Repeated infusions of escalating doses of expanded and activated autologous natural killer cells in minimal residual disease‐positive Ph+ acute lymphoblastic leukemia patients. A GIMEMA phase 1 trial
Authors: Giovanni Fernando Torelli, Sabina Chiaretti, Nadia Peragine, Walter Barberi, Laura Santodonato, Giuseppina D'Agostino, Elisabetta Abruzzese, Maria Ilaria Del Principe, Alessandra Mancino, Mabel Matarazzo, Mahnaz Shafii Bafti, Marco Mancini, Monica Messina, Luciano Castiello, Anna Guarini, Robin Foà
Journal: American Journal of Hematology (2022)

To the Editor: The prognosis of Philadelphia (Ph)+ acute lymphoblastic leukemia (ALL) patients has significantly improved with the advent of tyrosine kinase inhibitors (TKIs).1 TKIs now represent the gold standard frontline treatment for these patients,2 in combination3 or not4 with systemic chemotherapy, followed by a consolidation contemplating high-dose chemotherapy and, at least for patients who remain minimal residual disease (MRD)-positive, allogeneic transplant. Due to age and comorbidities, many patients are ineligible to receive these treatments, and there is an urgent need to develop less toxic but effective protocols. In this respect, immunotherapy appears an excellent alternative on consolidation chemotherapy. Our group has recently reported the results on the chemo-free Scheme D-ALBA5 based on dasatinib plus steroids in induction followed by blinatumomab as consolidation for patients of all ages. This strategy was associated with a high rate of MRD negativity (60%) and with overall and disease-free survival rates of 95% and 88% at 18 months, respectively. In addition to the extremely promising results, the D-ALBA trial underlined the potential of immunotherapy for Ph+ ALL patients, documented by a marked in vivo modulation of the host immune system induced by blinatumomab.6 Considerable interest has been raised over the years by natural killer (NK) cells.7 Our group reported the significantly increased expression in Ph+ ALL blast cells of some of the ligands recognized by the activating receptors expressed on the NK cell surface,8 which correlates with the degree of susceptibility to lysis by expanded NK cells. We developed a good manufacturing practice (GMP) compliant-protocol for the ex vivo expansion of autologous NK cells in the presence of autologous plasma, interleukin (IL)-2, IL-15, and irradiated autologous feeder cells9 to be used for clinical studies. Hereby, we report the results of the GIMEMA LAL2013 trial (EudraCT 2013-002547-29, ClinicalTrials.gov Identifier: NCT02185781), an open-label, multicenter, phase 1 study designed to assess the feasibility of an adoptive immunotherapy strategy with enriched and expanded autologous NK cells for patients with Ph+ ALL in complete hematologic remission (CHR) but with persistent/recurrent MRD, ≥ 60 years or not eligible for other post-CHR treatments (namely chemotherapy and/or allogeneic transplant). The primary endpoint of the study was to determine the maximum tolerated dose and the recommended dose for subsequent investigations. Secondary endpoints included the assessment of the safety and tolerability of the treatment, the immunologic modifications induced by the procedure, and the clinical response to treatment in terms of MRD control by quantitative real-time polymerase chain reaction (qRT-PCR). The trial was designed with a sample size of 6 patients. Inclusion and exclusion criteria for patients' enrolment are summarized in Table S1. The protocol was approved by the Ethical Committee of the Sapienza University of Rome, Italy, and by the Agenzia Italiana del Farmaco (AIFA). Patients underwent one or more procedure(s) of unstimulated leukapheresis at the Blood Bank of the Policlinico Umberto I, Sapienza University of Rome. Following apheresis, cells were processed in the GMP facility FaBioCell at the Istituto Superiore di Sanità of Rome under clean room conditions. Sequential CD3+ cell depletion and CD56+ NK cells-positive selection were performed using the Miltenyi CliniMACS apparatus, approved according to the European conformity requirements (CE-mark class 1). Enriched NK cells were co-cultured for 14 days at 37°C with autologous irradiated mononuclear feeder cells (mononuclear cell:NK cell ratio = 2.5:1) in CellGro® SCGM serum-free medium (CellGenix, Freiburg, Germany) supplemented with 5% autologous plasma, 500 U/mL IL-2 (Aldesleuchina, Proleukin®, kindly provided by Novartis Farma S.p.A., Varese, Italy) and 50 ng/mL IL-15 (CellGro®, CellGenix). Equal amounts of IL-2 and IL-15 were also added to the culture medium during the last 24 h of the expansion period. After 14 days of culture, cells were harvested, counted, and resuspended in a freezing medium at the final concentration of 20 × 106 cells/ml.9 NK cells presented a 12.3 mean fold expansion. At the end of the expansion, the immunophenotypic analysis showed an increase in the percentage of NK cells expressing Nkp30, Nkp44, and Nkp46 activating receptors (Figure S1A) and an overall upregulation of all activating receptors (Figure S1B). Expanded NK cells also displayed measurable cytotoxicity against K562 cells (Figure S2A) and primary blasts, both in the allogeneic and autologous setting (Figure S2B). The 6 patients enrolled in the trial underwent repeated intravenous infusions (a maximum of 5 infusions was allowed) of escalating doses of expanded autologous NK cells, at a cell dose ranging from 1 × 106/kg to a maximum of 50 × 106/kg of body weight (BW). Patients were allowed to continue TKI maintenance. The patients' characteristics are reported in Table S2. Five patients received the NK cell infusion in 1st CHR, while 1 patient was in 2nd CHR. Three patients received 5 NK cell infusions, 2 patients received 4 infusions, and 1 patient received 2 infusions (Table S3). For 3 patients, the maximum administered NK cell dose was 5 × 107 cells/kg of BW, for 2 patients 1 × 107 cells/kg of BW, for 1 patient (the one who received only 2 infusions) 5 × 106 cells/kg of BW. The median time between subsequent infusions was 28 days (range 28–294). No patient experienced infusion-related toxicities. Two adverse events (AEs) were recorded, both judged not serious (grade 1 and 2) and resolved by stopping the TKI (imatinib). Both AEs were not related to study treatment. Immunophenotypic analyses of peripheral blood cells performed one week after the infusion of expanded NK cells revealed a persistent significant increase in the expression of the activation markers Nkp30 and Nkp44 after the infusion of the highest NK dose (5 × 107/kg) (Figure 1A). The infusion of higher doses of NK cells was also associated with a significantly higher TNFα production by NK and T cells (Figure 1B) and a higher IFN-γ production by T cells (Figure 1C). The trend of cytokine production by the different lymphocyte subsets (NK, T4, T8, and NKT) one week after the infusion of expanded NK cells is shown in Figure S3 and confirms the general increased production by all lymphocyte subsets with the infusion of increasing doses of NK cells. In 2 cases, the increased cytokine production capability was also observed over time from the last infusion: Figure S4 reports the TNF-α production recorded 2.2 years after the last infusion in patient 1 (A) and the IFN-γ production 1.8 years after the last infusion in patient 5 (B). The increased expression of the activation markers Nkp30 and Nkp44 and the increased cytokine production capacity recorded one week after the infusion of the highest doses of NK cells paralleled the significant increased cytotoxic capacity of peripheral blood mononuclear cells against the K562 cell line (Figure 1D). Data on the molecular and clinical monitoring of enrolled patients are reported in Figure 1E. All patients but 1 presented a progressive reduction of the MRD levels during treatment. One patient reached a complete molecular response (CMR), 3 patients became positive not-quantifiable (PNQ), while the patient who received 2 infusions of NK cells presented a 1 log MRD reduction. On the contrary, the only patient who was enrolled in 2nd CHR showed an increase in MRD followed by a hematologic relapse. The reduction of the MRD level of the 5 patients who received the NK cell treatment in 1st CHR persisted over time; at a median follow-up of 30.8 months from the last infusion, 2 patients are in CMR, and 3 are PNQ; 1 patient (PNQ) subsequently experienced an increase in MRD and required additional treatment (inotuzumab and blinatumomab) after NK cell adoptive immunotherapy. The treatment planned in the study design was completed by 5/6 patients; the absence of serious AEs and the feasibility of the program lead to recommend the dose of 5 × 107 NK cells/Kg of recipient BW for future studies. In conclusion, this phase 1 study demonstrates that the infusion of ex vivo expanded autologous NK cells is safe and represents a tolerable and feasible model of immunotherapy that can be employed as consolidation treatment in Ph+ ALL adult patients in CHR who display a positive MRD and who are ≥60 years or not eligible for other consolidation treatments. The protocol for NK cell manipulation has proven capable of generating highly pure effector cells that can effectively proliferate and exert cytotoxic and secretory functions. The results of this study show that the infusion of the higher NK cell doses leads to a greater NK cell activity and host immune modulation. Some patients remain in CMR or PNQ after a long observation time; this leads to hypothesize that the immune system of these patients, when properly educated, may be able to maintain a prolonged control of the disease even if persistently detectable at the molecular level. This study represents the basis for further investigations, which are needed to substantiate this clinical program and to perform the necessary clinico-biologic correlations. The authors declare that there is no conflict of interest. The protocol was approved by the Ethical Committee of the Sapienza University of Rome, Italy, and by the Agenzia Italiana del Farmaco (AIFA), Rome, Italy. Each patient gave his/her written informed consent to participate to the study. The data that support the findings of this study are available from the corresponding author upon reasonable request. The data that support the findings of this study are available from the corresponding author upon reasonable request. Figure S1. Characterization of Philadelphia (Ph)+ acute lymphoblastic leukemia (ALL) patients' expanded natural killer (NK) cell phenotype. The surface expression levels of activating receptors on NK cells from Ph+ ALL patients before and after expansion were assessed by multicolor flow cytometry. The analysis was performed on the receptor positive subsets within the total NK cell population. Results are expressed as mean percentage ± SD of receptor-positive NK cells (A) and mean fluorescent intensity (MFI) ± SD of each receptor examined (B). Figure S2. Characterization of Philadelphia (Ph)+ acute lymphoblastic leukemia (ALL) patients' expanded natural killer (NK) cell lytic activity. Cytolytic activity of expanded NK cells against (A) the K562 cell line and (B) against Ph+ ALL primary blasts (bone marrow leukemic cells from patient 2 at diagnosis) was measured by flow cytometry using the NKTEST cytotoxicity assay. Data are presented as percentage of lysis ± SD. Figure S3. Trend of Philadelphia (Ph)+ acute lymphoblastic leukemia (ALL) patients' cytokine production by different immune cell subsets at +D7 from the infusion at different natural killer (NK) cell doses. Data are presented as mean percentage ± SD of tumor necrosis factor (TNF)-α and interferon (IFN)-γ T4, T8, NKT, and NK producing cells from all patients studied. Figure S4. Natural killer (NK) cell cytokine production over time in Philadelphia (Ph)+ acute lymphoblastic leukemia (ALL) patients 1 (A) and 5 (B). Data are presented as percentage of tumor necrosis factor (TNF)-α and interferon (IFN)-γ producing NK cells before the NK cell infusion (T0), at +D7 from the infusion at different NK cell doses and 2.2 and 1.8 years after the last NK cell infusion in patients 1 and 5, respectively. Data S1. Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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