CD56 Enrichment Research Articles

Automated and closed clinical-grade manufacturing protocol produces potent NK cells against neuroblastoma cells and AML blasts

Natural killer (NK) cells are a promising allogeneic immunotherapy option due to their natural ability to kill tumor cells, and due to their apparent safety. This study describes the development of a GMP-compliant manufacturing protocol for the local production of functionally potent NK cells tailored for high-risk acute myeloid leukemia (AML) and neuroblastoma (NBL) patients. Moreover, the quality control strategy and considerations for product batch specifications in early clinical development are described. The protocol is based on the CliniMACS Prodigy platform and Natural Killer Cell Transduction (NKCT) (Miltenyi Biotec). NK cells are isolated from leukapheresis through CD3 depletion and CD56 enrichment, followed by a 12-hour activation with IL-2 and IL-15 cytokines. Three CliniMACS Prodigy processes demonstrated the feasibility and consistency of the modified NKCT process. A three-step process without expansion, however, compromised the NK cell yield. T cells were depleted effectively, indicating excellent safety of the product. Characterization of the NK cells before and after cytokine activation revealed a notable increase in the expression of activation markers, particularly CD69, consistent with enhanced functionality. Intriguingly, the NK cells exhibited increased killing efficacy against patient-derived CD33 + AML blasts and NBL cells in vitro, suggesting a potential therapeutic benefit in AML and NBL.

Cytokines impact natural killer cell phenotype and functionality against glioblastoma in vitro.

Natural killer (NK) cells are a part of the innate immune system and first-line defense against cancer. Since they possess natural mechanisms to recognize and kill tumor cells, NK cells are considered as a potential option for an off-the-shelf allogeneic cell-based immunotherapy. Here, our objective was to identify the optimal cytokine-based, feeder-free, activation and expansion protocol for cytotoxic NK cells against glioblastoma in vitro. NK cells were enriched from human peripheral blood and expanded for 16 days with different activation and cytokine combinations. The expansion conditions were evaluated based on NK cell viability, functionality, expansion rate and purity. The cytotoxicity and degranulation of the expanded NK cells were measured in vitro from co‑cultures with the glioma cell lines U‑87 MG, U‑87 MG EGFR vIII, LN-229, U-118 and DK-MG. The best expansion protocols were selected from ultimately 39 different conditions: three magnetic cell‑selection steps (Depletion of CD3+ cells, enrichment of CD56+ cells, and depletion of CD3+ cells followed by enrichment of CD56+ cells); four activation protocols (continuous, pre-activation, re-activation, and boost); and four cytokine combinations (IL-2/15, IL‑21/15, IL‑27/18/15 and IL-12/18/15). The expansion rates varied between 2-50-fold, depending on the donor and the expansion conditions. The best expansion rate and purity were gained with sequential selection (Depletion of CD3+ cells and enrichment of CD56+ cells) from the starting material and pre-activation with IL‑12/18/15 cytokines, which are known to produce cytokine-induced memory-like NK cells. The cytotoxicity of these memory-like NK cells was enhanced with re-activation, diminishing the donor variation. The most cytotoxic NK cells were produced when cells were boosted at the end of the expansion with IL-12/18/15 or IL-21/15. According to our findings the ex vivo proliferation capacity and functionality of NK cells is affected by multiple factors, such as the donor, composition of starting material, cytokine combination and the activation protocol. The cytokines modified the NK cells' phenotype and functionality, which was evident in their reactivity against the glioma cell lines. To our knowledge, this is the first comprehensive comparative study performed to thisextent, and these findings could be used for upscaling clinical NK cellmanufacturing.

Rapid production of clinical-grade SARS-CoV-2 specific T cells.

ObjectivesTo determine whether the frequencies of SARS‐CoV‐2‐specific T cells are sufficiently high in the blood of convalescent donors and whether it is technically feasible to manufacture clinical‐grade products overnight for T‐cell therapy and assessment of COVID‐19 immunity.MethodsOne unit of whole blood or leukapheresis was collected from each donor following standard blood bank practices. The leukocytes were stimulated using overlapping peptides of SARS‐CoV‐2, covering the immunodominant sequence domains of the S protein and the complete sequence of the N and M proteins. Thereafter, functionally reactive cells were enriched overnight using an automated device capturing IFNγ‐secreting cells.ResultsFrom 1 × 109 leukocytes, a median of 0.98 × 106 (range 0.56‐2.95) IFNγ + T cells were produced from each of the six donors, suggesting a high frequency of SARS‐CoV‐2 reactive T cells in their blood, even though only one donor had severe COVID‐19 requiring mechanical ventilation whereas the other five donors had minor symptoms. A median of 57% of the enriched T cells were IFNγ+ (range 20%‐74%), with preferential enrichment of CD56+ T cells and effector memory T cells. TCRVβ‐spectratyping confirmed distinctively tall oligoclonal peaks in final products. With just six donors, the probability that a recipient would share at least one HLA allele with one of the donors is >88% among Caucasian, >95% among Chinese, >97% among Malay, and >99% among Indian populations.ConclusionsHigh frequencies of rapid antigen‐reactive T cells were found in convalescent donors, regardless of severity of COVID‐19. The feasibility of clinical‐grade production of SARS‐CoV‐2‐specific T cells overnight for therapeutics and diagnostics is revealed.

Fully automated integrated process for depletion of T cells with or without subsequent purification of NK cells from apheresis using the CliniMACS Prodigy

Background & Aim Donor-derived natural killer (NK) cells mediate graft-versus-leukemia effects low risk in inducing alloreactive graft-versus-host-disease and are used as component of T cell-depleted donor lymphocyte infusion (DLI) or purified. As clinical grade purification of NK cells requires two semi-automated separation procedures, including multiple manual hands-on steps, we developed an integrated fully automated process enabling either preparation of T cell-depleted DLI or clinical grade purification of NK cells within a single automated process on the CliniMACS Prodigy. Methods, Results & Conclusion The novel integrated CliniMACS Prodigy LP-3-56 System uses the CliniMACS Prodigy Tubing Set 320 for an fully automated procedure with integrated labeling and washing steps for both, CD3 and CD56 separation enabled through an integrated lytic rinsing sequence allowing subsequent CD56 enrichment in the same tubing set. By users choice the depletion of CD3+ cells will be performed with or without subsequent enrichment of CD56+ cells. For both strategies target cells are prepared with user-chosen formulation solution (e. g. sodium chloride or NK MACS medium). The new application software effectively depletes 9.6 × 109 CD3+ cells, and enriches 4.5 × 109 CD56+ cells from up to 40 × 109 total white blood cells. Inhouse evaluation runs with leukapheresis products (n= 29 in total, n=17 with CD3 depletion, n=12 with CD3 depletion and CD56 enrichment) were performed. After CD3 depletion, mean log depletion of T cells was 4.0. The mean recovery of NK cells was 89% with an average NK:T ratio-increase of 9509 fold. After CD3 depletion and CD56 enrichment, mean log depletion of T and B cells was 4.2 and 2.7, respectively. Purity of NK cells was 90.7% on average with a mean recovery of 49%. The majority of non-NK cells after the process were monocytes with a mean frequency of 5.9%. Functionality of purified NK cells was proven by their ability to lyse AML cells, to secrete cytokines after co-culture, and to perform antibody dependent cell-mediated cytotoxicity (ADCC) against Raji cells, using human CD20 antibody. In summary, the integrated fully automated CliniMACS Prodigy LP-3-56 System is capable of efficiently deplete CD3+ cells with or without subsequent CD56 enrichment to purify functional NK cells from leukapheresis products and provides target cells in formulation solution. The performance verification and submission to an European notified body for CE mark are the important next steps.

GMP-compliant, automated process for generation of CAR NK cells in a closed system for clinical use

Background & Aim Major advances have been made in harnessing natural killer (NK) cells in cancer immunotherapy in recent years. Regulated by their germ-line encoded activating and inhibitory receptors, NK cells can recognize and eliminate tumor cells rapidly without prior sensitization. Clinical evidence has shown that donor-derived NK cells have low risk in inducing graft-versus-host-disease (GvHD) as well as a reduced risk of life-threatening cytokine storms during therapy. Both properties make NK cells ideal for allogeneic application. To enhance the anti-tumor specificity and efficiency, NK cells can be further modified by chimeric antigen receptors (CARs). Promising clinical outcome of CAR-engineered NK cells has been recently reported for patients with relapsed or refractory B cell malignancies. Methods, Results & Conclusion In this study purified NK cells were cultivated with a feeder cell-free protocol including cytokines in NK MACS GMP Medium. For generation of CAR NK cells we used a Baboon endogenous virus (BaEV)-pseudotyped lentiviral vector system to efficiently transduce NK cells with various CARs and routinely obtained transduction efficiencies between 30-70%. To facilitate the clinical application of CAR NK cells, we implemented this protocol into a highly efficient, automated process to generate CAR NK cells under good manufacturing practice (GMP)-compliant conditions in a closed system by using CliniMACS Prodigy device. The process covers the complete procedure of NK cell manufacturing, including separation, activation, gene modification, and expansion/cultivation. High purity of NK cells (mean 94%) could be achieved by CD3 depletion followed by subsequent CD56 enrichment, resulting in a mean log depletion of T cells of 4.3. Inhouse evaluation runs with leukapheresis products (n=11) achieved a mean transduction efficiency of 52.0% CAR+ cells, resulting in 5.2 – 17.9 x 108 (mean 7.17 x 108) CAR+ NK cells after 14 days of culture. In summary, we developed a novel process for automated NK cell purification, transduction, and cultivation in a closed GMP-compatible system. The high level of automation enables standardized, consistent, and operator independent genetic engineering of NK cells for therapeutic applications.

Optimization of Human NK Cell Manufacturing: Fully Automated Separation, Improved Ex Vivo Expansion Using IL-21 with Autologous Feeder Cells, and Generation of Anti-CD123-CAR-Expressing Effector Cells.

The administration of ex vivo expanded natural killer (NK) cells as potential antitumor effector cells appears to be suitable for effector cell-based immunotherapies in high-risk cancer patients. However, good manufacturing practice (GMP)-compliant manufacturing of clinical-grade NK cells at sufficiently high numbers represents a great challenge. Therefore, previous expansion protocols for those effector cells were improved and optimized by using newly developed culture medium, interleukin (IL)-21, and autologous feeder cells (FCs). Separation of primary human NK cells (CD56+CD3-) was carried out with the CliniMACS Prodigy® in a single process, starting with approximately 1.2 × 109 leukocytes collected by small-scale lymphapheresis or from buffy coats. Enriched NK cells were adjusted to starting cell concentrations within approximately 1 × 106 effector cells/mL and cultured in comparative expansion experiments for 14 days with IL-2 (1,000 IU/mL) in different GMP-compliant media (X-VIVO™10, CellGro®, TexMACS™, and NK MACS®). After medium optimization, beneficial effects for functionality and phenotype were investigated at the beginning of cell expansion with irradiated (25 Gy) autologous FCs at a ratio of 20:1 (feeder: NK) in the presence or absence of IL-21 (100 ng/mL). Additionally, expanded NK cells were gene modified to express chimeric antigen receptors (CARs) against CD123, a common marker for acute myeloid leukemia (AML). Cytotoxicity, degranulation, and cytokine release of transduced NK cells were determined against KG1a cells in flow cytometric analysis and fluorescent imaging. The Prodigy manufacturing process revealed high target cell viabilities (median 95.4%), adequate NK cell recovery (median 60.4%), and purity of 95.4% in regard to CD56+CD3- target cells. The process in its early phase of development led to a median T-cell depletion of log 3.5 after CD3 depletion and log 3.6 after the whole process, including CD3 depletion and CD56 enrichment steps. Manually performed experiments to test different culture media demonstrated significantly higher NK cell expansion rates and an approximately equal distribution of CD56dimCD16pos and CD56brightCD16dim&neg NK subsets on day 14 with cells cultivated in NK MACS® media. Moreover, effector cell expansion in manually performed experiments with NK MACS® containing IL-2 and irradiated autologous FCs and IL-21, both added at the initiation of the culture, induced an 85-fold NK cell expansion. Compared to freshly isolated NK cells, expanded NK cells expressed significantly higher levels of NKp30, NKp44, NKG2D, TRAIL, FasL, CD69, and CD137, and showed comparable cell viabilities and killing/degranulation activities against tumor and leukemic cell lines in vitro. NK cells used for CAR transduction showed the highest anti-CD123 CAR expression on day 3 after gene modification. These anti-CD123 CAR-engineered NK cells demonstrated improved cytotoxicity against the CD123pos AML cell line KG1a and primary AML blasts. In addition, CAR NK cells showed higher degranulation and enhanced secretion of tumor necrosis factor alpha, interferon gamma, and granzyme A and B. In fluorescence imaging, specific interactions that initiated apoptotic processes in the AML target cells were detected between CAR NK cells and KG1a. After the fully automated NK cell separation process on Prodigy, a new NK cell expansion protocol was generated that resulted in high numbers of NK cells with potent antitumor activity, which could be modified efficiently by novel third-generation, alpha-retroviral SIN vector constructs. Next steps are the integration of the manual expansion procedure in the fully integrated platform for a standardized GMP-compliant overall process in this closed system that also may include gene modification of NK cells to optimize target-specific antitumor activity.

A Novel Tool Using Marrow Natural Killer (NK) Cell Percentages to Predict Collection Requirements of Peripheral Blood Mononuclear Cell (MNC), Apheresis Products Used in NK Cell Adoptive Immunotherapy

▪Our institution is involved in two clinical trials that require a single infusion of non-expanded Natural Killer (NK) cells in recipients of haploidentical marrow transplants. NK cells are selected from non-mobilized Mononuclear cells (MNC), apheresis products collected 6 days after HPC, Marrow harvest then processed and infused the following day. The two-step processing procedure requires initial depletion of CD3+ T cells followed by enrichment of CD56+ NK cells. Results from 27 patients treated thus far show an average CD3-CD56+ NK cell recovery of 54.5 ± 8.3% with 5.46 ± 0.74 log depletion of CD3+CD56- T cells. The NK cell dose level target is now 5.0 x 106 NK cells/kg and is limited to ≤1.0 x 104 T cells/kg in these mixed pediatric and adult patients. For 21 of 26 patients (80.7%) whose adult donors underwent a standard 2-blood volume (BV) apheresis procedure, a dose level >5.0 x 106 NK cells/kg was achievable. Processing a larger BV would likely improve the percentage achieving the desired target dose but would also increase the likelihood of adverse reactions, particularly citrate toxicity. It is believed that mature lymphocytes and monocytes in marrow harvested for clinical transplant come primarily from contaminating peripheral blood. Given this, we hypothesized that NK cell content in marrow MNCs would reflect NK cell content within the apheresis product and allow us to predict when a larger BV collection is needed. Immunophenotype data from 18 donors of both marrow and MNC, Apheresis products was evaluated to determine the correlation of the percentage of NK cells within the Marrow MNC population with that within the apheresis products. The % of NK cells within the MNC population was quite variable for marrow (6.1 ±3.7%, range 2.4 to 17.7%) and apheresis products (7.3± 4.6%, range 2.4 to 26.1%) but were highly correlated for individual donors (R2=0.85, p <0.0001). Based on this data, we developed a prediction workform tool requiring 3 data points from the marrow immunophenotype: 1) % of marrow lymphocytes (defined by flow cytometry as CD45bright, CD14-), 2) the % of marrow monocytes (defined as CD45bright, CD14+), and 3) the % of CD3-CD56+ NK cells within the marrow lymphocyte gate. These data were used to calculate NK cell content in the marrow MNC fraction. That data together with the desired NK cell dose/kg, patient weight, and average post-processing NK recovery (54.5%) was used to calculate the number of MNC cells required in the apheresis product to meet target NK cell doses. The BV to be processed was then estimated using the % MNC in the peripheral blood at marrow harvest and the known MNC collection efficiency of the device used (a conservative estimate of ~35% MNC collection efficiency was used). The prediction workform indicated that additional BV needed to be processed for one of the 3 donors for whom it was used indicated. This resulted in sufficient NK cells to meet the target infusion dose. Calcium infusion was used prophylactically to prevent citrate toxicity. Similar information could be obtained using a peripheral blood sample to determine NK cell content the day of collection. However, having information in advance from the marrow harvest allows for planning on the part of the donor and the collection team when additional collection time is needed. In conclusion, when available data from a marrow harvest performed prior to MNC, Apheresis is a useful tool to estimate the BVs needed to be processed meet infusion cell target dose. DisclosuresNo relevant conflicts of interest to declare.

Characteristics of products for NK-cell infusion prepared by CD3 depletion followed by CD56 enrichment: requirements for rare event analysis

functionality we re-incubated 3-days rested cells with antigens. Induction of IFN-g in up to 100% of T cells was observed demonstrating cells maintained their in vivo imprinted physiological role. We also monitored CD45RA, CD28, and CCR7 expression on pp65-peptide specific CD8+ T cells before and directly after the enrichment. The enrichment process did not induce phenotypic changes. Thus, the newly developed automated manufacturing process provides a product, where the original phenotypic and functional characteristics of virus-specific T cells are conserved. Hence this cellular product is expected to fight viral infections effectively upon adoptive transfer.

Immunomagnetic selection or irradiation eliminates alloreactive cells but also reduces anti-tumor potential of cytokine-induced killer cells: implications for unmanipulated cytokine-induced killer cell infusion

Background aimsCytokine-induced killer (CIK) cells may offer a novel therapeutic approach for patients with malignancies relapsing after allogeneic stem cell transplantation. Although CIK cells display negligible alloreactivity and cause minimal graft versus-host-disease (GVHD), high CIK cell doses required during relapse may pose a risk for severe GVHD, specifically in the mismatched or haploidentical transplantation setting. Manipulation of CIK cells may reduce risk for GVHD without affecting the anti-tumor potential. MethodsIn this pre-clinical study, we provide a detailed functional comparison of conventional and irradiated, CD56-enriched or T-cell receptor α/β-depleted CIK cells. ResultsIn vitro analysis showed retained anti-leukemic and anti-tumor potential after CIK cell manipulation. Even being sequentially infused into immunodeficient mice grafted with malignant cells, cytotoxic effects were fewest after irradiation but were improved by CD56 enrichment and were best with conventional CIK cells. Hence, considering the proliferative capacity of inoculated malignancies and effector cells, a single dose of conventional CIK cells resulted in prolonged disease-free survival and elimination of rhabdomyosarcoma cells, whereas sequential infusions were needed to achieve comparable results in leukemia-bearing mice. However, this mouse model has limitations: highly effective conventional CIK cells demonstrated both limited xenogenic GVHD and low alloreactive potential in vitro. ConclusionsOur study revealed that conventional CIK cells demonstrate no significant alloreactive potential but provide the strongest anti-tumor efficacy compared with manipulated CIK cells. Conventional CIK cells may therefore be tested in high numbers and short-term intervals in patients with impending relapse even after mismatched transplantation.

CliniMACS® Cell Enrichment Using NKp46 — A Large Scale, Single-Step NK Cell Isolation Method.

Natural Killer (NK) cells show potent anti-leukemic activity in vitro and thus NK cells isolated using the CliniMACS® Cell Separation System are currently under clinical evaluation as part of a treatment for acute myeloid leukemia (AML). (Uharek L et al., 2003, Gentilini C et al., 2005; Koehl U et al., 2004, 2005; Passweg, JR et al., 2005, 2006; Miller J et al., 2005). Currently, the processing of highly purified, clinical-grade NK cells requires a two-step method using the CliniMACS Instrument: Depletion of CD3+ cells followed by enrichment of CD56+ cells (Iyengar R et al., 2003). This procedure shows excellent performance (reviewed in Passweg et al., 2005) with yields of 30–60%, purities of >85%, > 99.97%/3.5 log T cell depletion but is time consuming. Simpler one-step procedures have thus been tried to reduce the processing time (CD3 depletion, CD3/CD19 depletion, CD6 depletion, CD56 enrichment) but result in a more heterogeneous final cell product that usually contains less than 60% NK cells. NKp46 (CD335) has been demonstrated to be primarily expressed by NK cells and may thus be a suitable selection marker for one-step NK cell enrichment. We evaluated an NKp46 enrichment strategy using the autoMACS™ Pro Separator, NKp46-Biotin, and Anti-Biotin MicroBeads. We could enrich CD56+CD3- cells from peripheral blood mononuclear cells (PBMCs) to a purity of 90% with a yield of 40–60% and a >99.92%/3.1 log depletion of T cells (n = 2). These results encouraged the evaluation of this procedure on a clinical scale by using the CliniMACS Cell Separation System. Using the CliniMACS Plus Instrument, NK cells could be enriched to 73–87% purity (n = 3) from Buffy Coats derived from whole blood. T cells were efficiently depleted (>99.92%/3.0 log) to such a degree that NKp46-selected NK cell products may fulfil the release criteria for NK cell donor lymphocyte infusions (NK-DLI) in the context of haploidentical stem cell transplantation. While CD56+ CD3- NK cells are not activated by antibody binding during cell separation, magnetic labelling using anti-NKp46 antibodies may trigger NK cell activation. NKp46-mediated NK cell activation may be synergistically enhanced by the use of IL-2. Functional studies of NK cells isolated by different methods will be required to further evaluate their feasibility for use in NK-DLIs.

High Numbers of NK Cell Clones Can Be Generated by Limiting Dilution in SCGM Medium Supplemented with IL-2, IL-15 and Anti-NKp46 Antibody 9E2.

NK cell clones obtained by limiting dilution have frequently been used for functional studies and as an analytical tool e.g. for assessment of alloreactivity in the context of haploidentical stem cell transplantation [Velardi et al., Blood 2007]. NK cell clones can be used in co-culture experiments with allogeneic target cells to determine the number of alloreactive clones and therefore to quantify NK cell alloreactivity. This method provides additional quantitative data, while PCR based methods for HLA and KIR typing only provide qualitative results of potential alloreactivity. Generation of NK cell clones usually is difficult and cloning efficiency is low (< 5% [Yssel et al., J. Immunol. Methods 1984]). Protocols have been developed to increase cloning efficiency [Wang et al., J. Immunol. Methods 2005] We evaluated whether cloning efficiency can be further increased by optimization of cell processing prior to culture and/or of medium components. Published reference methods use media supplemented with Interleukin-2 (IL-2). These conditions also stimulate proliferation of T cells, which usually overgrow NK cells in culture. Thus depletion of T cells prior to incubation with IL-2 may facilitate identifation of proliferating NK cells. T cells can be actively depleted upon CD3 magnetic labelling, by use of an NK cell isolation kit or passively by CD56 enrichment. CD 56 enrichment efficiently depletes CD3+CD56- T cells and residual CD3+CD56+ NKT cells do not expand under the chosen culture conditions. IL-15 is capable of preventing NK cell apoptosis at low concentrations [Caligiuri et al., J. Clin. Invest. 1997; Carson et al., J Clin Invest. 1997; Puzanov et al., J Immunol. 1996)] and plays a role in proliferation and survival of NK cells. Cross linking of NKp46 leads to NK cell activation [Long et al., Blood 2005]. As simultaneous engagement of two independent activating NK cell receptors (IL-2/IL-15 receptors and NKp46) may enhance NK cell activation and proliferation, we evaluated different combinations of IL-2, IL-15 and the anti-NKp46 antibody 9E2. Optimal results were obtained when CD56 selected cells were used (compared to CD3 depletion, NK cell enrichment, KIR2D selection). Supplementation of SCGM medium (Cell Genix) with IL-2 (500u/mL), IL-15 (10ng/mL) and anti NKp46 antibody (2μg/mL, CD335, clone 9E2) increased the number of NK cell clones by a factor of 2.3–2.5 (n=3) compared to medium and IL-2 alone. 37 NK cell clones with yields of > 2E6 cells per clone have been generated using this optimised method and will now be used for Killer Immunoglobulin-like receptor mRNA analyses and antibody based KIR phenotyping (CD158a/h, CD158b, CD158e, CD158i, anti KIR2D). KIR analysed NK cell clones may be used to generate novel highly specific anti KIR antibodies for flowcytometric, quantitative KIR phenotyping. Such a panel can be helpful for optimization of donor selection for allogeneic stem cell transplantation.

Phase I-II Trial of Adoptive Immunotherapy with Haploidentical KIR Ligand-Mismatched Natural Killer Cells in High Risk Acute Myeloblastic Leukemia Patients.

Natural killer (NK) cells play a critical role in innate immunity and are relevant for the immune control of tumors. Their effector function is regulated by a number of activating and inhibitory receptors, termed killer immunoglobulin receptors (KIRs). In haploidentical T-cell depleted transplantation the donor/recipient KIR mismatch significantly impacts on tumor cell killing, particularly in acute myeloid leukemia (AML). Therefore, haploidentical KIR-mismatched NK cells may be used as adoptive immunotherapy for high risk AML patients in non-transplantation settings. Thirty-one high risk AML patients entered a phase I-II study of NK-cell based immunotherapy and were screened for the avalilability of one haploidentical KIR ligand mismatched donor. Ten of them resulted as having one suitable donor. NK cells were enriched from steady-state leukaphereses by using a double-step immunomagnetic separation system (Miltenyi Biotec, Germany), consisting in1.depletion of CD3+ T cells followed by2.positive selection of CD56+ NK cells, and then cryopreserved.CD56+ NK cells were enriched from 8.28±3.7% to 85.9±3.2% (recovery 67.9±15%, viability >92%). So far, 8 patients (1 partial remission, 4 progressions and 3 complete remissions) received NK cell infusion which was preceeded by immunosuppressive chemotherapy, including fludarabine 125 mg/m2 and cyclophosphamide 4g/m2. AML patients received 6 subcutaneous injections of interleukin 2 (IL-2) after adoptive immunotherapy. The median number of reinfused NK cells was 3.6 × 106/Kg and contaminating CD3+ T cells were always less than 1×105/Kg. NK cells were capable to kill NK-sensitive K562 cells in a flow-cytometry-based cytotoxicity assay. The procedure was well-tolerated and no significant toxicity, including GVHD, related to NK cell infusion was observed. The patient in partial remission obtained a complete remission, which lasted for 6 months; among the 4 patients with active/progressive disease 2 patients showed the persistence of disease and one patient died during the aplastic phase. The 3 patients in complete remission are stable after 5, 3 and 4 months of follow up. By using a RT-PCR-based chimerism assay, we demonstrated the presence of adoptively transferred NK cells in 2/4 patients with a peak in circulating NK cell number at day 12 after infusion. Functional biological studies demonstrated the presence of alloreactive NK cell clones in all the donors (at the frequencies of 11 ± 4%) and in 3/4 patients (at the frequences of 16 ± 5%) only at day 3 after infusion of NK cells. In summary, a two-step enrichment of CD56+ NK cells allows the collection of a suitable number of target cells to be used as adoptive immunotherapy in AML patients. Infusion of cryopreserved NK cells is feasible and safe and adoptively transferred NK cells can be detected in the peripheral blood after infusion.

IL-2 activated NK cell immunotherapy of three children after haploidentical stem cell transplantation

Natural killer (NK) cells are thought to be of benefit in HLA-mismatched hematopoietic transplantation (H-SCT). Therefore, we developed a protocol for clinical-use expansion of highly enriched and IL-2-stimulated NK cells. Purification of unstimulated leukaphereses by a two-step T cell depletion with a final CD56 enrichment procedure leads to a mean purity of 95% CD56 +CD3 − NK cells with a four- to five-log depletion of T cells. So far, three pediatric patients with multiply relapsed acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML) were treated with repeated transfusions post-H-SCT. Directed killer immunoglobulin-like receptor (KIR) mismatches were demonstrated in all three cases. Although all patients showed blast persistence at the time of transplant, they reached complete remission and complete donor chimerism within 1 month post-H-SCT. NK cell therapy was tolerated well without graft-versus-host disease (GvHD) induction or other adverse events. The AML patient died of early relapse on day +80, while the ALL patients died of thrombotic-thrombocytopenic purpura and atypical viral pneumonia on days +45 and +152, respectively. This initial trial showed the feasibility of good manufacturing practice (GMP)-compliant NK cell isolation and expansion for clinical applications. We now launch a clinical phase I trial with activated NK cells post-H-SCT.