scholarly journals Decrease post-transplant relapse using donor-derived expanded NK-cells

Leukemia ◽  
2021 ◽  
Author(s):  
Stefan O. Ciurea ◽  
Piyanuch Kongtim ◽  
Doris Soebbing ◽  
Prashant Trikha ◽  
Gregory Behbehani ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Disease Free Survival ◽  
Dependent Manner ◽  
Post Transplant ◽  
Haploidentical Transplantation ◽  
High Doses ◽  
High Level

AbstractIn this phase I/II clinical trial, we investigated the safety and efficacy of high doses of mb-IL21 ex vivo expanded donor-derived NK cells to decrease relapse in 25 patients with myeloid malignancies receiving haploidentical stem-cell transplantation (HSCT). Three doses of donor NK cells (1 × 105–1 × 108 cells/kg/dose) were administered on days −2, +7, and +28. Results were compared with an independent contemporaneously treated case-matched cohort of 160 patients from the CIBMTR database.After a median follow-up of 24 months, the 2-year relapse rate was 4% vs. 38% (p = 0.014), and disease-free survival (DFS) was 66% vs. 44% (p = 0.1) in the cases and controls, respectively. Only one relapse occurred in the study group, in a patient with the high level of donor-specific anti-HLA antibodies (DSA) presented before transplantation. The 2-year relapse and DFS in patients without DSA was 0% vs. 40% and 72% vs. 44%, respectively with HR for DFS in controls of 2.64 (p = 0.029). NK cells in recipient blood were increased at day +30 in a dose-dependent manner compared with historical controls, and had a proliferating, mature, highly cytotoxic, NKG2C+/KIR+ phenotype.Administration of donor-derived expanded NK cells after haploidentical transplantation was safe, associated with NK cell-dominant immune reconstitution early post-transplant, preserved T-cell reconstitution, and improved relapse and DFS. TRIAL REGISTRATION: NCT01904136 (https://clinicaltrials.gov/ct2/show/NCT01904136).

scholarly journals Safety and Feasibility of Administration of High Doses of Ex Vivo Expanded NK Cells for Prevention of Disease Relapse after Transplantation for Patients with Myeloid Malignancies - Final Results of a Phase I Clinical Trial

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 500-500 ◽  
Author(s):  
Stefan O. Ciurea ◽  
Dean A. Lee ◽  
Cecele Denman ◽  
Jolie Schafer ◽  
Roland L Bassett ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Conditioning Regimen ◽  
Advisory Board ◽  
Post Transplant ◽  
Equity Ownership ◽  
High Doses

Abstract Background: Disease relapse remains the most common cause of treatment failure after transplantation. To enhance anti-tumor effect of the graft we explored the use of high doses of donor-derived ex vivo expanded NK cells administered after haploidentical stem cell transplantation (HaploSCT) with the ultimate goal to decrease relapsed rate post-transplant. Methods: We aimed to study safety and determine the maximum tolerated dose (MTD) of high doses of mbIL-21 ex vivo expanded NK cells in a phase I clinical trial (clinicaltrials.gov NCT01904136) administered post-transplant in patients with myeloid malignancies (AML, CML, MDS). We hypothesized that infusion of mature NK cells would compensate for the lower NK-cell numbers and poor function previously observed by our group in the first month post-transplant. Patients received conditioning with melphalan 140mg/m2, fludarabine 160mg/m2 and 2GyTBI, and GVHD prophylaxis with post-transplant cyclophosphamide, tacrolimus and mycophenolate (Gaballa S, et al. Cancer. 2016). All patients had a bone marrow graft. NK cells were generated from peripheral blood mononuclear cells of the same donor with infusions on Days -2, +7 and on/after +28. The first infusion was with fresh and the other two were with cryopreserved NK cells. Dose escalation was planned in cohorts of 2 patients starting at 1x105 to 1x109 NK cells/Kg. Having an NK-cell alloreactive donor or certain KIR genotype was not a requirement to participate in this study, although these characteristics were evaluated in all patients. Results: Thirteen patients were enrolled. Eight patients had AML (7 in CR1, 4 with high-risk cytogenetics, 3 had primary induction failure AML and 3 had +minimal residual disease (MRD) at transplant, and one FLT3+ AML in CR2 with persistent MRD by flow cytometry), and 5 had CML (4 in second chronic phase, 2 with prior CNS disease, one had associated MDS with monosomy 7, and 2 who failed multiple TKIs). The median age was 41.5 years (range 18-60). Five patients were males and 8 females. The NK-cell dose escalation was as follows: 1x105/kg (N=2), 1x106/kg (N=3), 1x107/kg (N=3), 3x107/kg (N=2) and 1x108/kg (N=2). One patient was treated with 1x104/kg (Dose -1). All patients received the 3 planned infusions except one. Median purity of the NK-cell product was 98.98%. All patients achieved primary engraftment (100%) with 100% donor chimerism except one patient (who received 1x104/kg) who had secondary graft failure with concurrent parainfluenza pneumonia and died of treatment-related mortality (TRM). The median time to neutrophil and platelet engraftment was 18 and 26 days, respectively. Of 12 patients evaluable for aGVHD, the maximum grade was II in 7 patients. No grade III-IV aGVHD or cGVHD was observed. Only 5/12 patients had CMV reactivation (41.6% compared with 71% in retrospective data with the same treatment without NK cells), while none developed BK virus hemorrhagic cystitis. All patients (N=12) achieved remission with negative MRD after transplant. One patient treated NK cells at 1x105/kg/dose relapsed, received salvage treatment and was alive at last follow-up. All other patients are alive and in complete remission (N=11) after a median follow-up of 12.8 months (range 6-25.1). Compared with patients treated on a previous clinical trial with the same conditioning without NK cells (AML in CR1/2 and CML in CP), the patients who received NK cells had marked improvement in NK-cell function and cytotoxicity, as well as a significantly increase in INF gamma and TNF alpha production. A lower relapse rate and improved survival was observed compared with the retrospective cohort of patients treated without NK cells, although not statistically significant (p=0.21) (Figure 1). Conclusions: Doses up to 3x108/kg total dose of mbIL-21 ex vivo expanded NK cells obtained from the same donor can be safely administered in the early post-transplant period after HaploSCT. This was the maximum feasible dose to be manufactured in the current system, while no toxicity or increase in GVHD was observed. Three infusions of 1x108 NK cells per kg will be administered in the phase II study Figure 1 Relapse and survival for patients treated with the same conditioning regimen with and without NK cells. Figure 1. Relapse and survival for patients treated with the same conditioning regimen with and without NK cells. Disclosures Ciurea: Spectrum Pharmaceuticals: Other: Advisory Board; Cyto-Sen Therapeutics: Equity Ownership. Lee:Sanofi: Consultancy; Cyto-Sen Therapeutics: Equity Ownership, Other: Board of Directors; Intellia Therapeutics: Other: Advisory Board; Courier Therapeutics: Other: Advisory Board; Intrexon: Consultancy, Patents & Royalties; Shire: Other: Advisory Board; Ziopharm: Consultancy, Patents & Royalties. Bashir:Spectrum: Consultancy; Celgene: Research Funding; Takeda: Research Funding; Takeda: Consultancy. Champlin:Intrexon: Equity Ownership, Patents & Royalties; Ziopharm Oncology: Equity Ownership, Patents & Royalties.

Enhanced antitumor effect and lower viral reactivation with high doses of ex vivo expanded NK cells administered after haploidentical transplantation.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 74-74
Author(s):  
Stefan O. Ciurea ◽  
Rima Saliba ◽  
Doris Soebbing ◽  
Gabriela Rondon ◽  
Kai Cao ◽  
...  
Keyword(s):  
Nk Cells ◽  
Relapse Rate ◽  
Nk Cell ◽  
Ex Vivo ◽  
Viral Reactivation ◽  
T Cell Subsets ◽  
Matched Pair ◽  
Post Transplant ◽  
Antiviral Effects ◽  
High Doses

74 Background: NK cells have potent antitumor and antiviral effects. We hypothesized that multiple infusions of high doses of mb-IL21 ex vivo expanded NK cells administered peri-transplant will enhance graft-versus-leukemia effect and decrease viral reactivation. Methods: In the phase 1 dose escalation study patients received NK cells starting at 1x105 to 1x108/Kg/dose (N = 13). No dose limiting toxicities were observed. In the phase 2 study, 12 patients with myeloid malignancies received mbIL-21 ex vivo expanded NK cells at dose 1x108NK cells/kg with a K562 feeder cell system and with 41BB. NK cells were generated from peripheral blood mononuclear cells of the same donor and infused fresh on day -2, and cryopreserved on day +7 and +28. Conditioning regimen consisted of melphalan, fludarabine and TBI. All patients had bone marrow graft. Here we report updated results of all patients treated to date on this study. Results: Twenty-five patients (18 AML/MDS and 7 CML) were treated to date. The median age was 45 years, the median follow-up of survivors was 28 months. 16/18 AML/MDS patients had high-risk disease. No adverse effects occurred. Engraftment was achieved in all 24 evaluable patients after a median of 19 days. The cumulative incidence (CI) of grade 2 aGVHD was 38% at day 100. No chronic GVHD was observed. The CI of TRM was 21% at 2 years. The CI of relapse at 1 year was 8%. The 2-years PFS was 66%. Ten patients reactivated CMV. Immunologic reconstitution of T cell subsets showed a 5.7 fold higher number of NK cells on day 28 in patients who received highest NK cell doses vs. others. A matched-pair analysis done with an independent CIBMTR cohort of patients who received either MAC (N = 61) or RIC (N = 57), showed a significantly improvement in relapse rate and PFS in favor of patients treated on trial. Conclusions: Administration of higher doses of NK cells was associated with higher NK cell numbers early post-transplant, suggesting that a dose-dependent effect could be achieved. A low relapse rate and lower viral reactivation post-transplant continues to be seen, suggesting that NK cells exert both anti-tumor and antiviral effects in this setting. Clinical trial information: NCT01904136.

scholarly journals Phase I Trial of IL-21 Ex Vivo Expanded NK Cells Administration to Prevent Disease Relapse after Haploidentical Stem-Cell Transplantation for Myeloid Leukemias

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 102-102 ◽  
Author(s):  
Stefan O. Ciurea ◽  
Dean A. Lee ◽  
Kai Cao ◽  
Gabriela Rondon ◽  
Julianne Chen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Phase I ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Disease Relapse ◽  
Post Transplant ◽  
Equity Ownership

Abstract Background: As outcomes of haploidentical stem cell transplantation (HaploSCT) have improved, disease relapse represents the most common cause of treatment failure. Methods: We initiated a phase I clinical trial (clinicaltrials.gov NCT01904136) using peripheral blood-derived NK cells expanded ex vivo for 14 days with K562 antigen presenting cells expressing membrane-bound (mb) IL21 to prevent disease relapse after HaploSCT for patients with myeloid malignancies (AML,CML,MDS). We hypothesized that infusion of expanded and activated NK cells would compensate for the lower NK cell function in the early post-transplant period, and higher NK cell numbers would enhance anti-tumor effects of the graft. The primary endpoints were safety and determining the maximum tolerated dose (MTD). Patients were treated with a melphalan-based conditioning regimen (Figure). All patients had a primary bone marrow graft. NK cells were generated from peripheral blood mononuclear cells of the same donor obtained prior to marrow harvest and infused on days -2, +7 and on/after +28. The first infusion was with fresh and the other two were with cryopreserved NK cells. The dose escalation was planned in cohorts of 2 patients starting at 1x105/kg up to 1x109/kg or more if MTD will not be reached. Predictive NK alloreactivity and/or donor KIR B genotyping was preferred but not required to participate on study, however, was evaluated in all patients (Table). Results: Ten patients have been enrolled and treated to date. Of these, 8 patients were beyond Day+30 and were evaluated, 5 with AML (3 in CR1 with intermediate and 1 with high-risk cytogenetics, and 1 in CR2 FLT3+ with persistent MRD by flow cytometry) and 3 with CML (2 in second chronic phase, one with clonal evolution who failed multiple TKIs). The median age was 39 years (range 18-59). Four patients were males and 4 females. The NK cells dose escalation was as follows: 1x105/kg (N=2), 1x106/kg (N=3) and 1x107/kg (N=2). One patient was treated with 1x104/kg before full evaluation of 1x105/kg was completed. All patients achieved primary engraftment (100%). All patients except the one who received the lowest dose (1x104/kg) had sustained engraftment and 100% donor chimerism on Day 30 post-transplant. The median time to neutrophil engraftment was 18 days and to platelet engraftment was 26 days. The first patient had a mixed chimerism, developed secondary graft failure and concurrent parainfluenza pneumonia. He was re-transplanted with a different donor but died of treatment-related mortality (TRM). Of 7 patients evaluable for aGVHD, the maximum aGVHD grade was gr II in 4 patients. No gr III-IV aGVHD or cGVHD was observed. Only 3/7 patients had CMV reactivation (43% compared with 71% in retrospective data with the same treatment without NK cells), 2 requiring a brief period of treatment of approximately 1 month. None developed BK virus hemorrhagic cystitis. All patients achieved CR after transplant. One patient (#2) treated at 1x105/kg NK cell dose relapsed, received salvage treatment and is alive at last follow-up. All other patients are alive and in remission (N=6) after a median follow-up of 6 months (range 1-12.5). NK cell phenotype and function early post-transplant will be presented at the meeting. Conclusions: Doses up to 1x107/kg of ex vivo expanded NK cells using the mbIL-21 method can be safely administered after HaploSCT. Administration of these cells in this setting in not associated with a higher incidence of aGVHD. There was a low rate of viral reactivation, suggesting that the infused NK cells may provide antiviral activity. MTD has not been reached, the study is ongoing. Table. PT Nr Initials NK cell dose (/kg) Pt KIR Ligand Donor Donor KIR Ligand NK Allo-reactivity Donor KIR Haplotype # Cen-B/B KIR Score KIR Centromeric KIR2DS1 Outcome 1 RB 1x104 C2/C2, Bw4 Son C2, Bw4 No A/A 0 Neutral Cen-A/A - Died 2 FM 1x105 C1/C2, Bw4 Son C1, Bw4 No A/B 2 Better Cen-A/B - Relapsed +120 3 RG 1x105 C1/C2, Bw4 Daughter C1, C2, Bw4 No A/A 0 Neutral Cen-A/A - CR +374 4 GM 1x106 C1/C1, Bw4 Sister C1, C2, Bw4 Yes A/B 2 Better Cen-A/B - CR +168 5 DS 1x106 C1/C1 Brother C1, C2, Bw4 Yes A/A 0 Neutral Cen-A/A - CR +166 6 MH 1x107 C1/C2, Bw4 Sister C1, Bw4 No A/B 2 Best Cen-B/B + CR +91 7 JG 1x106 C1/C2, Bw4 Sister C1, C2, Bw4 No A/A 0 Neutral Cen-A/A - CR +35 8 DD 1x107 C1/C1, Bw4 Father C1, Bw4 No A/A 0 Neutral Cen-A/A - CR +87 9 RR 3x107 C1/C1 Brother C1, C2 Yes A/B 2 Better Cen-A, Cen/Tel-B - NE 10 JC 3x107 C2/C2, Bw4 Son C1/C2, Bw4 Yes A/B 2 Better Cen-A/B + NE Figure 1. Figure 1. Disclosures Lee: Intrexon: Equity Ownership; Ziopharm: Equity Ownership; Cyto-sen: Equity Ownership. Rezvani:Pharmacyclics: Research Funding.

scholarly journals Impact of Early Immune Reconstitution after Haploidentical Stem Cell Transplantation with Post-Transplant Cyclophosphamide

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2118-2118
Author(s):  
Nieves Dorado ◽  
Ana Pérez-Corral ◽  
Mi Kwon ◽  
Laura Solán ◽  
Rebeca Bailén ◽  
...  
Keyword(s):  
Nk Cells ◽  
Lymphocyte Count ◽  
Immune Reconstitution ◽  
Conflicts Of Interest ◽  
Conditioning Regimen ◽  
Prognostic Significance ◽  
Disease Free Survival ◽  
Haploidentical Transplantation ◽  
The Impact

Abstract Introduction: Immune reconstitution (IR) has a significant impact in HSCT outcome with a role against opportunistic infections and in disease control. In the setting of unmanipulated haploidentical transplantation (Haplo-HSCT), some groups have identified the absolute leukocyte count on day +30 (ALC30) as an independent prognostic factor in terms of overall survival (OS), disease free survival (DFS) and infection related mortality (IM). The aim of this study was to evaluate the impact of early IR on different HSCT outcomes in patients who underwent Haplo-HSCT with postransplant cyclophosphamide (PTCy) at our institution. Patients and methods: Eighty-eight patients received a Haplo-HSCT from 2011 to 2016. Thirty-six percent of the patients received myeloablative conditioning regimen and 64% received reduced intensity regimen. Graft-versus-host disease (GVHD) prophylaxis was based on PTCy, cyclosporine and mycophenolate mofetil. Early IR was assessed through the analysis of different lymphocyte subpopulations at days +30 and +90 after transplantation, including ALC30 (cellular analyzer DXH, Beckman Coulter®); CD3+ lymphocyte count and their different subpopulations (CD4+ and CD8+ lymphocytes, naive and memory T cells) and NK cells count. Lymphocytes subpopulations were determined by multiparametric flow cytometry (FC500 and Navios, Beckman Coulter®). ROC curves were used to determine the optimal cut-off values for each of the studied variables. Results: Eighty-one patients were studied, excluding 7 who died before day +30. Median follow-up was 26 months (10-43). Patient´s characteristics are shown in Table 1. CMV reactivation was documented in 76% (62) of the patients, 4% (3) developed a proven invasive fungal infection, and 31% (25) presented hemorrhagic cystitis. Median OS and DFS were 26 months (10-43) and 24 months (9-39), respectively. IM rate and NRM rate were 10% and 24%, respectively, at the end of follow up. Median lymphocyte populations counts at day +30 are shown in Table2. ALC30 below 100 cells/uL (p= 0.027) and CD4+ naïve lymphocytes below 12 cells/uL (p=0.033) (both corresponding to the 25 percentile) were associated with lower OS compared to patients with higher counts at day +30 (Figure 1). Patients with ALC30 lower than 300 cells/uL (p=0.026) showed significantly higher NRM; CD8+ count lower than 20 cells/uL (p=0.022) also showed higher NRM. NK cells counts at day +30 lower than 14 cells/uL (p=0.014), near to percentile 25, predicted higher IM (62.5% vs 37.5%). We did not identify any lymphocyte subpopulation that could predict DFS. Patients with acute GVHD grades II-IV showed values of ALC30 lower than 200 cells/uL (p=0.051), although non-statistically significant. No relationship was found between lymphocytes subpopulations at day +90 and HaploSCT outcomes. Conclusions: Our study supports the prognostic significance of early IR after unmanipulated haploidentical transplantation with PTCy, as previously described by other groups. ALC30, CD4+ naïve lymphocytes, and CD8+ lymphocyte count at day +30 may be good early predictors for OS and NRM in this setting. On the other hand, low NK cells counts (lower than percentile 25) predicted higher IM. Patients with very low lymphocyte counts should be monitored closely as they are at high risk for infectious complications, NRM and OS. Disclosures No relevant conflicts of interest to declare.

Early NK Cell Proliferation After Umbilical Cord Blood Transplantation Is Associated With Superior Disease-Free Survival Due To Reduced Leukemia Relapse

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4610-4610
Author(s):  
Rachel Joy Bergerson ◽  
Sarah Cooley ◽  
Julie Curtsinger ◽  
Ryan Shanley ◽  
Claudio Brunstein ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cord Blood ◽  
Nk Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Nk Cell ◽  
Disease Free Survival ◽  
Free Survival ◽  
Disease Free

Compared to traditional chemotherapy, allogeneic hematopoietic cell transplantation (allo-HCT) has the potential of triggering graft vs. leukemia (GVL) reactions that make this procedure uniquely curative. Despite this, relapse remains the most common cause of treatment failure. Early after allo-HCT the donor immune system reconstitutes within the host and natural killer (NK) cells are the earliest lymphocyte population to recover. Previous studies by us and other investigators have linked rapid lymphocyte recovery and/or high NK cell numbers early after transplant with less relapse. Mechanistically, the reasons for this are unknown. We hypothesized that NK cell proliferation would be associated with allo-HCT outcomes. In a large data set with short-term follow-up we compared stem cell source to NK cell proliferation at Day 28 after transplantation (using Ki67+ staining). In patients undergoing autologous (n=117), sibling (n=57), single umbilical cord blood (sUCB) (n=62) or double umbilical cord blood (dUCB) (n=50) transplantation there were significant differences in the median NK cell proliferation (2.1%, 3.3%, 3.8%, and 19.2%, respectively, p<0.0001). These results suggest that NK cell proliferation is increased when patients are transplanted with stem cell sources that have an increased number of HLA mismatches (dUCB). We next examined factors extrinsic to the NK cells to determine whether differences in the four patient groups were due to these factors. Regulatory T cells (Tregs) are known to negatively regulate the proliferation and activation of a variety of cell types, including NK cells. We utilized the percentage and absolute number of Tregs (defined as CD4+CD25highFoxP3+CD127low) at Day 28 (available for a subset of the above patient samples) and correlated it with the percentage of proliferating NK cells at Day 28 after allogeneic transplant (n=212). The median percentage of Tregs within the lymphocyte fraction was 1.49%. In patients with higher than 1.49% Tregs, the mean NK cell proliferation was 10.8% +/-16.4. In contrast, patients with lower than 1.49% Tregs had a mean NK cell proliferation of 21.2% +/-24.3 (p=0.0004). A nearly identical trend was observed for the absolute numbers of Tregs, suggesting that Tregs may play an extrinsic role in NK cell proliferation after allo-HCT. To further address differences in lymphocyte proliferation and clinical outcomes, we used Ki67 staining to assess T cell (CD4+ and CD8+) and NK cell (CD3-CD56+) proliferation at Day 28 in a different subset of patients undergoing dUCB transplant with acute leukemia or MDS (80%) or other malignant disorders using either myeloablative (42%) or reduced intensity conditioning (58%) and a median of one year follow-up (n=53). There was no association of transplant outcomes with T cell (CD4 or CD8) proliferation. In the NK cell compartment there was a wide range in the percentage of proliferating NK cells (0-86%), with a median of 20%. Patients were segregated into high (>20%) and low (<20%) NK cell proliferating groups and assessed for cytokine levels and transplant outcomes. At Day 28 soluble IL15 levels were not different between high and low NK cell proliferating patients. There was no significant association between NK cell proliferation and the probability or time to neutrophil recovery (p=0.15) or treatment related mortality (p=0.88). Excluding the 14 patients who developed aGVHD prior to day of NK cell assessment (Day 28), the high NK proliferating group had a trend toward a higher cumulative incidence of aGVHD (46% vs. 25%, p=0.17). Using multivariable analysis to control for a variety of patient and disease-associated variables, patients with high NK cell proliferation had significantly better disease-free survival (HR=0.29, 95%CI=0.12-0.71, p=0.01) (Figure 1). Strikingly, patients with high NK cell proliferation were 4-times less likely to relapse (HR=0.24, 95%CI=0.08-0.77, p=0.02). Collectively, these studies show that early NK cell proliferation is associated with superior outcomes after dUCB transplantation, due to reduced relapse, and that this is partly modulated by Tregs. Prospective strategies to increase NK cell proliferation may be of therapeutic benefit to improve transplant outcomes.Figure 1Disease Free Survival for patients undergoing dUCB transplant (n=53) based on low (blue) vs high (red) NK cell proliferation at Day 28 post transplant.Figure 1. Disease Free Survival for patients undergoing dUCB transplant (n=53) based on low (blue) vs high (red) NK cell proliferation at Day 28 post transplant. Disclosures: Wagner: Novartis: Research Funding. Miller:Coronado Biosciences: Scientific Advisory Board Other.

Serotonin Shapes the Migratory Potential of NK Cells – An in vitro Approach

2017 ◽  
Vol 38 (11) ◽  
pp. 857-863 ◽  
Author(s):  
Philipp Zimmer ◽  
Wilhelm Bloch ◽  
Markus Kieven ◽  
Lukas Lövenich ◽  
Jonas Lehmann ◽  
...  
Keyword(s):  
Nk Cells ◽  
Acute Exercise ◽  
Cell Function ◽  
Nk Cell ◽  
Ex Vivo ◽  
Killer Cell ◽  
Dependent Manner ◽  
The Impact ◽  
Dose Dependent

AbstractIncreased serotonin (5-HT) levels have been shown to influence natural killer cell (NK cell) function. Acute exercise mobilizes and activates NK cells and further increases serum 5-HT concentrations in a dose-dependent manner. The aim of this study was to investigate the impact of different serum 5-HT concentrations on NK cell migratory potential and cytotoxicity. The human NK cell line KHYG-1 was assigned to 4 conditions, including 3 physiological concentrations of 5-HT (100, 130 or 170 µg/l 5-HT) and one control condition. NK cells were analyzed regarding cytotoxicity, migratory potential and expression of adhesion molecules. No treatment effect on NK cell cytotoxicity and expression of integrin subunits was detected. Migratory potential was increased in a dose dependent manner, indicating the highest protease activity in cells that were incubated with 170 µg/l 5-HT (170 µg/l vs. control, p<0.001, 170 µg/l vs. 100 µg/l, p<0.001; 170 µg/l vs. 130 µg/l, p=0.003; 130 µg/l vs. control, p<0.001, 130 µg/l vs. 100 µg/l, p<0.001). These results suggest that elevated 5-HT serum levels play a mediating role in NK cell function. As exercise has been shown to be involved in NK cell mobilization and redistribution, the influence of 5-HT should be investigated in ex vivo and in vivo experiments.

scholarly journals Improved Outcomes for Patients Receiving High-Doses of IL-21 Ex Vivo Expanded NK Cells after Haploidentical Transplantation (haploSCT): Long-Term Follow-up of a Phase 1/2 Clinical Trial with Comparison to CIBMTR Controls

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 700-700
Author(s):  
Stefan O. Ciurea ◽  
Roland Bassett ◽  
Doris Soebbing ◽  
Gabriela Rondon ◽  
Kai Cao ◽  
...  
Keyword(s):  
Nk Cells ◽  
Board Of Directors ◽  
Research Funding ◽  
Phase 1 ◽  
Advisory Committees ◽  
Post Transplant ◽  
Long Term Follow Up ◽  
High Doses

Background: Disease relapse following allogeneic stem-cell transplantation remains unacceptably high and there is an urgent need for new therapies that decrease relapse rates and improve survival post-transplant. Natural killer (NK) cells have potent antitumor effects, particularly those expended with mb-IL21 from peripheral blood. Preliminary data from a phase-1 dose-escalation study of up to 1x108 NK cells/Kg/dose and multiple dosing yielded promising results and a favorable safety profile (Ciurea SO.Blood.2017;130:18657). This report presents long-term follow-up from a phase-1/2 clinical trial in patients with high-risk myeloid malignancies (AML/MDS/CML) (clinicaltrials.gov NCT01904136) and a comparison with CIBMTR controls. Methods: Patients received conditioning with fludarabine 160 mg/m2, melphalan 140 mg/m2 and 2GyTBI, post-transplant cyclophosphamide-based GVHD prophylaxis and bone marrow graft from a haploidentical donor. Ex vivo expanded NK cells were generated from peripheral blood mononuclear cells of the same donor with a K562 feeder cells expressing mb-IL21 and 41BB and infused fresh on Day-2, and cryopreserved on Day+7 and +28 (up to Day+90). 1x108/Kg/dose was chosen for the phase 2 trial. An independent matched-pair analysis was done using controls from the CIBMTR database stratified by conditioning intensity. Results: 24/26 patients treated to date were evaluable (one short follow-up and one excluded as ineligible). 80% (19/24) of patients received all 3 doses of NK cells. The median age was 45 years (range 18-59), median follow-up was 43.6 months (range 15.1-60.9). Thirteen patients (54%) were females. 5 patients had donor-specific anti-HLA antibodies (DSA). The median HCT-CI was 2 (range 0-8), 12 patients (50%) had HCT-CI&gt;3. 17 patients (72%) had AML/MDS and 7 (28%) advanced CML. Of AML/MDS patients, 10 (59%) had high-risk cytogenetics, 7 (41%) had measurable residual disease, 9 (53%) had intermediate/adverse-risk ELN2017 and 5 (29.4%) had primary induction failure. No infusion reactions or significant adverse events were observed to date. All patients (100%) achieved engraftment after a median of 19 days (range 14-42). The cumulative incidence (CI) of grade 2-4 aGVHD was 29.2% at Day100 and 41.7% at 1-year post-transplant. Only one patient developed severe grade 3-4 aGVHD and one patient had extensive cGVHD. Only one patient relapsed (a patient with DSA who did not receive desensitization prior to transplantation), 1-year CI of relapse was 5.9%. The CI of TRM at 1-year for patients without DSA was 21%. The median overall survival and progression-free survival (PFS) were not reached. The 1-year and 3-year PFS for all patients and patients without DSA was 70.8% and 66.1%, and 79% and 72.9% for patients without DSA, respectively (Figure 1). One-year and 3-years GRFS for all patients and patients without DSA was 70.8% and 66.1%, and 79% and 72.9%, respectively. An independent matched-pair analysis (at least 1:1) was conducted by CIBMTR after the first 18 patients treated on study in 07/2018 with RIC (N=57) or MAC (N=61) controls. The relapse was 1/18 vs 25/57 for RIC (p=0.037) and 15/61 for MAC (p=0.07), while the 1-year PFS was 82% vs 49% for RIC and 64% for MAC (p=0.21) (Figure 1). Updated results of this analysis will be presented at the meeting. Conclusions: Results from this long-term follow-up analysis confirm very low relapse rate and excellent GRFS after haploSCT for patients treated with high-doses of NK cells expanded with mbIL21 stimulation. A prospective multi-center phase 2 BMTCTN study will evaluate the safetly and efficacy of high doses of NK cells for the prevention of relapse in patents with AML/MDS receiving haploSCT. Figure 1 Disclosures Ciurea: Miltenyi: Research Funding; Kiadis Pharma: Membership on an entity's Board of Directors or advisory committees, Other: stock holder; MolMed: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees. Bashir:Kite: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; StemLine: Research Funding; Acrotech: Research Funding; Celgene: Research Funding; Imbrium: Membership on an entity's Board of Directors or advisory committees; Spectrum: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Pasquini:Novartis: Research Funding; Kite Pharmaceuticals: Research Funding; BMS: Research Funding; Medigene: Consultancy; Amgen: Consultancy; Pfizer: Consultancy. Lee:Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

scholarly journals Natural Killer (NK) Alloreactivity Seems Not to Play a Role in Preventing Leukemia Relapse in Unmanipulated Haploidentical Bone Marrow Transplantation with Post-Transplant Cyclophosphamide

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2033-2033 ◽  
Author(s):  
Federica Galaverna ◽  
Fabio Guolo ◽  
Daniela Pende ◽  
Alida Dominietto ◽  
Raffaella Meazza ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Disease Free Survival ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Free Survival ◽  
Post Transplant ◽  
Leukemia Relapse ◽  
Disease Free

Abstract Background Natural Killer (NK) cells have been widely studied due to their non-major histocompatibility complex (MHC)-restricted cytotoxicity towards transformed or virally infected target cells. In the setting of hematopoietic stem cell transplantation (HSCT), donor NK cells may be "alloreactive" as their killer immunoglobuline-like receptors (KIRs) do not recognize their ligands on recipient human leukocyte antigen (HLA) class I molecules (i.e. KIR-ligands), leading to NK activation. NK alloreactivity can often occur in haploidentical HSCT (Haplo-HSCT), by means of KIR/KIR-L mismatch in graft versus host (GvH) direction, contributing to graft-versus leukemia (GvL) effect, clearing residual leukemic blasts. In the last decade, several studies have shown that NK cells alloreactivity plays a role in T-depleted Haplo-HSCT leading to higher disease free survival rates for patients transplanted from NK-alloreactive donors; recent studies have also shown that donors having KIR B haplotypes (characterized by the presence of more activating KIR) or expressing KIR2DS1 correlated with a better clinical outcome of transplantation. Thus, these NK cell features might be positively considered in the donor selection strategy. Materials and Methods: We analyzed NK-alloreactivity in the setting of unmanipulated Haplo-HSCT with post-transplant cyclophosphamide for patients affected by acute myeloid leukemia or myelodisplastic syndromes. 101 consecutive patients transplanted from September, 2010 to October, 2014 were enrolled, with the big majority of donors and patients studied for HLA-genotype and KIR. Results: Disease status at HSCT was the most relevant factor affecting outcome (p <0.0001), with 3 y 78% overall survival (OS) and 76% disease free survival (DFS) rates for "early" patients (CR1+CR2, n=61) versus 33% OS and 28% DFS rates for "advanced" patients (CR3 or active disease, n=40). Nor NK-alloreactivity nor the presence of donor KIR-B haplotype nor the presence of donor KIR2DS1 seemed to play a role in preventing leukemia relapse. NK alloreactive patients had DFS rate similar to non-NK alloreactive group (62% vs 59%, p 0.47) with a better, still non-significant trend in OS (72% vs 60%, p 0.14) for NK-alloreactive patients. Similarly, Haplo-HSCT from donors with KIR-B haplotype (with B-content score>=2) or who had KIR2DS1 was not associated with better outcome (p 0.67 and p 0.89, respectively). We observed an high expression of CD56 and inhibitory receptors such as NKG2A on surface of NK cells in post-HSCT samples, suggesting that NK-cell function could be inhibited in unmanipulated haploidentical setting. Conclusions NK alloreactivity seems not to play a role in preventing leukemia relapse in unmanipulated haploidentical transplantation with post-transplantation. The different immunosuppressive approach of this Haplo-HSCT setting compared to T-depleted Haplo-HSCT, with concomitant use of cyclosporine from early transplant days, which has been shown to interact and possibly inhibit NK cells in vivo, and post transplant cyclophosphamide effects, selectively killing activated T-cell and inducing long-term tolerance, could affect NK efficacy. Further studies are needed to better understand the complexity of this intriguing issue, leading to a more complete definition of NK cell functions in this Haplo-HSCT setting. Disclosures No relevant conflicts of interest to declare.

Patients Receiving IL-2 Activated Donor NK Cells Show Lower Incidence of Severe GvHD after Haploidentical SCT.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 354-354 ◽  
Author(s):  
Chiara Gentilini ◽  
Urte Hilbers ◽  
Volker Huppert ◽  
Christoph Loddenkemper ◽  
Arne Müßig ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Ex Vivo ◽  
Striking Difference ◽  
Cell Mediated Immune Response ◽  
Significant Difference ◽  
Grade Ii ◽  
High Doses

Abstract To improve the antileukemic effectiveness of haploidentical stem cell transplantation (HSCT) and strengthen the cell mediated immune response, we have adoptively transferred high numbers of alloreactive donor NK cells during the early phase after transplantation. In addition, we activated the transferred NK-cell population ex vivo in short term cultures with high doses of IL-2 in order to enhance the antileukemic activity and inhibit the occurrence of severe GvHD. Method: In a phase-II study, 17 patients (10 AML, 1 MDS, 1 HD, 2 CML, 3 ALL, median age 37 yrs, range 17–48 yrs) were transplanted in late phases of their disease (6 pts. as 2nd transplantation) and received purified NK cells from their haploidentical donors at day +2 after HSCT. Conditioning consisted of 12 Gy fTBI, Thiotepa (10mg/kg), Fludarabine (5 x 30 mg/qm) and OKT3 (day −4 to +2). NK cells were isolated from the CD34- fraction using an automated two-step procedure of CD3+ depletion and subsequent CD56+ selection. Seven patients received activated NK cells and 10 patients received unstimulated NK cells. Cells were activated by 16h incubation with IL-2 (500U/1x107 cells). Results: After selection and subsequent overnight activation of the NK cells with IL-2 (7 out of 17 patients), a mean number of 8.3 x 106/kg CD56+CD3− NK cells was transferred at day 2 after transplantation. The purity was 76%, due to contaminating CD56+ monocytes. The mean number of contaminating CD3+ cells in the transfused NK product was 2.1 x 104/kg. Activation of donor NK cells with IL-2 resulted in an increase of cytotoxic activity, when cells were tested against target cell lines. No differences in yield or number of contaminating T cells were observed between IL-2 activated and not activated NK cells. No severe acute toxicity attributable to NK cell infusion was observed in both groups of patients. Comparing the rate of high-grade GvHD revealed an interesting result. Whereas only one patient developed GvHD ≥ grade II after treatment with IL-2 activated NK cells, seven out of ten patients showed GvHD ≥ grade II after transfer of non-activated NK cells (p<0.05). When the correlation between GvHD and the presence of a KIR mismatch was analyzed no significant difference was observed. Moreover immunocytometric analysis of lymphocyte subpopulations at different time points after transplant revealed a long-lasting cellular immunodeficiency in all patients, with slow recovery of CD4+ lymphocytes. As for the incidence of GvHD, there was also a striking difference in immune recovery between the patients receiving IL2-activated and those treated with non-activated NK cells. Patients receiving activated NK cells showed significantly lower numbers of NK- and T cells during the first months post transplant, whereas no differences in the number of granulocytes were present between the two groups. Based on these findings we can assume that the use of IL-2 for NK cell activation could play a role in reducing the incidence of severe GvHD after haploidentical HSCT.

scholarly journals Phase 1 Trial of Prophylactic Donor-Derived IL-2 Activated NK Cell Infusion after Allogeneic Hematopoietic Stem Cell Transplantation from a Matched Sibling Donor

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-7
Author(s):  
Raynier Devillier ◽  
Boris Calmels ◽  
Sophie Guia ◽  
Cyril Fauriat ◽  
Sabine Furst ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Phase 1 ◽  
Immunosuppressive Treatment ◽  
Sibling Donor ◽  
Matched Sibling ◽  
Ancillary Studies ◽  
Nk Cell Therapy

Background: Donor lymphocyte infusion (DLI) is commonly used to enhance T-cell mediated graft-versus-tumor (GVT) effect after allogeneic stem cell transplantation (allo-HSCT). It was showed that prophylactic DLI can produce promising outcome but is also associated with a high rate of graft-versus-host disease (GVHD), limiting its use as maintenance treatment. By contrast with T cells, there are clinical and biological evidences that NK cells can provide strong GVT effect without inducing GVHD, making post allo-HSCT NK cell-based immunotherapy appealing. Thus, we designed a phase 1 clinical trial evaluating the safety of donor-derived ex vivo IL-2 activated NK cells (DLI-NK) as maintenance immunotherapy after allo-HSCT (DLI-NK: NCT01853358). Methods: We included patients who were in morphologic remission of hematological cancers 2 months after allo-HSCT from a matched sibling donor after reduced intensity conditioning regimen, without history of acute GVHD. NK cells were purified from the donor (CD3 depletion / CD56 positive selection) and cultured ex vivo during 7 days with IL-2 before prior to infusion in patients between day+60 and day+90 after allo-HSCT. The study included a first phase of dose escalation (3+3 method) to determine the maximal tolerated dose (MTD) using 3 dose levels: 1.0 (dose 1), 5.0 (dose 2), 5.0-50 (dose 3) million cells/kg. A second phase allowed the treatment of subsequent patients to reach a total amount of 10 patients treated at the MTD. As ancillary studies, we performed: 1) preclinical evaluation of donor-derived IL-2 stimulated NK cell therapy in mice; 2) phenotypical and functional characterization of the NK cell therapy product before and after 7-day IL-2 stimulation, and 3) longitudinal immunomonitoring of NK cell phenotype in patients. Results: We treated 16 patients without observing any dose-limiting toxicity (grade 3-4 adverse events related to DLI-NK within 30 days after infusion). During the follow up, 4 patients developed chronic GVHD (1 mild, 2 moderate and 1 severe forms). All of them were in complete remission of GVHD without immunosuppressive treatment at last contact. We observed one non-relapse death due to idiopathic pneumonia. From the four patients who relapsed (3 AML and 1 CLL), 2 died from their disease and 2 were in complete response after salvage therapy. At last follow up, 11 out of 16 treated patients were alive in CR without GVHD features and immunosuppressive treatment. Ancillary studies showed that IL-2 stimulated allogeneic NK cells did not induce GVHD and displayed strong antitumor effect in mice against YAC-1 tumor cells, supporting the efficacy of this approach. In human, we observed that IL-2-activated NK cells expressed high levels of activating receptors (DNAM-1, NKG2A, NKp46, NKp30) and of the inhibitory receptor NKG2A, and exhibited increased degranulation and cytokine production when challenged by K562 tumor cells, as compared to freshly isolated NK cells. Immunomonitoring of patient NK cells after DLI-NK do not reveal significant phenotypic or functional dysfunction, with a pattern of NK cell recovery that is similar to what was previously observed after allo-HSCT. Conclusion: We showed that prophylactic DLI-NK after matched sibling donor allo-HSCT was feasible and safe. We observed low incidence of GVHD as compared to previous reports of conventional prophylactic DLI, reinforcing the rationale of NK cell role in GVHD prevention. In addition, promising disease-free survival supports the design of future clinical trials evaluating the efficacy of DLI-NK as disease relapse prevention. Disclosures Harbi: Sanofi: Honoraria. Olive:Imcheck Therapeutics: Other: Co-Founder & shareholder; Emergence Therapeutics: Other: Co-Founder & shareholder; Alderaan Biotechnology: Other: Co-Founder & shareholder. Blaise:Jazz Pharmaceuticals: Honoraria.

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