Genetic modification and effector functions of natural killer cells in acute myeloid leukemia

Acute myeloid leukemia (AML) is characterized by a poor long-term outcome in the majority of patients following conventional treatment with chemotherapy. Even after allogeneic or autologous hematopoietic stem cell transplantation (HSCT) the patients are prone to relapse, indicating that the leukemic blasts escape elimination by the immune system. Natural Killer (NK) cells have emerged as a major component of the innate immunosurveillance of AML and were identified to participate in the graft versus leukemia effect following allogeneic HSCT. The effector functions of NK cells are regulated by the balanced engagement of activating receptors and inhibitory receptors. Triggering of activating receptors by the corresponding ligands on target cells counteracts the signalling pathways of inhibitory receptors and thereby elicits target cell lysis. In AML the leukemic blasts were shown to express low amounts of ligands for the NK cell activating receptor NKG2D and the natural cytotoxicity receptors (NCRs), while the expression of HLA class I molecules, the ligands for inhibitory receptors, is mostly retained at normal levels. This predominance of inhibitory signalling together with a putative deficiency in the expression of NK cell activating receptors may result in an insufficient stimulation of cytolytic NK cell responses against leukemic blasts. To investigate the mechanisms of impaired recognition and lysis of leukemic cells, we evaluated the phenotypic and functional properties of NK cells from AML patients (AML-NK cells). We examined the cytolytic activity against the autologous leukemic blasts in vitro and in vivo in the NOD/SCID transplantation mouse model, in order to exploit their potential in cellular immunotherapy of leukemia. Further we explored the feasibility to overexpress the NCR NKp46 in NK cells by lentivirus-mediated gene transfer. This approach was intended to test the hypothesis of shifting the receptor balance in AML-NK cells towards a status that favours NK cell activation and thereby increases the anti-tumor activity. The results demonstrated a significant, about ten-fold, reduction in the content of NK cells from patients with newly diagnosed or relapsed AML as compared to healthy individuals (donor-NK cells). Nevertheless, AML-NK cells retained a high proliferative capacity and could be efficiently expanded in vivo in response to NK cell specific growth factors. Also, the expression pattern of NK cell receptors and activation markers by AML-NK cells did not differ from donor-NK cells. AMLNK cells were fully functional in terms of IFN-γ production in response to the activation with IL-12 and IL-18 and displayed a high cytolytic activity against the NK cell sensitive erythroleukemia cell line K562 in vitro. Also in vivo, the adoptive transfer of AML-NK cells to NOD/SCID mice engrafted with K562 cells lead to an efficient suppression of tumor formation. The cytolytic activity of AML-NK cells against autologous leukemic blasts in vitro was in general below 10% at the E:T ratio of 10:1. The antibody-mediated block of inhibitory interactions could enhance the killing responses to about 70%, indicating that AML-NK cells are able to recognize autologous blasts through activating receptors. Cytolytic activity of AML-NK cells was also seen in NOD/SCID mice engrafted with human leukemia. Adoptive NK cell transfer resulted in reduction of tumor load from 31% to an average of about 10% of human blasts in the BM of treated mice. This high in vivo activity of AML-NK cells might be due to an increased expression of the ligands for NKG2D and the NCRs. Taken together, our results showed that AML-NK cells do not differ from healthy donor derived NK cells; they can be isolated and efficiently expanded to high cell numbers in vitro and display the same expression pattern of the major activating receptors. AML-NK cells have a normal cytokine producing ability, preserve their cytolytic activity throughout the process of in vitro expansion and display a strong anti-leukemic effect against autologous blasts in vivo in NOD/SCID mice repopulated with human leukemia. These results suggest that escape of AML blasts from the immunosurveillance by NK cells may be due to the reduction of the NK cell compartment and the predominance of signals elicited by the inhibitory receptors. We used HIV-derived lentiviral vectors for the gene transfer of the GFP marker and the NKp46 receptor to NK cell lines, primary NK cells and NK cells generated in vitro from CD34+ hematopoietic progenitor cells. Both single-gene and bicistronic vectors expressing these transgenes were prepared. Through the FACS sorter based enrichment of transduced cells 100% transgenic lines and primary NK cell populations were generated with a transgene expression that remained stable during in vitro expansion. We demonstrated that GFP+ NK cells can be generated by the in vitro differentiation of lentiviral transduced CD34+ progenitors, representing a highly efficient approach to produce large amounts of modified NK cells. However, a sustained expression of the exogenous NKp46 receptor was only achieved in NK cell lines. Except for a high pseudotransduction that resulted in the transient expression of NKp46, no stable expression of transgenic NKp46 was obtained in primary NK cells, neither in cells generated from the progenitors nor in peripheral blood-derived mature NK cells. Moreover, exogenous NKp46 in NK cell lines and transiently transduced primary cells had no ability to trigger the cytokine release or cytotoxic responses, and further studies are required to achieve the overexpression of functional NKp46. Our results demonstrated that lentiviral vectors are suitable to obtain genetically modified NK cell lines and primary NK cells. Transgenic NK cells can be expanded to high numbers without loosing the transgene expression, thus indicating the possibility to use genetically modified and expanded patient-derived NK cells for the adoptive transfer in the immunotherapy of AML. So far, the lentivirus-based approach was successful with the GFP marker transgene, but requires further optimisation for transfer of the NKp46-encoding gene. The over-expression of tumor-specific activatory receptors would be of importance in an immunotherapeutic approach to direct NK cell effector functions specifically towards the diseased cells, thereby contributing to a graft-versus-leukemia activity against residual malignant cells

Human acute myeloid leukemia CD34+CD38− stem cells are susceptible to allorecognition and lysis by single KIR-expressing natural killer cells

In this study, the authors have investigated the anti-leukemic action of alloreactive single KIR positive natural killer cells on CD34+CD38− AML cells, showing that the HDAC inhibitor valproic acid augments this activity

Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients

Alloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use

Clinical-grade purification of natural killer cells in haploidentical hematopoietic stem cell transplantation

BACKGROUND: Because of a high risk of graft-versus-host disease (GVHD), donor lymphocyte infusions with unmodified lymphapheresis products are not used after haploidentical hematopoietic stem cell transplantation. Natural killer (NK) cells have antitumor activity and may consolidate engraftment without inducing GVHD. Production of NK cells under good manufacturing practice (GMP) conditions in a sufficient number is difficult. STUDY DESIGN AND METHODS: Twenty-four apheresis procedures and subsequent NK-cell enrichment from 14 haploidentical donors were performed. NK-cell enrichment was performed using a GMP suitable immunomagnetic procedure. Factors influencing the NK-cell recovery, purity, and NK-cell dose were analyzed. RESULTS: A median number of 4.9 x 10(8) NK cells were obtained and median NK-cell recovery was 58 percent. Median T-cell depletion was 4.32 log. The absolute NK-cell number in the final product after processing significantly correlated with the preharvest NK-cell content of the peripheral blood (p = 0.002, r = 0.867). The NK-cell recovery was inversely correlated to the absolute NK-cell number in the apheresis product (p = 0.01, r = -0.51). The NK-cell dose per kg of body weight of the patient was inversely correlated to the weight of the patient (p = 0.007, r = -0.533). CONCLUSION: Donors with a high NK-cell count in peripheral blood are likely to provide NK-cell products with the highest cell number. However, maximal NK-cell dose is limited and high NK-cell doses may only be obtained for patients with a low body weight, making children and young adults the best candidates for NK-cell therapy

NKG2D ligand expression in AML increases in response to HDAC inhibitor valproic acid and contributes to allorecognition by NK-cell lines with single KIR-HLA class I specificities

This study exploited alloreactivity of natural killer (NK) cells for augmenting the recognition of human acute myeloid leukemia (AML). To circumvent the inhibitory effect of killer immunoglobulin receptor (KIR) signaling, we generated NK-cell lines with single KIR specificities for major human leukocyte antigen (HLA) class I allotypes. We demonstrated efficient cytolysis of KIR-HLA class I-mismatched primary AML blasts even at low effector-to-target ratios. To define the impact of tumor-associated activating NKG2D-ligands (NKG2D-L), 66 AML patients at diagnosis were analyzed. NKG2D-L were selectively expressed on monoblastic cells in AML M4 and M5 yet absent or weakly expressed on myeloblastic cells in all AML subtypes. Paucity of cell-surface NKG2D-L was not the result of shedding because levels of soluble ULBP1 ligand measured in AML plasma were in the normal range. Notably, purified NKG2D-L(+) monoblastic cells were more susceptible to NK-mediated killing than NKG2D-L(-) myeloblastic cells. Accordingly, induction of cell-surface NKG2D-L by treatment with the histone deacetylase inhibitor, valproic acid, rendered cells more sensitive to NK cytolysis. These data suggest that adoptive transfer of selected populations of alloreactive HLA class I-mismatched NK cells in combination with pharmacologic induction of NKG2D-L merits clinical evaluation as novel approaches to immunotherapy of human AML

Nab-paclitaxel plus gemcitabine versus nab-paclitaxel plus gemcitabine followed by FOLFIRINOX induction chemotherapy in locally advanced pancreatic cancer (NEOLAP-AIO-PAK-0113): a multicentre, randomised, phase 2 trial

Background The optimal preoperative treatment for locally advanced pancreatic cancer is unknown. We aimed to compare the efficacy and safety of nab-paclitaxel plus gemcitabine with nab-paclitaxel plus gemcitabine followed by fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX) as rnultidrug induction chemotherapy regimens in locally advanced pancreatic cancer. Methods In this open-label, multicentre, randomised phase 2 study, done at 28 centres in Germany, eligible patients were adults (aged 18-75 years) with an Eastern Cooperative Oncology Group performance status of 0 or 1 and histologically or cytologically confirmed, treatment-naive locally advanced pancreatic adenocarcinoma, as determined by local multidisciplinary team review. After two cycles of nab-paclitaxe1125 mg/m(2) plus gemcitabine 1000 mg/m(2) (administered intravenously on days 1,8, and 15 of each 28-day cycle), patients without progressive disease or unacceptable adverse events were randomly assigned (1:1) to receive either two additional cycles of nab-paclitaxel plus gemcitabine (nab-paclitaxel plus gemcitabine group) or four cycles of sequential FOLFIRINOX (oxaliplatin 85 mg/m(2), leucovorin 400 mg/m(2), irinotecan 180 mg/m(2), fluorouracil 400 mg/m(2) by intravenous bolus followed by a continuous intravenous infusion of 2400 mg/m(2) for 46 h on day 1 of each 14-day cycle; sequential FOLFIRINOX group). Randomisation was done by the clinical research organisation on request of the trial centre using a permuted block design (block size 2 and 4). Patients, investigators, and study team members were not masked to treatment allocation. The primary endpoint was surgical conversion rate (complete macroscopic tumour resection) in the randomised population by intention-totreat analysis, which was assessed by surgical exploration in all patients with at least stable disease after completion of induction chemotherapy. This trial is registered with ClinicalTrials.gov, NCT02125136. Findings Between Nov 18, 2014, and April 27,2018,168 patients were registered and 130 were randomly assigned to either the nab-paclitaxel plus gemcitabine group (64 patients) or the sequential FOLFIRINOX group (66 patients). Surgical exploration after completed induction chemotherapy was done in 40 (63%) of 64 patients in the nabpaclitaxel plus gemcitabine group and 42 (64%) of 66 patients in the sequential FOLFIRINOX group. 23 patients in the nab-paclitaxel plus geincitabine group and 29 in the sequential FOLFIRINOX group had complete macroscopic tumour resection, yielding a surgical conversion rate of 35.9% (95% CI 24.3-48-9) in the nab-paclitaxel plus gemcitabine group and 43.9% (31.7-56.7) in the sequential FOLFIRINOX group (odds ratio 0.72 [95% CI 0.35-145]; p=0.38). At a median follow-up of 24.9 months (95% CI 21.8-27.6), median overall survival was 18.5 months (95% CI 14.4-21.5) in the nab-paclitaxel plus gemcitabine group and 20.7 months (13.9-28.7) in the sequential FOLFIRINOX group (hazard ratio 0.86 [95% CI 0-55-1.36]; p=0-53). All other secondary efficacy endpoints, such as investigator-assessed progression-free survival, radiographic response rate, CA 19-9 response rate, and RO resection rate, were not significantly different between the two treatment groups except for improved histopathological downstaging in evaluable resection specimens from the sequential FOLFIRINOX group (ypN2 stage: 20 [69%] of 29 patients in the sequential FOLFIRINOX group vs four [17%] of 23 patients in the nabpaclitaxel plus gemcitabine group, p=0.0003; ypN0 stage: 15 [52%] of 29 patients in the sequential FOLFIRINOX group vs four [17%] of 23 patients in the nab-paclitaxel plus gemcitabine group, p=0.02). Grade 3 or higher treatmentemergent adverse events during induction chemotherapy occurred in 35 (55%) of 64 patients in nab-paclitaxel plus gemcitabine group and in 35 (53%) of 66 patients in the sequential FOLFIRINOX group. The most common of which were neutropenia (18 [28%] in nab-paclitaxel plus gemcitabine group, 16 [24%] in the sequential FOLFIRINOX group), nausea and vomiting (two [3%] in nab-paclitaxel plus gemcitabine group, eight [12%] in the sequential FOLFIRINOX group), and bile duct obstruction with cholangitis (six [9%] in nab-paclitaxel plus gemcitabine group, seven [1.1%] in the sequential FOLFIRINOX group). No deaths were caused by treatment-related adverse events during the induction chemotherapy phase. Interpretation Our findings suggest that nab-paclitaxel plus gemcitabine is similarly active and safe as nab-paclitaxel plus gemcitabine followed by FOLFIRINOX as multidrug induction chemotherapy regimens for locally advanced pancreatic cancer. Although conversion to resectability was achieved in about a third of patients, additional evidence is required to determine whether this translates into improved overall survival. Copyright (C) 2020 Elsevier Ltd. All rights reserved

Intraplatform reproducibility and technical precision of gene expression profiling in 4 laboratories investigating 160 leukemia samples: the DACH study

BACKGROUND: Gene expression profiling has the potential to offer consistent, objective diagnostic test results once a standardized protocol has been established. We investigated the robustness, precision, and reproducibility of microarray technology. METHODS: One hundred sixty individual patient samples representing 11 subtypes of acute and chronic leukemias, myelodysplastic syndromes, and nonleukemia as a control group were centrally collected and diagnosed as part of the daily routine in the Munich Leukemia Laboratory. The custom AmpliChip Leukemia research microarray was used for technical analyses of quadruplicate mononuclear cell lysates in 4 different laboratories in Germany (D), Austria (A), and Switzerland (CH) (the DACH study). RESULTS: Total-RNA preparations were successfully performed in 637 (99.5%) of 640 cases. Mean differences between pairs of laboratories in the total-RNA yield from the same sample ranged from 0.02 mug to 1.03 mug. Further processing produced 622 successful in vitro transcription reactions (97.6%); the mean differences between laboratories in the cRNA yield from the same sample ranged from 0.40 mug to 6.18 mug. After hybridization to microarrays, a mean of 47.6%, 46.5%, 46.2%, and 46.4% of probe sets were detected as present for the 4 laboratories, with mean signal-intensity scaling factors of 3.1, 3.7, 4.0, and 4.2, respectively. In unsupervised hierarchical cluster and principal component analyses, replicates from the same patient always clustered closely together, with no indications of any association between gene expression profiles due to different operators or laboratories. CONCLUSIONS: Microarray analysis can be performed with high interlaboratory reproducibility and with comparable quality and high technical precision across laboratories