Natural Killer Cell Infusion Research Articles

NK cells suppress CD8+ T cell immunity via NKG2D in severe aplastic anemia

The roles of natural killer (NK) cells in shaping the immune system had raised wide interests. Here we intended to explore the regulatory functions of NK cells on CD8+ T cells in severe aplastic anemia (SAA) using human participants and lymphocyte infusion-induced bone marrow failure (BMF) mouse model. In SAA patients, NK cells had over-expressions of NKG2D and NKp46, under-expression of NKG2A and enhanced cytotoxicity. NK cells limited autologous CD8+ T cell immunity in an effector/target ratio manner. The suppression was dependent on the existence of NKG2D. We also observed upregulated MICA expression on activated CD8+ T cells, which were susceptible to NK cell mediated lysis in SAA. Animal model concurred with the data from patients. Infusion of NK cells suppressed the proliferation of CD8+ T cells and decreased IFN-γ production. In conclusion, NK cells served NKG2D-dependent immunoregulatory roles by attenuating autologous CD8+ T cell response in SAA.

Cytomegalovirus: an unlikely ally in the fight against blood cancers?

Cytomegalovirus (CMV) infection is a potentially fatal complication in patients receiving haematopoietic stem cell transplantation (HSCT), but recent evidence indicates that CMV has strong anti-leukaemia effects due in part to shifts in the composition of natural killer (NK) cell subsets. NK cells are the primary mediators of the anti-leukaemia effect of allogeneic HSCT, and infusion of allogeneic NK cells has shown promise as a means of inducing remission and preventing relapse of several different haematological malignancies. The effectiveness of these treatments is limited, however, when tumours express human leucocyte antigen (HLA)-E, a ligand for the inhibitory receptor NKG2A, which is expressed by the vast majority of post-transplant reconstituted and ex-vivo expanded NK cells. It is possible to enhance NK cell cytotoxicity against HLA-Epos malignancies by increasing the proportion of NK cells expressing NKG2C (the activating receptor for HLA-E) and lacking the corresponding inhibitory receptor NKG2A. The proportion of NKG2Cpos /NKG2Aneg NK cells is typically low in healthy adults, but it can be increased by CMV infection or ex-vivo expansion of NK cells using HLA-E-transfected feeder cells and interleukin (IL)-15. In this review, we will discuss the role of CMV-driven NKG2Cpos /NKG2Aneg NK cell expansion on anti-tumour cytotoxicity and disease progression in the context of haematological malignancies, and explore the possibility of harnessing NKG2Cpos /NKG2Aneg NK cells for cancer immunotherapy.

Haploidentical IL-15/41BBL activated and expanded natural killer cell infusion therapy after salvage chemotherapy in children with relapsed and refractory leukemia

Primary refractory or relapsed pediatric leukemia yield significant morbidity and mortality, with long-term survival rates <40%. Here we present a post-hoc analysis assessing safety and efficacy of infusing activated and expanded Natural Killer cells (NKAE) from haploidentical donors in patients from 2 clinical trials. In total, 18 children, adolescents and young adults with relapse or refractory acute leukemia were treated with two cycles of rescue chemotherapy followed by fresh NKAE cells infusions and low doses of IL-2. The overall response rate, complete remission achievement at the end of the study, was 72% (13 of 18). We infused 52 NKAE cell products containing a median of 6.76 × 106 NK cells/kg (0.7–34.16) and 0.49 × 106 T cells/kg (0–11). All infusions were well tolerated with no graft versus host disease nor other serious adverse events. Among the 14 patients who completed treatment, 4 of them are alive and leukemia-free more than 750 days post-transplant. We conclude that infusion of fresh NKAE cell therapy is feasible and safe in heavily pretreated pediatric population, and should be further investigated in advanced-phase clinical trials as well as a consolidation therapy to decrease relapse in patients with high-risk leukemia.Trials registration: Registered at www.clinicaltrials.gov as NCT01944982 and NCT02074657.

Decitabine enhances targeting of AML cells by CD34+ progenitor-derived NK cells in NOD/SCID/IL2Rgnull mice

AbstractCombining natural killer (NK) cell adoptive transfer with hypomethylating agents (HMAs) is an attractive therapeutic approach for patients with acute myeloid leukemia (AML). However, data regarding the impact of HMAs on NK cell functionality are mostly derived from in vitro studies with high nonclinical relevant drug concentrations. In the present study, we report a comparative study of azacitidine (AZA) and decitabine (DAC) in combination with allogeneic NK cells generated from CD34+ hematopoietic stem and progenitor cells (HSPC-NK cells) in in vitro and in vivo AML models. In vitro, low-dose HMAs did not impair viability of HSPC-NK cells. Furthermore, low-dose DAC preserved HSPC-NK killing, proliferation, and interferon gamma production capacity, whereas AZA diminished their proliferation and reactivity. Importantly, we showed HMAs and HSPC-NK cells could potently work together to target AML cell lines and patient AML blasts. In vivo, both agents exerted a significant delay in AML progression in NOD/SCID/IL2Rgnull mice, but the persistence of adoptively transferred HSPC-NK cells was not affected. Infused NK cells showed sustained expression of most activating receptors, upregulated NKp44 expression, and remarkable killer cell immunoglobulin-like receptor acquisition. Most importantly, only DAC potentiated HSPC-NK cell anti-leukemic activity in vivo. Besides upregulation of NKG2D- and DNAM-1–activating ligands on AML cells, DAC enhanced messenger RNA expression of inflammatory cytokines, perforin, and TRAIL by HSPC-NK cells. In addition, treatment resulted in increased numbers of HSPC-NK cells in the bone marrow compartment, suggesting that DAC could positively modulate NK cell activity, trafficking, and tumor targeting. These data provide a rationale to explore combination therapy of adoptive HSPC-NK cells and DAC in patients with AML.

Recipient T Cell Exhaustion and Successful Adoptive Transfer of Haploidentical Natural Killer Cells

Natural killer (NK) cells mediate surveillance for malignancy. In some chemotherapy refractory myeloid leukemia patients, adoptive transfer of NK cells from haploidentical donors can induce remission. We have previously shown that remission induction is linked to NK cell persistence at day +14, but the factors influencing NK cell persistence are unknown. To address this question, patient samples from a phase I trial of National Cancer Institute (NCI) IL-15 in whom either did or did not show NK cell expansion were compared with healthy donor control subjects. Before lymphodepleting chemotherapy, high absolute CD3+ count was predictive of patients who failed to expand their haploidentical NK cell graft. Interestingly, both groups had elevated expression of inhibitory receptors and decreased cytokine production compared with control subjects, suggestive of T cell exhaustion among all patients before haploidentical NK cell infusion. At day +14, however, haploidentical NK cell expanders had persistence of recipient CD8+ T cells with the most exhausted inhibitory phenotype (either PD-1high or dual PD-1+Tim-3+) and elevated expression of T-bet and Eomes compared with NK cell nonexpanders and control subjects. This suggested that maintenance of an exhausted T cell state at day +14 permits haploidentical NK cell expansion and supports further efforts to selectively deplete recipient T cells or modulate their dysfunction.

An important discovery on combination of irreversible electroporation and allogeneic natural killer cell immunotherapy for unresectable pancreatic cancer.

PurposeTo study the safety and clinical efficacy on combination of irreversible electroporation and allogeneic natural killer cell therapy for treating Stage III/IV pancreatic cancer, evaluating median progression free survival (PFS), and overall survival (OS).ResultsAdverse events of all patients were limited to grades 1 and 2, including local (mainly tussis 13.4%, nausea and emesis 7.1%, pain of puncture point 29.6% and duodenum and gastric retention 4.3%) and systemic (mainly fatigue 22.3%, fever 31.6%, and transient reduction of intraoperative blood pressure 25.1% and white cell count reduction 18.3%) reactions, fever was the most frequent. The serum amylase level at 24 h and 7 d after IRE was not significantly changed compared to those before IRE (P > 0.05). CA19–9 value was lower in IRE-NK group than in IRE at 1 month after treatment (P < 0.05). After a median follow-up of 7.4 months (3.6–11.2 months): in stage III group, median PFS was higher in IRE-NK group (9.3 months) than in IRE group (8.1 months, P = 0.0465), median OS was higher in IRE-NK (13.2 months) than in IRE (11.4 months, P = 0.0411), and median PFS was higher in who received multiple NK than single NK (9.8 months vs.8.1 months, P = 0.0423, respectively), median OS who received multiple NK was higher than single NK (13.9 months vs.12.3 months, P = 0.0524, respectively), the RR in IRE-NK (63.2%) was higher than in IRE (50.0%, P < 0.05); in stage IV group, median OS was higher in IRE-NK (9.8 months) than in IRE (8.7 months, P = 0.0397), the DCR in IRE-NK (66.7%) was higher than in IRE (42.9%, P < 0.05).Materials and MethodsBetween July 2016 and May 2017, we enrolled 71 patients who met the enrollment criteria. The patients were divided into stage III (32 patients, 17 patients received only IRE and 15 patients received IRE-NK (Irreversible electroporation- natural killer): 8 patients underwent a course of NK and 7 patients underwent ≥ 3 courses) and stage IV (39 patients, 22 patients received only IRE and 17 patients received IRE-NK: 9 patients underwent a course of NK and 8 patients underwent ≥ 3 courses). The safety and short-term effects were evaluated firstly, then the median PFS, median OS, response rate (RR) and disease control rate (DCR) were assessed.ConclusionsCombination of irreversible electroporation and allogeneic natural killer cell immunotherapy significantly increased median PFS and median OS in stage III pancreatic cancer and extended the median OS of stage IV pancreatic cancer. Multiple allogeneic natural killer cells infusion was associated with better prognosis to stage III pancreatic cancer.

Genetic engineering of human NK cells to express CXCR2 improves migration to renal cell carcinoma

BackgroundAdoptive natural killer (NK) cell transfer is being increasingly used as cancer treatment. However, clinical responses have so far been limited to patients with hematological malignancies. A potential limiting factor in patients with solid tumors is defective homing of the infused NK cells to the tumor site. Chemokines regulate the migration of leukocytes expressing corresponding chemokine receptors. Various solid tumors, including renal cell carcinoma (RCC), readily secrete ligands for the chemokine receptor CXCR2. We hypothesize that infusion of NK cells expressing high levels of the CXCR2 chemokine receptor will result in increased influx of the transferred NK cells into tumors, and improved clinical outcome in patients with cancer.MethodsBlood and tumor biopsies from 14 primary RCC patients were assessed by flow cytometry and chemokine analysis. Primary NK cells were transduced with human CXCR2 using a retroviral system. CXCR2 receptor functionality was determined by Calcium flux and NK cell migration was evaluated in transwell assays.ResultsWe detected higher concentrations of CXCR2 ligands in tumors compared with plasma of RCC patients. In addition, CXCL5 levels correlated with the intratumoral infiltration of CXCR2-positive NK cells. However, tumor-infiltrating NK cells from RCC patients expressed lower CXCR2 compared with peripheral blood NK cells. Moreover, healthy donor NK cells rapidly lost their CXCR2 expression upon in vitro culture and expansion. Genetic modification of human primary NK cells to re-express CXCR2 improved their ability to specifically migrate along a chemokine gradient of recombinant CXCR2 ligands or RCC tumor supernatants compared with controls. The enhanced trafficking resulted in increased killing of target cells. In addition, while their functionality remained unchanged compared with control NK cells, CXCR2-transduced NK cells obtained increased adhesion properties and formed more conjugates with target cells.ConclusionsTo increase the success of NK cell-based therapies of solid tumors, it is of great importance to promote their homing to the tumor site. In this study, we show that stable engineering of human primary NK cells to express a chemokine receptor thereby enhancing their migration is a promising strategy to improve anti-tumor responses following adoptive transfer of NK cells.

Effect of entinostat on NK cell-mediated cytotoxicity against osteosarcoma cells and osteosarcoma lung metastasis

ABSTRACTThere is a crucial need for a new therapeutic approach for osteosarcoma (OS) lung metastasis since this disease remains the main cause of mortality in OS. We previously demonstrated that natural killer (NK) cell therapy has minimal efficacy against OS metastasis. This study determined whether the histone deacetylase inhibitor entinostat could immunosensitize OS cells to NK cell lysis and increases the efficacy of NK cell therapy for OS lung metastasis. Entinostat upregulated ligands for NK cell-activating receptors (major histocompatibility complex [MHC] class I polypeptide-related chain A [MICA] and B [MICB]; UL16 binding proteins 1, 2, 5, and 6; and CD155) on OS cells both in vitro and in vivo and led to more susceptibility to NK cell-mediated cytotoxicity in vitro. Importantly, entinostat did not change NK cell viability, receptor expression, or function within the 24-h treatment. We also demonstrated two potential mechanisms by which entinostat enhanced expression of MICA and MICB on OS cells. Although entinostat upregulated ligands for the NK cell activating receptor on OS lung metastasis, it failed to augment the efficacy of NK cell therapy in our nude mouse human OS lung metastasis model. This can be partly explained by our finding that although the infused NK cells were active and functional and could penetrate into the lungs, they failed to infiltrate into the lung nodules. These challenges regarding cellular immunotherapy against solid tumors may be overcome by combination therapy, such as adding a NK cell-activating cytokine (IL-2 or IL-21).

Abstract CT033: Phase I study of intraventricular infusions of autologous exvivo expanded NK cells in children with recurrent/refractory malignant posterior fossa tumors of the central nervous system

Abstract Scientific background/rationale: The prognosis of recurrent/refractory medulloblastoma (MB), ependymoma (EP) and atypical teratoid/rhabdoid neoplasms (AT/RT), which constitutes the majority of malignant posterior fossa tumors (MPFT) in children, remain dismal with no defined effective therapy. Current treatment strategies have significant toxicities and thus profiling newer trials with clinical efficacy and minimal side-effects are imperative. Advantages of direct infusion of natural killer (NK) cells into the ventricles could enhance therapeutic efficacy by maximally concentrating them in the central nervous system (CNS) near the tumor area, bypassing the blood brain barrier and alleviating toxicity as seen with systemic chemotherapy. We have shown the efficacy of activated and propagated NK cells to lyse and kill the MPFT in vitro and tumor explants in mice models. Also, in a first-time human in vivo pilot study, we demonstrated the feasibility of infusions of biologic agents into the fourth ventricle. Infusing biologic agents through catheters placed in the lateral ventricles are well established in neurooncology. The current ongoing study is the first-in-human, loco-regional infusion of NK cells directly into the brain evaluating safety with intent to avoid the significant side-effects of systemic therapy. This novel trial will hopefully be able to provide therapeutic efficacy and a significant increase in the survival of these children. The current format of this trial incorporates changes including different dose, schedule, routes of intraventricular infusions of NK cells from the previously presented version of the trial, and thus submitted. Correlative studies: Cerebrospinal fluid (CSF) drawn during infusions of NK cells will be evaluated for the immunophenotype, function, persistence of NK cells and influx or presence of any other immune cells and correlating with tumor response. Methods: This trial (NCT02271711-recruiting) will proceed with infusions of NK cells directly into the lateral or fourth ventricles, after placement of a catheter at that location. Recurrent or refractory MB, AT/RT and EP involving the brain and/or spine at original diagnosis or relapse with histological verification will be eligible. Three to six evaluable patients may be entered at a dose level in this study for determination of maximum tolerated dose (MTD). A minimum of nine patients and a maximum of twenty-four patients will be enrolled. After autologous expanded NK cell product have been manufactured as per good manufacturing practice (GMP) guidelines and released, patients will receive three cycles of NK cell infusions over 12 weeks. Each cycle consists of one infusion per week for three weeks followed by a rest week (week 4). Dosing is based on recipient body surface area (BSA), and three dose levels will be evaluated. Currently, the trial is in the second cohort, with no dose limiting toxicity (DLT) noted with the previous dose level. 35% of the minimal accrual has been met. Also as a secondary aim, we will evaluate the antitumor activity of the NK cell infusions within the confines of a phase I study Citation Format: Soumen Khatua, Vidya Gopalakrishnan, Laurence Cooper, Dean Lee, David I. Sandberg, Michael Rytting, Jason J. Johnson, Leena Ketonen, Diane Liu, Judy Moyes, Wafik Zaky. Phase I study of intraventricular infusions of autologous exvivo expanded NK cells in children with recurrent/refractory malignant posterior fossa tumors of the central nervous system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT033. doi:10.1158/1538-7445.AM2017-CT033

Cryoablation combined with allogenic natural killer cell immunotherapy improves the curative effect in patients with advanced hepatocellular cancer.

In this study, the clinical efficacy of cryosurgery combined with allogenic natural killer cell immunotherapy for advanced hepatocellular cancer was evaluated. From October 2015 to March 2017, we enrolled 61 patients who met the enrollment criteria and divided them into two groups: 1) the simple cryoablation group (Cryo group, n = 26); and 2) the cryoablation combined with allogenic natural killer cells group (Cryo-NK group, n = 35), the safety and short-term effects were evaluated firstly, then the median progression-free survival, response rate and disease control rate were assessed. All adverse events experienced by the patients were recorded, and included local (e.g., pain, pleural effusion, and ascites) and systemic (e.g., chills, fatigue, and fever) reactions, fever was more frequent. Other possible seriously side effects (e.g., blood or bone marrow changes) were not detected. Combining allogeneic natural killer cells with cryoablation had a synergistic effect, not only enhancing the immune function, improving the quality of life of the patients, but also reducing the expression of AFP and significantly exhibiting good clinical efficacy of the patients. After a median follow-up of 8.7 months (3.9 –15.1months), median progression-free survival was higher in Cryo-NK (9.1 months) than in Cryo (7.6 months, P = 0.0107), median progression-free survival who received multiple natural killer was higher than who just received single natural killer (9.7 months vs.8.4 months, P = 0.0011, respectively), the response rate in Cryo-NK (60.0%) was higher than in Cryo (46.1%, P < 0.05), the disease control rate in Cryo-NK (85.7%) was higher than in Cryo group (69.2%, P < 0.01). Percutaneous cryoablation combined with allogeneic natural killer cell immunotherapy significantly increased median progression-free survival of advanced hepatocellular cancer patients. Multiple allogeneic natural killer cells infusion was associated with better prognosis to advanced hepatocellular cancer.

The therapeutic potential of natural killer cells in treating systemic lupus erythematosus.

Abstract Systemic lupus erythematosus (SLE) is a chronic disease defined by rampant autoantibody production and multi-organ pathology. Standard treatments for SLE are broadly immunosuppressive and often ineffective, as ~38% of patients develop lupus nephritis (LN) that can result in kidney failure and death. Current therapeutics may lack efficacy because they do not target the specific immune processes underlying SLE. For example, lupus autoantibodies can arise from aberrant germinal centers (GCs) driven by dysregulated follicular helper T cells (TFH). We have shown that natural killer (NK) cells suppress activated TFH during immunization, resulting in blunted GCs and antibody production. We hypothesize that an effective new strategy to treat SLE would involve harnessing of this NK cell-mediated humoral regulation. To assess whether therapeutic elevation of NK cell numbers can ameliorate disease, we used IL-2/αIL-2 complexes to expand endogenous populations of NK cells in the bm12 cGVHD mouse model of SLE-like disease. We also evaluated ex vivo activation, expansion, and infusion of NK cells as an immunotherapeutic means to ameliorate disease in mouse models of SLE. Finally, we are engineering human NK cells expressing a programmed death-ligand 1 (PD-L1)-based chimeric antigen receptor (CAR) that will facilitate cytolytic elimination of TFH and B cells that highly express programmed cell death protein 1 (PD-1). We plan to evaluate the capacity of these CAR NK cells to selectively kill PD-1+ targets and to reduce autoantibody-mediated LN in humanized mouse models of SLE. These CAR NK cells represent an innovative means to curtail autoantibody production, particularly during disease flares, and thereby promote disease remission in SLE.

Interferon Gamma Induces Changes in Natural Killer (NK) Cell Ligand Expression and Alters NK Cell-Mediated Lysis of Pediatric Cancer Cell Lines.

Natural killer (NK) cells have therapeutic potential for cancer due to their capacity for targeting tumor cells without prior sensitization. Our laboratory has developed an NK cell expansion protocol that generates large quantities of NK cells for therapeutic infusion that secret 20 times the amount of interferon gamma (IFNγ) than resting NK cells. IFNγ can upregulate major histocompatibility complex (MHC)-class I, an inhibitory ligand for NK cells, but can also upregulate intercellular adhesion molecule 1 (ICAM-1) which promotes NK:target cell interaction for an efficient lysis. Due to the opposing effects reported for IFNγ on tumor sensitivity to NK cells, we evaluated a panel 22 tumor cell lines from the pediatric preclinical testing program corresponding to different tumor types. We determined the impact of IFNγ on their expression of NK cell activating and inhibitory ligands, death receptors, and adhesion molecules using mass cytometry. We also evaluated the effect of IFNγ on their sensitivity to NK cell-mediated lysis. Our results show upregulation of PD-L1, ICAM-1, MHC-class I, HLA-DR, CD95/FasR, and CD270/HVEM after IFNγ treatment, this upregulation is variable across different tumor types. We also observed a variable impact of IFNγ in NK cell-mediated lysis. For six of the cancer cell lines IFNγ resulted in increased resistance to NK cells, while for three of them it resulted in increased sensitivity. Modeling of the data suggests that the effect of IFNγ on NK cell-mediated tumor lysis is mostly dependent on changes in MHC-class I and ICAM-1 expression. For three of the cell lines with increased resistance, we observed higher upregulation of MHC-class I than ICAM-1. For the cell lines with increased sensitivity after IFNγ treatment, we observed upregulation of ICAM-1 exceeding MHC-class I upregulation. ICAM-1 upregulation resulted in increased conjugate formation between the NK cells and tumor cells, which can contribute to the increased sensitivity observed. However, the effects of MHC-class I and ICAM-1 are not readily predictable. Due to the high IFNγ secretion of NK cell infusion products, a better understanding of the NK ligands on tumor cells and how they are affected by IFNγ is essential to optimize NK cell immunotherapy.

Prophylactic Natural Killer Cell Immunotherapy Following HLA-Haploidentical Hematopoietic Cell Transplantation Prevents Relapse and Improves Survival in Patients with High-Risk Hematological Malignancies

Background: Regimens using post-transplant cyclophosphamide (CY) have been developed to provide potent in vivo T cell depletion for patients undergoing human leukocyte antigen (HLA)-haploidentical hematopoietic cell transplantation (HCT). Luznik, O'Donnell and colleagues (BBMT 2008) reported that when this immune suppression strategy is coupled with non-myeloablative conditioning (fludarabine 150 mg/m2, CY 29 mg/kg, 2 Gy total body irradiation) followed by marrow transplantation, it was well-tolerated with low rates of non-relapse mortality (NRM; 15% at 1 year). However, the 2 year overall survival (OS) and event-free survival were low at 36% and 26%, respectively, due to high relapse rates (51% at 1 year). One explanation could be that while post-HCT CY promoted low rates of acute graft-versus-host disease (GVHD), it also eliminated early T and natural killer (NK) cell clones important for disease surveillance. Based on this hypothesis, we developed a next-generation Phase I/II clinical trial incorporating a boost of donor NK cells on day +7 as an attempt to prevent relapse after transplant. In this study, CY 50 mg/kg as a single dose on day +3 was used for T cell depletion.Methods: Forty patients (pediatric, n=14; adult, n=26) with median age of 45 (range 8-75) years having ALL (n=11), AML (n=9), MDS (n=6), HL (n=6), MM (n=4), NHL (n=3), and CLL (n=1) underwent non-myeloablative transplantation using related, HLA-haploidentical marrow donors on this prospective clinical trial. Patients were high risk due to underlying disease, potential for relapse, and/or risk for transplant-related mortality (TRM). Most patients were heavily pre-treated, with median time from cancer diagnosis to transplant being 2 (0.3 - 12.1) years, including 18 patients having 26 prior HCTs (auto, n=14; allo, n=12). Twenty-five patients (63%) had HCT-CI scores ≥ 3 indicating high risk for TRM.In order to obtain NK cells, non-mobilized peripheral blood mononuclear cells were collected from donors on day +6 using apheresis and stored overnight. NK cells were isolated on day +7 using the Miltenyi CliniMACS system (CD3 depletion followed by CD56 selection) and were administered as a single, fresh infusion that same day without prior culturing or expansion. The Phase I dose-finding study (n=11) enrolled at 2 NK doses [2.5 or 5 x 106/kg +/- 20%, respectively], with extended enrollment at the 2nd dose level for Phase II (n=29) with 83% of patients meeting NK dose parameters. NK cell products had a median log T cell depletion of 5.4 (4.1-7.1), median NK recovery of 54% (33-68%), and median NK purity of 92% (74-99%). Excellent viability (>95%) was seen in all NK products.Results: One patient developed chest pain associated with NK cell infusion; otherwise all other patients tolerated their NK cell infusions well without fevers or other adverse reactions. Full donor chimerism (>95% CD3) was seen in 83% of patients at last follow-up, while 18% and 10% experienced graft rejection or graft failure, respectively. Cumulative incidence of grades 2-3 and grade 3 (no grade 4 seen) acute GVHD occurred in 36% and 8% of patients, respectively, at day +100. Of the 39 evaluable patients, 16% developed chronic extensive GVHD at 1 year. Relapse or progression occurred in 31% of patients by 1 year after HCT. With a median follow-up of 1.5 years (range, 0.1 - 4.9 years), 14 patients have died from relapse/progression (n=11) or infection/VOD (n=3), giving a probability of OS, relapse/progression-free survival (PFS), and NRM at 1 year of 73%, 62%, and 8%, respectively, and 2 year OS and relapse/PFS of 63% and 46%, respectively (Fig 1).Summary: We have demonstrated the safety of infusing donor NK cells early after HCT in a group of heavily-treated patients with high-risk hematological malignancies. In many patients, disease-free survival was possible with the aid of this prophylactic infusion of donor NK cells in combination with allogeneic HCT. These results provide a promising platform to further augment NK cell alloreactivity in the post-HCT setting to prevent relapse and disease progression. [Display omitted] DisclosuresHari:Merck: Research Funding; BMS: Honoraria.

Infusion of Umbilical Cord Blood-Derived Natural Killer Cells in Immune-Deficient Mice for the Treatment of Resistant Acute Myeloid Leukaemia

Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against acute myeloid leukaemia (AML). Success of this NK cell immunotherapy is dependent on obtaining high numbers of functional NK cells that have the potential to survive in vivo. The use of umbilical cord blood (UCB) CD34+ cells as a source of allogenic NK cells is an interesting method that can generate a readily available, non-invasive, off the shelf cellular product.We developed a cytokine-based culture method for the generation of NK cell products derived from CD34+hematopoietic progenitor cells (HPC) isolated from fresh UCB units. Immuno-phenotyping of ex vivo expanded NK cells showed typical inhibitory and activating NK receptors except for CD16 and the KIR receptors.UCB-derived NK cells displayed good cytolytic activity against NK-sensitive K562 cells with a percentage of specific lysis varying from 30 to 50%. Cytolysis was directly correlated to CD94 expression since CD94-sorted NK cells were responsible for all the in vitro cytolytic function of differentiated NKs against K562 cells. There was an inconstant susceptibility of patient-derived primary AML cells to UCB-derived NK lysis in vitro with a specific lysis ranging from 0 to 25%.We further characterized UCB-derived NK cells by investigating their toxicity, biodistribution, and anti-leukemic potential in vivo. As adoptive transfer of NK cells is an attractive approach for treating refractory leukemia, immune deficient mice were engrafted with a patient derived AML strain resistant to NK-mediated lysis and doxorubicin.After successful engraftment and randomization, leukemic mice were injected with either UCB- derived NK cells or NK cells from healthy donors (NKhds) or doxorubicin, with one control group that didn't receive any treatment. Mice were sacrificed after 2 weeks of treatment and leukemia load along with NK distribution were evaluated by flow cytometry in the blood, bone marrow (BM) and spleen.There was no evidence of toxicity of UCB-derived or healthy donors NK cells in mice. Both types of cells were preferentially found in the blood and in the spleen, even though NKhds reached much higher levels than UCB-derived NKs.As for treatment efficacy, none of our treatment showed anti-leukemic potential based on the absence of decrease of leukemic cells in BM, blood, and spleen. In vivo microenvironment didn't overcome resistance of the patient derived AML cell to NK lysis or to doxorubicin.Remarkably, all of the UCB derived NK cells founded in vivo expressed the CD94 whereas not more than 20% of the injected cells were positive for this marker. Whether it was by in vivo selection or by in vivo differentiation must be investigated.Interestingly, a small cell population with CD56 and CD34 double staining was distinguished in UCB-derived NK and NK healthy donor treated leukemic mice suggesting in vivo interaction between leukemic and NK cells. Further characterization of this population may help to understand the molecular mechanism of leukemic recognition by NK cells and resistance of leukemic cells to cytolysis.In conclusion, UCB-derived NK generation is feasible. Investigation of the role of CD94 in these cells is needed, as cell sorting by CD94 selection in addition to the CD56 could be an interesting approach in the future to select highly functional expanded NK cells before therapeutic use. Furthermore, infusion of UCB-derived NK cells into immune-deficient mice is achievable and non-toxic. However, in vivo environment didn't overcome primary in vitro resistance of AML cells despite an established interaction. Additional elucidation of AML resistance mechanisms to NK lysis is mandatory before therapeutic application. DisclosuresNo relevant conflicts of interest to declare.

Inhibiting TGF-Beta Signaling in the Tumor Micro-Environment Enhances the Cytotoxic Function of Adoptive NK Cells

Introduction: The tumor micro-environment poses a limitation to the efficacy of adoptive NK cell therapy due to several immunosuppressive cytokines. TGF-beta, produced in excess by tumor cells, regulatory T cells and stromal cells, facilitates epithelial to mesenchymal transformation thereby promoting metastasis and fibrosis. This high TGF-beta milieu in cancer patients also impairs innate natural killer (NK) cell mediated cancer immunity through various mechanisms.Adoptive transfer of healthy donor NK cells will have limited clinical efficacy when highly activated NK cells are introduced into the immunosuppressive TGF-beta rich tumor microenvironment of cancer patients. We are testing several small molecule inhibitors of the TGF-beta receptor in combination with healthy donor NK cells with a goal of enhancing adoptive NK cell therapy in various disease models.Methods: Human NK cells isolated from healthy donor peripheral blood were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540). Cell culture media was supplemented with IL-2 (50mU/mL).For in vitro assays, at the beginning of expansion week 3 the culture conditions for NK cells was continued unchanged, supplemented with TGF-beta 1 ligand (TGF-B1) at 5ng/mL or 10ng/mL either alone or in combination with EW7197 (a TGF-beta Type 1 receptor inhibitor) or with EW7197 alone. These NK cells were tested with Calcein-AM release cytotoxicity assays at various time points from addition of TGF-B1 (0h, 36h, 72h and 96h). Briefly, NK cells were co-cultured with OCI-AML cells labeled with Calcein-AM at a ratio of 5NK:1 OCI-AML. At the end of 4 hours co-incubation, cytotoxicity was measured by relative fluorescence of calcein release into the culture supernatant compared with Triton-X induced complete target cell lysis.In a murine liver metastases model using the colon cancer cell line HCT116, 105 HCT116 cells were surgically implanted into NSG mouse spleens (following hemi-splenectomy). Mice in 2 groups (3/group) subsequently received 5 x 106 NK cells each (tail vein), weekly for two weeks, starting 10 days post-op. Another control group received vehicle infusions alone while a fourth control group received the TGF-beta inhibitor LY2157299 by oral gavage twice daily at 75mg/kg for two weeks. One group of mice receiving NK cells also received twice daily LY2157299 for two weeks starting with the first NK cell infusion. Mice receiving NK cells also received IL2 (75,000U IP) three times a week for two weeks.Results: At TGF-beta levels similar to that found in AML patients (5ng/ml), NK cell killing of OCI cells was markedly impaired progressively from 36h to 96h exposure. This reduced cytotoxic activity correlated with a 3-4 fold decrease in expression of NKG2D most marked at 96h(Figure 1). CD16 expression was also significantly reduced by 2-3 fold on TGF-beta exposed NK cells after 96h. The TGF-beta inhibitor EW7197 maintained NK cell killing as well as NKG2D and CD16 expression in the presence of TGF-B1 (Figure 1).At 32 days post-splenic implantation, vehicle mice (those that received HCT116 either alone or in combination with LY2157299 alone) appeared moribund and had grossly distended abdomens with ascites. All mice were autopsied at this time point. All control mice had grossly enlarged livers with significant metastases and absence of viable liver tissue (Fig 2). In contrast, mice treated with two weekly infusions of NK cells and LY2157299 for 4 weeks had no ascites and predominantly healthy livers. All mice that received NK cells alone had some evidence of liver metastases with various degrees of disease burden. Composite H&E stains of liver sections from 3 mice/group showed <10% liver metastases microscopically in mice treated with a combination of NK cells and LY2157299. This was significantly reduced compared to 80-90% in the untreated or LY2157299 alone mice and 60-70% in the NK cell alone group (Fig 3).Discussion: Our preliminary results indicate that TGF-beta inhibition in combination with adoptive NK cell therapy can mitigate the immunosuppressive tumor microenvironment conferred by TGF-beta signaling. We are in the process of validating this approach in other preclinical models of both hematologic malignancies and solid tumors. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Continuous IL-15 Signaling Leads to Functional Exhaustion of Human Natural Killer Cells through Metabolic Changes That Alters Their In Vivo Anti-Tumor Activity

Natural Killer (NK) cells represent an exciting immunotherapeutic approach to treat cancer. We have shown that in vivo expansion and activation of donor NK cells supported by administration of IL-2 induces remission in patients with refractory AML. Recent clinical studies by our group have shown that IL-15 is superior to IL-2 to support NK cell persistence 14 days after adoptive transfer. However, only 36% of patients treated with 12 consecutive days of IL-15 had NK cell expansion to the level of ≥100 donor derived NK cells/µL blood compared to 10% in subjects treated with IL-2 (p=0.02). This leads us to conclude that we might not know the optimal route and interval to administer in vivo IL-15. We hypothesized that daily uninterrupted IL-15 dosing could lead to exhaustion or NK cellular stress. Therefore we designed an in vitro model system in which enriched NK cells are treated with three 3-day cycles of continuous IL-15 (IL-15cont) or were rested with a "gap" (skipping the middle cycle [IL-15gap]) before returning to the last cycle of IL-15. IL-15cont treatment yielded more proliferation and higher cell numbers compared to IL-15gap (4.8±0.44 vs. 1.9±0.26 million cells/ml, p < 0.0001) when cells were analyzed at the end of the three cycles (on day 9, where all in vitro measurements were taken). However, NK cell death, measured by flow cytometry, in the IL-15cont group was higher (18.9±2.2 vs 14.9±1.7 % cell death, p = 0.035) and this group also had an enrichment in genes involved in cell cycle checkpoint/ arrest, perhaps indicating more cellular stress in the IL-15cont. In an in vitro flow cytometric functional assay, the IL-15cont group had decreased activation when compared to the IL-15 gap group against K562 targets (43.6±2.1 vs 55.6±2.7 % CD107a [degranulation], p < 0.0001; 1.9±0.41 vs 7.1±0.93 % IFNg [inflammatory cytokine production], p = 0.0055). The decrease in NK cell activation correlated with a strong decrease in tumor target killing in an in vitro chromium release assay (Figure 1A) measuring killing of acute promyelocytic leukemia (HL-60) cell targets, in which the IL-15cont NK cells were potently outperformed by the IL-15gap cells (6.4±2.6 vs 51.5±4.8 % killing at 2.5:1 effector:target ratio, p < 0.0001). We used an in vivo xenogeneic model of AML, where conditioned NSG (NOD scid gamma) mice are engrafted with HL-60luc tumor targets 3 days prior to infusion with nothing, IL-15cont or IL-15gap human NK cells prepared within our 9 day culture system. Only the IL-15gap NK group mediated statistically significant tumor control when compared to tumor alone at two weeks following NK cell infusion (Figure 1B). To probe deeper into the functional defect we evaluated signaling after these treatments and noted decreased phosphorylation of several proteins in the IL-15cont group. These data led us to explore proteins involved in metabolism and we noted that CPT1A, a critical enzyme involved in fatty acid oxidation (FAO), was strongly increased in the IL-15gap treated NK cells (protein MFI of 15,759±2,603 [IL-15gap] vs 5,273±744 [IL-15cont], p = 0.009). Metabolic analysis using a Seahorse XFe24 analyzer showed an increased mitochondrial spare respiratory capacity (SRC) in the IL-15gap group, denoting better capability of the IL-15gap NK cells to respond to energetic demands (Figure 1C). In a separate experiment the groups were treated with etomoxir to inhibit CPT1A, and the SRC phenotype was reversed, with the IL-15gap group containing lower SRC than the IL-15cont group. To test these findings in a functional assay we repeated the IL-15cont treatment in combination with rapamycin, which can induce CPT1A through inhibition of mTORC1, and saw restoration of function to levels similar to IL-15gap (40.8±2.0 vs 49.3±2.9 % CD107a in the IL-15cont vs IL-15cont + rapamycin, p = 0.005; 2.4±0.47 vs 4.8±1.0 % IFNg in the IL-15cont vs IL-15cont + rapamycin, p = 0.03). These data indicate that NK cell functional exhaustion via continuous IL-15 signaling is mediated by a decrease in FAO. Intermittent IL-15 dosing or altering metabolism through other mechanisms may overcome this competition. These findings could impact ongoing clinical trials through simple alterations in dosing strategies in order to minimize NK cell exhaustion in the immunotherapeutic setting. [Display omitted] DisclosuresCooley:Fate Therapeutics: Research Funding. Miller:Oxis Biotech: Consultancy, Other: SAB; Fate Therapeutics: Consultancy, Research Funding.

Natural Killer Cell Reconstitution after Haploidentical Hematopoietic Stem Cell Transplantation with Post-Transplant Cyclophosphamide: Elimination of Donor-Derived Mature Alloreactive NK Cells, but Favorable Conditions for Adoptive Immunotherapy

INTRODUCTIONThe use of high-dose cyclophosphamide as post-transplant Graft versus Host Disease (GvHD) prophylaxis has revolutionized haploidentical hematopoietic stem cell transplantation (haplo-HSCT), allowing the safe infusion of T cell replete grafts. The efficacy of post-transplant cyclophosphamide (PT-Cy) has its basis in its capacity to selectively eliminate proliferating cells, including alloreactive T cells. It is however to date unknown whether PT-Cy affects the reconstitution of Natural Killer (NK) cells, whose alloreactivity is known to play a major role in T cell-depleted haplo-HSCT.PATIENTS AND METHODSWe analyzed the grafts and serial peripheral blood (PB) and bone marrow (BM) samples from 14 patients who received T cell replete haplo-HSCT followed by PT-Cy at the San Raffaele Scientific Institute, Milan (n=10, OSR) or the Johns Hopkins University, Baltimore (n=4, JHU). OSR patient received a myeloablative conditioning, PB stem cell grafts, and sirolimus and mycophenolate as pharmacological GvHD prophylaxis. JHU patients received the "classical" Baltimore nonmyeloablative conditioning, unmanipulated BM grafts, and tacrolimus and mycophenolate as pharmacological GvHD prophylaxis.To characterize NK cells reconstitution, we monitored absolute counts and employed a 27-marker flow cytometry panel with high dimensional single-cell analysis using the bh-SNE algorithm.We used intracellular staining to determine the frequency of Ki67+ proliferating cells and expression of Aldehyde Dehydrogenase (ALDH), known to confer resistance to PT-Cy. Interleukin-15 (IL-15) serum concentration was quantified using the Bio-Plex Pro Human Cytokine 4-plex assay.To directly assess the effect of PT-Cy on proliferating NK cells we exposed graft NK cells to IL-15 and mafosfamide, a cyclophosphamide analogue active in vitro. Functional assays against leukemic cell lines and primary AML blasts were performed measuring CD107A degranulation on NK cells and Annexin V on targets.RESULTSAll patients received high numbers of mature donor NK cells as part of the graft (OSR median 17x106/kg, JHU median 7.25x106/kg ), and donor-derived NK cells were detectable as early as day 3 after HSCT and throughout the entire follow-up.At day 3 after HSCT, all subsets of NK cells, including single KIR+ alloreactive cells, were actively proliferating (mean 61.23% of Ki-67+ cells for OSR patients, and 58% for JHU patients), possibly driven by the high levels of IL-15 detected in patient serum after conditioning (Fig.1A).After PT-Cy, a marked reduction in the frequency and counts of proliferating NK cells was evident irrespectively of the transplantation platform, suggesting selective killing of dividing cells by PT-Cy.In line with this hypothesis, NK cells from the graft and from patient PB at day 3 after HSCT showed no detectable ALDH expression, and NK cells prompted to proliferate in vitro were killed in a dose-dependent manner by mafosfamide (Fig.1B).The phenotype of NK cells also changed upon PT-Cy: whereas before the infusion they resembled their mature counterparts from the graft, after PT-Cy an immature phenotype, CD62L+NKG2A+KIR-, became prevalent, suggesting derivation from donor HSCs rather than from infused NK cells (Fig.1C). Accordingly, bhSNE maps demonstrated differential clustering of NK cells from the graft and analyzed 30 days after HSCT (Fig.1D).In line with these features, we detected very low numbers of putatively alloreactive single KIR+ NK cells both in the PB and in the BM of patients at day 30 after HSCT, and these NK cells displayed impaired cytotoxic potential against leukemic targets.Finally, consistent with these observations, when we analyzed the impact of predicted NK alloreactivity in an extended series of 99 patients who received myeloablative haplo-HSCT with PT-Cy, we detected no significant difference in progression-free survival (Fig.1E).CONCLUSIONOur data suggest that the majority of mature NK cells infused with unmanipulated grafts are eliminated upon PT-Cy administration and that in this transplantation platform NK cell alloreactivity might be blunted by the elimination of donor single KIR+ NK cells and by the competition between reconstituting NK and T cells. Still, the high levels of IL-15 detected in patients' sera at early time-points might provide a biological rationale for the infusion of mature donor NK cells early after PT-Cy administration. [Display omitted] DisclosuresBonini:TxCell: Membership on an entity's Board of Directors or advisory committees; Molmed SpA: Consultancy. Ciceri:MolMed SpA: Consultancy.

Haploidentical Natural Killer Cell Infusion Induces Remission in Non-Hodgkin Lymphoma and Overcomes Resistance to Rituximab

Patients with chemotherapy refractory non-Hodgkin lymphoma (NHL) have poor prognosis. We report the results of a phase 2 clinical trial testing the efficacy of haploidentical donor NK cell infusion with rituximab for refractory NHL. Sixteen patients were enrolled, and 14 were evaluable.Treatment included IV lymphodepleting chemotherapy (pentostatin 3.75mg x 2 days; n=6 or fludarabine 25mg/m2 x 4 days; n=10) with cyclophosphamide (60mg/kg IV day -5; n=14). To enhance NK cell function and deplete regulatory T cells(Treg), we used the IL-2 receptor-targeting agent denileukin diftitox (18 μg/kg/day IV day -1; n=5) or methylprednisolone (1 mg/kg day -2 through day +9; n=9). Donor lymphocytes were CD3- and CD19-depleted and overnight stimulated with 1000 U/ml IL-2. The cellular product at a dose of 1.5-8 x 107 NK cells/kg was infused IV on day 0 followed by subcutaneous infusion of IL-2 at 9 million IU x 6 doses. Peripheral blood (PB) donor NK cells were evaluated by short tandem repeats and/or flow cytometry analyzing donor-specific HLA alleles.Patients characteristics included median age of 52 years (range 48-67); diffuse large B cell lymphoma was the most common diagnosis (n=11); all patients were rituximab and chemotherapy refractory.Of 14 evaluable patients at 2 months post-infusion, 4 achieved remissions (2 complete, 2 partial; 28%). Three patients in continuous remission proceeded to allogeneic donor transplantation at 2,3 and 9 months after NK cell therapy.Compared to healthy controls, NK cells in patients prior to therapy had reduced function (CD107a, INFγ and TNFα production), lower expression of natural cytotoxic receptors NKp46, NKp44 and DNAM (but higher NKp30) and reduced expression of suppressor receptors TIGIT, PD1 and TIM3.In all evaluable patients (n=9), donor NK cells persisted in the PB for at least 7 days after infusion with median 18% (range 0.5-97%) of the NK cells were of donor origin. In one responding patient, donor NK cells sustained beyond day 28. Responding patients (R) had significantly lower levels of absolute number of Treg (Foxp3+CD25+CD127-) at baselineand 14 days after therapy (Figure) as compared to non-responders (NR), however neither denileukin diftitox nor methylprednisolone resulted in PB Treg depletion. Expectedly, lower PB Tregs correlated with low serum levels of IL-10 (R2=0.7; p<0.001; n=12), suggestive of attenuated immuno-suppressive milieu. In addition, frequencies of PB myeloid derived suppressor cells (MDSC) were remarkably low in responders as compared to non-responders at baseline (4.5%±0.05% vs. 11%±6%) and after therapy (day 14: 3.8%±1%; vs. 9.4%±5%).Endogenous Inteleukin-15 (IL-15) serum levels peaked at the day of NK cell infusion (range 11-33pg/dl). Responders had higher IL-15 levels compared to non-responders at day 0 (Figure). IL-15 levels also correlated with functional NK cell killing at day 14 as measured by expression of CD107a (R2=0.8; p=0.008; n=5), suggesting that higher endogenous IL-15 improved NK cell cytotoxic capacity.We observed expected, but transient hematologic toxicity. Therapy was associated with grade 3 adverse effects (tumor lysis syndrome, Pneumocystis pneumonia; Clostridium difficile colitis, neutropenic fever; thrombotic microangiopathy). One unevaluable patient developed sepsis prior to NK cell infusion, and the second died of sepsis a week after therapy.In summary, our observation supports further investigation of donor NK cellular therapies for advanced NHL as a strategy to overcome chemoresistance. NK cell expansion and therapeutic efficacy might be further improved by administration of IL-15. [Display omitted] DisclosuresCooley:Fate Therapeutics: Research Funding. Miller:Oxis Biotech Scientific Advisory Board: Membership on an entity's Board of Directors or advisory committees.

Repopulation of Lymphocytes and Natural Killer Cells on Day 15 Following First Autologous Stem Cell Transplantation in Myeloma Patients Correlates with the Number of Reinfused Lymphocytes and Natural Killer T Cells in the Autologous Graft

IntroductionAutologous hematopoietic stem cell transplantation (aHSCT) is still considered standard of care for eligible myeloma patients following induction treatment. Beside the effect of a high dose melphalan, several studies proposed a beneficial effect of immune graft versus myeloma effect following aHSCT. The higher number of lymphocytes and natural killer (NK) cells on day 15 after aHSCT correlated with a better longterm outcome. We have previously published the data on the influence of different mobilization regimens on the number of collected lymphocytes and NK cells and their number on day 15 post aHSCT. In this abstract we present the data on the correlation between the dose of reinfused lymphocytes, NK cells and CD34+ cells and the number of lymphocytes and NK cells on day 15 after aHSCT.MethodsWe prospectively enrolled 48 newly diagnosed myeloma patients following induction treatment with bortezomib and dexamethasone. They were mobilized with either filgrastim (10 mcg/kg body weight daily), pegfilgrastim (12 mg one-time injection) or cyclophosphamide (4 g/m2) followed by filgrastim (10 mcg/kg body weight daily).The lymphocyte and NK cell (CD16+/56+) counts in the stem cell concentrate and on day 15 post aHSCT in peripheral blood were determined with the flow cytometer. Pearson r correlation was used to associate the dose of infused cells and their number on day 15 after aHSCT in peripheral blood. Scatter plots and linear regression were used for the data presentation. SPSS Statistics v21 (IBM, USA) was used for the analysis. The study complied with the Declaration of Helsinki and was approved by the local ethical committee. All patients signed a written informed consent.ResultsWe established a correlation between the dose of infused lymphocytes in the autologous graft and their number in the peripheral blood on day 15 after aHSCT (Figure 1; Pearson r = 0.27, p = 0.04). There was also a correlation between the dose of infused NK cells and their number in the peripheral blood on day 15 post aHSCT (Figure 2; Pearson r = 0.6, p < 0.01). However, there was no correlation between the number of reinfused CD34+ cells and the number of lymphocytes on day 15 after aHSCT (Figure 3).ConclusionThe early immune repopulation is important for better overall survival after the aHSCT in multiple myeloma and lymphoma patients. The data from our study showed that the higher numbers of reinfused lymphocytes and NK cells, but not the total number of CD 34+ cells, significantly impact earlier lymphocyte and NK cell engraftment. We concluded that with the aim to achieve the best possible outcomes of aHSCT for our patients, we must make sure to reinfuse not only sufficient number of CD 34+ cells, but also of lymphocytes and NK cells. [Display omitted] [Display omitted] [Display omitted] DisclosuresSkopec:amgen: Consultancy, Honoraria; novartis: Honoraria.

Donor-Derived Natural Killer Cell Infusion after Human Leukocyte Antigen–Haploidentical Hematopoietic Cell Transplantation in Patients with Refractory Acute Leukemia

The optimum method of donor natural killer cell infusion (DNKI) after allogeneic hematopoietic cell transplantation (HCT) remains unclear. Fifty-one patients (age range, 19 years to 67 years) with refractory acute leukemia underwent HLA-haploidentical HCT and underwent DNKI on days 6, 9, 13, and 20 of HCT. Median DNKI doses were .5, .5, 1.0, and 2.0 × 108/kg cells, respectively. During DNKI, 33 of the 45 evaluated patients (73%) developed fever (>38.3°C) along with weight gain (median, 13%; range, 2% to 31%) and/or hyperbilirubinemia (median, 6.2 mg/dL; range, 1.0 mg/dL to 35.1 mg/dL); the toxicity was reversible in 90% of patients. After transplantation, we observed cumulative incidences of neutrophil engraftment (≥500/µL), grade 2 to 4 acute graft-versus-host disease (GVHD), chronic GVHD, and nonrelapse mortality of 84%, 28%, 30%, and 16%, respectively. The leukemia complete remission rate was 57% at 1 month after HCT and 3-year cumulative incidence of leukemia progression was 75%. When analyzed together with our historical cohort of 40 patients with refractory acute leukemia who underwent haploidentical HCT and DNKI on days 14 and 21 only, higher expression of NKp30 (>90%) on donor NK cells was an independent predictor of higher complete remission (hazard ratio, 5.59) and less leukemia progression (hazard ratio, .57). Additional DNKI on days 6 and 9 was not associated with less leukemia progression (75% versus 55%).