versus relapse, or baseline versus 48 hours post bortezomib. However, we also observed that TRAIL-R1 gene expression was down regulated in only 2 of the 4 resistant cell lines suggesting that there might not always be a tight correlation for TRAIL-R expression between GEP and flow cytometry. Next, we demonstrated that supernatants derived from both ENK cell-sensitive and -resistant MM cell lines could down modulate cell surface molecules either by ligand-shedding or reducing expression of receptors critical for activation such as CD54, NKp30, NKp80, NKG2D, CD26, CD69, CD70 and DNAM-1. Furthermore, the cytolytic activity of ENK cells was adversely affected, suggesting an additional NK cell suppressive effect mediated by soluble factors. The latter could be reversed by re-incubation of ENK cells in fresh medium with IL2 (Figure 1B). In summary, down regulation of the TRAIL-R deserves further exploration as a potential mechanism of resistance to ENK cell mediated lysis of MM cells and could be examined pre-and post-ENK cell therapy. MM cells may also evade ENK cell-mediated lysis via the production of suppressive factors. However, both mechanisms of resistance may be surmountable by NK cell sensitizing agents such as bortezomib or by generating the optimal cytokine milieu for infused ENK cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications.

Interest in natural killer (NK) cell-based immunotherapy has resurged since new protocols for the purification and expansion of large numbers of clinical-grade cells have become available. We have successfully adapted a previously described NK expansion method that uses K562 cells expressing interleukin (IL)-15 and 4-1 BB Ligand (BBL) (K562-mb15-41BBL) to grow NK cells in novel gas-permeable static cell culture flasks (G-Rex). Using this system we produced up to 19 × 10(9) functional NK cells from unseparated apheresis products, starting with 15 × 10(7) CD3(-) CD56 (+) NK cells, within 8-10 days of culture. The G-Rex yielded a higher fold expansion of NK cells than conventional gas-permeable bags and required no cell manipulation or feeding during the culture period. We also showed that K562-mb15-41BBL cells up-regulated surface HLA class I antigen expression upon stimulation with the supernatants from NK cultures and stimulated alloreactive CD8 (+) T cells within the NK cultures. However, these CD3 (+) T cells could be removed successfully using the CliniMACS system. We describe our optimized NK cell cryopreservation method and show that the NK cells are viable and functional even after 12 months of cryopreservation. We have successfully developed a static culture protocol for large-scale expansion of NK cells in the gas permeable G-Rex system under good manufacturing practice (GMP) conditions. This strategy is currently being used to produce NK cells for cancer immunotherapy.

Highly activated and expanded natural killer cells for multiple myeloma immunotherapy

Patients with gene expression profiling-defined high-risk myeloma in relapse have poor outcomes with current therapies. We tested whether natural killer cells expanded by co-culture with K562 cells transfected with 41BBL and membrane-bound interleukin-15 could kill myeloma cells with a high-risk gene expression profile in vitro and in a unique model which recapitulates human myeloma. OPM2 and high-risk primary myeloma tumors were grown in human fetal bone implanted into non-obese diabetic severe combined immunodeficiency mice with a deficient interleukin-2 receptor gamma chain. These mice are devoid of endogenous natural killer and T-cell activity and were used to determine whether adoptively transferred expanded natural killer cells could inhibit myeloma growth and myeloma-associated bone destruction. Natural killer cells from healthy donors and myeloma patients expanded a median of 804- and 351-fold, respectively, without significant T-cell expansion. Expanded natural killer cells killed both allogeneic and autologous primary myeloma cells avidly via a perforin-mediated mechanism in which the activating receptor NKG2D, natural cytotoxicity receptors, and DNAX-accessory molecule-1 played a central role. Adoptive transfer of expanded natural killer cells inhibited the growth of established OPM2 and high-risk primary myeloma tumors grown in the murine model. The transferred, expanded natural killer cells proliferated in vivo in an interleukin-2 dose-dependent fashion, persisted up to 4 weeks, were readily detectable in the human bone, inhibited myeloma growth and protected bone from myeloma-induced osteolysis. These studies provide the rationale for testing expanded natural killer cells in humans.

IL-15 administered by continuous infusion to rhesus macaques induces massive expansion of CD8+ T effector memory population in peripheral blood

AbstractIL-15 promotes activation and maintenance of natural killer (NK) and CD8+ T effector memory (TEM) cells, making it a potential immunotherapeutic agent for the treatment of cancer and immunodeficiency states. Here we report the immunologic effects of 3 different IL-15 dosing strategies in Rhesus macaques. IL-15 at a dose of 20 μg/kg/d administered by continuous intravenous infusion for 10 days resulted in a massive (100-fold) expansion of CD8+ TEM cells in the peripheral blood. In contrast, the administration of 20-40 μg/kg/d of IL-15 by subcutaneous injection resulted in a more modest (10-fold) expansion of CD8+ TEM cells. NK expansion was similar in both the continuous intravenous and daily subcutaneous treatment groups. The observation that IL-15 administered by continuous intravenous infusion is able to induce markedly greater expansions of CD8+ TEM cells than the same dose administered by other routes may have important implications for clinical development of this cytokine.