Monitoring of Circulating CAR T Cells: Validation of a Flow Cytometric Assay, Cellular Kinetics, and Phenotype Analysis Following Tisagenlecleucel.

Chimeric antigen receptor (CAR) T cell therapy is a potent new treatment option for relapsed or refractory hematologic malignancies. As the monitoring of CAR T cell kinetics can provide insights into the activity of the therapy, appropriate CAR T cell detection methods are essential. Here, we report on the comprehensive validation of a flow cytometric assay for peripheral blood CD19 CAR T cell detection. Further, a retrospective analysis (n = 30) of CAR T cell and B cell levels over time has been performed, and CAR T cell phenotypes have been characterized. Serial dilution experiments demonstrated precise and linear quantification down to 0.05% of T cells or 22 CAR T cell events. The calculated detection limit at 13 events was confirmed with CAR T cell negative control samples. Inter-method comparison with real-time PCR showed appreciable correlation. Stability testing revealed diminished CAR T cell values already one day after sample collection. While we found long-term CAR T cell detectability and B cell aplasia in most patients (12/17), some patients (5/17) experienced B cell recovery. In three of these patients the coexistence of CAR T cells and regenerating B cells was observed. Repeat CAR T cell infusions led to detectable but limited re-expansions. Comparison of CAR T cell subsets with their counterparts among all T cells showed a significantly higher percentage of effector memory T cells and a significantly lower percentage of naïve T cells and T EMRA cells among CAR T cells. In conclusion, flow cytometric CAR T cell detection is a reliable method to monitor CAR T cells if measurements start without delay and sufficient T cell counts are given.

Improving Clinical Manufacturing of IL-15 Activated Cytokine-Induced Killer (CIK) Cells.

Cytokine-induced killer (CIK) cells are an immunotherapeutic approach to combat relapse following allogeneic hematopoietic stem cell transplantation (HSCT) in acute leukemia or myelodysplastic syndrome (MDS) patients. Prompt and sequential administration of escalating cell doses improves the efficacy of CIK cell therapy without exacerbating graft vs. host disease (GVHD). This study addresses manufacturing-related issues and aimed to develop a time-, personal- and cost-saving good manufacturing process (GMP)-compliant protocol for the generation of ready-for-use therapeutic CIK cell doses starting from one unstimulated donor-derived peripheral blood (PB) or leukocytapheresis (LP) products. Culture medium with or without the addition of either AB serum, fresh frozen plasma (FFP) or platelet lysate (PL) was used for culture. Fresh and cryopreserved CIK cells were compared regarding expansion rate, viability, phenotype, and ability to inhibit leukemia growth. Cell numbers increased by a median factor of 10-fold in the presence of FFP, PL, or AB serum, whereas cultivation in FFP/PL-free or AB serum-free medium failed to promote adequate CIK cell proliferation (p < 0.01) needed to provide clinical doses of 1 × 106 T cells/kG, 5 × 106 T cells/kG, 1 × 107 T cells/kG, and 1 × 108 T cells/kG recipient body weight. CIK cells consisting of T cells, T- natural killer (T-NK) cells and a minor fraction of NK cells were not significantly modified by different medium supplements. Moreover, neither cytotoxic potential against leukemic THP-1 cells nor cell activation shown by CD25 expression were significantly influenced. Moreover, overnight and long-term cryopreservation had no significant effect on the composition of CIK cells, their phenotype or cytotoxic potential. A viability of almost 93% (range: 89-96) and 89.3% (range: 84-94) was obtained after freeze-thawing procedure and long-term storage, respectively, whereas viability was 96% (range: 90-97) in fresh CIK cells. Altogether, GMP-complaint CIK cell generation from an unstimulated donor-derived PB or LP products was feasible. Introducing FFP, which is easily accessible, into CIK cell cultures was time- and cost-saving without loss of viability and potency in a 10-12 day batch culture. The feasibility of cryopreservation enabled storage and delivery of sequential highly effective ready-for-use CIK cell doses and therefore reduced the number of manufacturing cycles.

Generation and flow cytometric quality control of clinical-scale TCRαß/CD19-depleted grafts.

BACKGROUND: The depletion of TCRαß+ T cells and CD19+ B cells is a graft purification method for haploidentical stem cell transplantation (HSCT) retaining stem cells, NK cells and TCRγδ+ T cells. To avoid treatment-related occurrence of severe GvHD a precise quantification of residual TCRαß+ T cells in the graft is of essential importance. METHODS: Nine stem cell grafts were purified immunomagnetically on a CliniMACS device and flow cytometric quality control (QC) was performed before and after TCRαß/CD19-depletion. RESULTS: As a challenge a new 10-color QC-panel was established, which enables accurate quantification of the graft composition. The binding sites of residual TCRαß+ T and CD19+ B cells were at least partly occupied by depletion antibodies impeding flow cytometric analysis. Based on respective controls and an assumed variation coefficient of 18%, the detection limit of residual TCRαß T cells was 1 cell/µl. and 0.002% of CD45+ cells. Log-depletion of TCRαß and CD19 cells was -3.9 and -3.3, respectively. The recovery was 82.1%, 67.1% and 72.7% for stem cells, NK cells and for TCRγδ+ T cells. CONCLUSIONS: In clinical use this method may help to improve transplantation outcome, due to the correct application of the desired stem cell and the limited T cell dose. The panel is designed for the QC following TCRαß/CD19-depletion but is adaptable to other depletion strategies as well. © 2015 International Clinical Cytometry Society.

Cytotoxic potential of IL-15-activated cytokine-induced killer cells against human neuroblastoma cells.

BACKGROUND: Neuroblastoma (NB) is the most common solid extracranial tumor in childhood. Despite advances in therapy, the prognosis is poor and optimized therapies are urgently needed. Therefore, we investigated the antitumor potential of interleukin-15 (IL-15)-activated cytokine-induced killer (CIK) cells against different NB cell lines. PROCEDURE: CIK cells were generated from peripheral blood mononuclear cells by the stimulation with interferon-γ (IFN-γ), IL-2, OKT-3 and IL-15 over a period of 10-12 days. The cytotoxic activity against NB cells was analyzed by nonradioactive Europium release assay before and after blocking of different receptor-ligand interactions relevant in CIK cell-mediated cytotoxicity. RESULTS: The final CIK cell products consisted in median of 83% (range: 75.9-91.9%) CD3+ CD56- T cells, 14% (range: 5.2-20.7%) CD3+ CD56+ NK-like T cells and 2% (range: 0.9-4.8%) CD3- CD56+ NK cells. CIK cells expanded significantly upon ex vivo stimulation with median rates of 22.3-fold for T cells, 58.3-fold for NK-like T cells and 2.5-fold for NK cells. Interestingly, CD25 surface expression increased from less than equal to 1% up to median 79.7%. Cytotoxic activity of CIK cells against NB cells was in median 34.7, 25.9 and 34.8% against the cell lines UKF-NB-3, UKF-NB-4 and SK-N-SH, respectively. In comparison with IL-2-stimulated NK cells, CIK cells showed a significantly higher cytotoxicity. Antibody-mediated blocking of the receptors NKG2D, TRAIL, FasL, DNAM-1, NKp30 and lymphocyte function-associated antigen-1 (LFA-1) significantly reduced lytic activity, indicating that diverse cytotoxic mechanisms might be involved in CIK cell-mediated NB killing. CONCLUSIONS: Unlike the mechanism reported in other malignancies, NKG2D-mediated cytotoxicity does not constitute the major killing mechanism of CIK cells against NB.

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 × 10(8) 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.

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 CD56CD3 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 CD16 and CD16 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-gamma, tumor necrosis factor-beta, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta) was observed after IL-2 stimulation compared with freshly isolated NK cells. However, NK cells of donors showing an initially enhanced cytotoxicity combined with NCR 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 CD56-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.