Jan Soerensen

Pre-emptive immunotherapy with purified natural killer cells after haploidentical SCT: a prospective phase II study in two centers

Adoptive immunotherapy with allogeneic purified natural killer (NK) cell products might exert graft-versus-tumor alloreactivity with little risk of GVHD. In a prospective phase II study in two centers, we administered purified NK cell products to high-risk patients treated with haploidentical T-cell-depleted SCT. Sixteen patients received a total of 29 NK cell infusions on days +3, +40 and +100 after transplantation. Median doses (and ranges) of infused NK- and T-cells per product were 1.21 (0.3–3.8) × 107/kg and 0.03 (0.004–0.72) × 105/kg, respectively. With a median follow-up of 5.8 years 4/16 patients are alive. Cause of death was relapse in five, GVHD in three, graft failure in three, and transplant related neurotoxicity in one patient. Four patients developed acute GVHD⩾grade II, all receiving a total of ⩾0.5 × 105 T cells/kg. Compared with historical controls, NK cell infusions had no apparent effect on the rates of graft failure or relapse. Adoptive transfer of allogeneic NK cells is safe and feasible, but further studies are needed to determine the optimal dose and timing of NK cell therapy. Moreover, NK cell activation/expansion may be required to attain clinical benefit, while careful consideration must be given to the number of T cells infused.

IL-2-driven regulation of NK cell receptors with regard to the distribution of CD16+ and CD16- subpopulations and in vivo influence after haploidentical NK cell infusion

To characterize natural killer (NK) cell subpopulations during activation, we analyzed the NK cell receptor repertoire and functionality of purified clinical scale CD56+CD3− donor NK cells during stimulation with 1000 U/mL interleukin (IL)-2 for up to 14 days. In a phase I/II trial, we investigated the efficacy and feasibility of nonidentical NK cell infusion in patients with neuroblastoma after haploidentical stem cell transplantation. After IL-2 stimulation, large differences in the distribution of CD16negative and CD16positive subpopulations were found in 12 donors. Thereby, surface expression for all natural cytotoxicity receptors (NCRs) and NKG2D increased. In addition, killer cell immunoglobulin-like receptor (KIR)+ NK cells were overgrown by KIR− proportion and the homing receptor CD62L was lost during stimulation. NK cell cytotoxicity against K562 and neuroblastoma cells increased and significantly higher cytokine secretion (eg, interferon-γ, tumor necrosis factor-β, macrophage inflammatory protein-1α, macrophage inflammatory protein-1β) was observed after IL-2 stimulation compared with freshly isolated NK cells. However, NK cells of donors showing an initially enhanced cytotoxicity combined with NCRbright and CD69 expression, seemed to be exhausted and did not favor a stimulation period over 9 days. When IL-2–stimulated NK cells were given to transplant recipients, they induced a decrease of peripheral blood NK, in particular of CD56bright-NK cells. Our data indicate that IL-2 stimulation increases the expression of activating receptors and emphasizes mechanisms beside KIR/human leukocyte antigen. Furthermore, the results suggest that the expansion period of purified NK cells has to be individualized to optimize NK cell immunotherapy.

IL-2-activated haploidentical NK cells restore NKG2D-mediated NK-cell cytotoxicity in neuroblastoma patients by scavenging of plasma MICA

NK group 2D (NKG2D)‐expressing NK cells exhibit cytolytic activity against various tumors after recognition of the cellular ligand MHC class I chain‐related gene A (MICA). However, release of soluble MICA (sMICA) compromises NKG2D‐dependent NK‐cell cytotoxicity leading to tumor escape from immunosurveillance. Although some molecular details of the NKG2D‐MICA interaction have been elucidated, its impact for donor NK (dNK) cell‐based therapy of solid tumors has not been studied. Within an ongoing phase I/II trial, we used allogeneic IL‐2 activated dNK cells after haploidentical stem cell transplantation for immunotherapy of patients with high‐risk stage IV neuroblastoma. NKG2D levels on activated dNK cells increased strongly when compared with freshly isolated dNK cells and correlated with enhanced NK‐cell cytotoxicity. Most importantly, elevated sMICA levels in patients plasma correlated significantly with impaired dNK‐cell‐mediated cytotoxicity. This effect could be reversed by high‐dose infusion of activated dNK cells, which display high levels of surface NKG2D. Our data suggest that the provided excess of NKG2D leads to clearance of sMICA and preserves cytotoxicity of dNK cells via non‐occupied NKG2D. In conclusion, our results identify this tumor immune escape mechanism as a target to improve immunotherapy of neuroblastoma and presumably other tumors.

Clinical Grade Purification and Expansion of NK Cell Products for an Optimized Manufacturing Protocol

Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, n = 18; D2.12depl, n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 × 108 CD56+CD3− cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56+CD3− NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.

Enrichment of cell subpopulations applying automated MACS technique: purity, recovery and applicability for PCR-based chimerism analysis.

Enrichment of cell subpopulations is a prerequisite for lineage-specific chimerism analysis (LCA), a frequent approach in follow-up after allo-SCT. An efficient enrichment technique is Magnetic Cell Sorting (MACS) using the AutoMACS separator. However, evaluation of purity, recovery and applicability for PCR-based chimerism analysis of MACS-enriched subpopulations from post-transplant peripheral blood, providing reduced cell numbers and/or unbalanced proportions of subpopulations, is currently unavailable. We performed enrichment of CD3-, CD14-, CD15-, CD19- and CD56-positive subpopulations using ‘Whole Blood MicroBeads’ and AutoMACS separator in 137 prospectively collected peripheral blood samples from 15 paediatric patients after allo-CD3-/CD19-depleted SCT. Purity was assessed by immune phenotyping. Recovery and applicability for chimerism analysis was evaluated. Excellent purity >90% was achieved in CD14-, CD15-positive cells in 81%, 95% of the isolates and in 86% of CD3 and CD19 isolates, if ACC was >400 cells per μl. Median purity of CD56-positive isolates was 78.9%. Recovery >90% was between 93 (CD56) and 37% (CD15). Conventional and real-time PCR-based chimerism analysis was feasible in virtually all samples. Isolation of cell subpopulations by automated cell enrichment in post-transplant peripheral blood is feasible and fast providing excellent purity and recovery for routine lineage-specific chimerism analysis.

Feasibility of IL-15-activated cytokine-induced killer cell infusions after haploidentical stem cell transplantation

Feasibility of IL-15-activated cytokine-induced killer cell infusions after haploidentical stem cell transplantation

Mesenchymal stromal cells generated from pooled mononuclear cells of multiple bone marrow donors as a rescue therapy for children with severe steroid-refractory graft versus host disease: a multicenter survey

To circumvent donor-to-donor heterogeneity which may lead to inconsistent results after treatment of acute graft-versus-host disease with mesenchymal stromal cells generated from single donors we developed a novel approach by generating these cells from pooled bone marrow mononuclear cells of 8 healthy “3rd-party” donors. Generated cells were frozen in 209 vials and designated as mesenchymal stromal cell bank. These vials served as a source for generation of clinical grade mesenchymal stromal cell end-products, which exhibited typical mesenchymal stromal cell phenotype, trilineage differentiation potential and at later passages expressed replicative senescence-related markers (p21 and p16). Genetic analysis demonstrated their genomic stability (normal karyotype and a diploid pattern). Importantly, clinical end-products exerted a significantly higher allosuppressive potential than the mean allosuppressive potential of mesenchymal stromal cells generated from the same donors individually. Administration of 81 mesenchymal stromal cell end-products to 26 patients with severe steroid-resistant acute graft-versus-host disease in 7 stem cell transplant centers who were refractory to many lines of treatment, induced a 77% overall response at the primary end point (day 28). Remarkably, although the cohort of patients was highly challenging (96% grade III/IV and only 4% grade II graft-versus-host disease), after treatment with mesenchymal stromal cell end-products the overall survival rate at two years follow up was 71±11% for the entire patient cohort, compared to 51.4±9.0% in graft-versus-host disease clinical studies, in which mesenchymal stromal cells were derived from single donors. Mesenchymal stromal cell end-products may, therefore, provide a novel therapeutic tool for the effective treatment of severe acute graft-versus-host disease.

Interleukin-15-activated cytokine-induced killer cells may sustain remission in leukemia patients after allogeneic stem cell transplantation: feasibility, safety and first insights on efficacy

Anecdotal clinical cases previously suggested the potential of cytokine-induced killer (CIK) cells as an anti-leukemic immunotherapy (IT). Here, we report the aggregate experience from three hematology-oncology centers regarding the feasibility, safety and efficacy of interleukin (IL)-15-activated

Treatment of Infantile Inflammatory Bowel Disease and Autoimmunity by Allogeneic Stem Cell Transplantation in LPS-Responsive Beige-Like Anchor Deficiency

Inflammatory bowel disease (IBD) in young children can be a clinical manifestation of various primary immunodeficiency syndromes. Poor clinical outcome is associated with poor quality of life and high morbidity from the complications of prolonged immunosuppressive treatment and malabsorption. In 2012, mutations in the lipopolysaccharide-responsive beige-like anchor (LRBA) gene were identified as the cause of an autoimmunity and immunodeficiency syndrome. Since then, several LRBA-deficient patients have been reported with a broad spectrum of clinical manifestations without reliable predictive prognostic markers. Allogeneic hematopoietic stem cell transplantation (alloHSCT) has been performed in a few severely affected patients with complete or partial response. Herein, we present a detailed course of the disease and the transplantation procedure used in a LRBA-deficient patient suffering primarily from infantile IBD with immune enteropathy since the age of 6 weeks, and progressive autoimmunity with major complications following long-term immunosuppressive treatment. At 12 years of age, alloHSCT using bone marrow of a fully matched sibling donor—a healthy heterozygous LRBA mutant carrier—was performed after conditioning with a reduced-intensity regimen. During the 6-year follow-up, we observed a complete remission of enteropathy, autoimmunity, and skin vitiligo, with complete donor chimerism. The genetic diagnosis of LRBA deficiency was made post-alloHSCT by detection of two compound heterozygous mutations, using targeted sequencing of DNA samples extracted from peripheral blood before the transplantation.

Pre-Emptive Immunotherapy for Clearance of Molecular Disease in Childhood Acute Lymphoblastic Leukemia after Transplantation

Monitoring of minimal residual disease (MRD) or chimerism may help guide pre-emptive immunotherapy (IT) with a view to preventing relapse in childhood acute lymphoblastic leukemia (ALL) after transplantation. Patients with ALL who consecutively underwent transplantation in Frankfurt/Main, Germany between January 1, 2005 and July 1, 2014 were included in this retrospective study. Chimerism monitoring was performed in all, and MRD assessment was performed in 58 of 89 patients. IT was guided in 19 of 24 patients with mixed chimerism (MC) and MRD and by MRD only in another 4 patients with complete chimerism (CC). The 3-year probabilities of event-free survival (EFS) were .69 ± .06 for the cohort without IT and .69 ± .10 for IT patients. Incidences of relapse (CIR) and treatment-related mortality (CITRM) were equally distributed between both cohorts (without IT: 3-year CIR, .21 ± .05, 3-year CITRM, .10 ± .04; IT patients: 3-year CIR, .18 ± .09, 3-year CITRM .13 ± .07). Accordingly, 3-year EFS and 3-year CIR were similar in CC and MC patients with IT, whereas MC patients without IT experienced relapse. IT was neither associated with an enhanced immune recovery nor an increased risk for acute graft-versus-host disease. Relapse prevention by IT in patients at risk may lead to the same favorable outcome as found in CC and MRD-negative-patients. This underlines the importance of excellent MRD and chimerism monitoring after transplantation as the basis for IT to improve survival in childhood ALL.