Allogeneic CD56 + cell infusion as a bridge to hematopoietic stem cell transplantation in relapsed/refractory acute myeloid leukemia: a phase I clinical trial

Abstract

Background and purpose

Acute myeloid leukemia (AML) is an aggressive disease with suboptimal overall survival, especially in relapsed/refractory patients. The primary goal of salvage therapy in this patient is to achieve optimal disease control, thereby allowing the transition to hematopoietic stem cell transplantation (HSCT), which remains the only curative option for a subset of these patients. Allogeneic KIR ligand-mismatched CD56⁺ NK/NKT-like cells have demonstrated antileukemic activity and represent a promising platform for the development of novel cellular therapies.

Study design

Relapsed/refractory non-M3 AML patients who were not HSCT candidates were included in this phase I clinical trial. Patients received the FLAG conditioning regimen followed by three escalating doses (1 × 10⁶, 3 × 10⁶, 5 × 10⁶ cells/kg) of CD56⁺ NK/NKT-like cells at 5-day intervals.

Results

A total of 11 patients with a median age of 41.5 years were enrolled in the study. On average, they received three lines of prior chemotherapy and showed 18% blasts in their bone marrow. The infusion of CD56⁺ NK/NKT-like cells was safe, with no serious toxicity or graft-versus-host disease (GVHD) observed in any patient. Following this treatment protocol, five patients (45.4%) achieved complete remission (CR), with or without hematologic count recovery. Four of these patients (36.3%) underwent successful HSCT and remained event-free to the end of the follow-up period.

Conclusion

Overall, these trials indicated that the FLAG regimen chemotherapy combined with allogeneic KIR ligand-mismatched CD56⁺ NK/NKT-like cell infusion is safe and may serve as an effective bridge to HSCT in 36.3% of patients with refractory/relapsed non-M3 AML.

Graphic Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s00262-025-04285-9.

Introduction

Acute myeloid leukemia (AML) is an aggressive malignancy caused by clonal expansion of hematopoietic stem cells resulting from several genetic and/or epigenetic aberrations [1, 2]. For decades, intensive chemotherapy using agents like daunorubicin and cytarabine, followed by allogeneic hematopoietic stem cell transplantation (HSCT), was the standard treatment for adult AML patients who are at high risk of relapse. Recent innovations in cellular genetics and molecular biology have revolutionized the understanding of the biological basis of AML, resulting not only in improvements to standard chemotherapy but also in the development of a new wave of targeted therapies [3]. Despite all therapeutic advances, overall outcomes remain poor, with a 5-year overall survival rate of only 32.9% (up to 50% in young patients and 8.3% in patients ≥ 60 years) [4–6].

New therapeutic approaches have focused on achieving complete remission with no sign of minimal residual disease (MRD) to reduce relapse rates and increase overall survival [7, 8]. Immunotherapy represents a novel therapeutic strategy to achieve this goal by harnessing the immune system’s capacity to identify and attack cancer cells through mechanisms distinct from those of chemotherapy drugs, including adoptive natural killer (NK) cell therapy.

NK cells are essential components of the innate immune system, characterized by the coexpression of CD16 and CD56, and provide the first line of defense against tumors and pathogens [9]. NK cells exhibit cytotoxic activity against leukemia cells through perforin/granzyme-dependent mechanisms and the secretion of antitumor cytokines (e.g., IFN-γ and TNF-α). The enhanced cytotoxicity of autologous NK cells has been demonstrated to be correlated with prolonged leukemia-free survival [10–12]. However, in AML patients, autologous NK cells exhibit functional and quantitative defects, which have a direct impact on their antitumor response, leading to disease recurrence [13]. These include a decrease in the number of NK cells, impaired cytotoxic function (decreased secretion of perforin and granzyme B), alterations in surface receptors (downregulation in NKG2D, NKp30, NKp44, and upregulation in NKG2A), and suppression by tumor cells and microenvironment through secretion of TGF-β and IL-10 [14, 15]. The presence of dysfunctional NK cells in AML patients provides a strong rationale for the implementation of NK cell-based immunotherapeutic approaches aimed at restoring antitumor activity, thereby contributing to improved clinical outcomes.

Initially, pioneers Miller et al. conducted a study of adoptively infused haploidentical NK cells in combination with a high-dose cyclophosphamide and fludarabine regimen, along with IL-2, in 19 high-risk AML patients with active disease; their results demonstrated a favorable safety and efficacy, with five patients achieving complete remission (CR) [16]. In a recent study, a less toxic lymphocyte-depleting regimen was employed to minimize the unwanted side effects associated with high-intensity chemotherapy. Additionally, subcutaneous IL-2 injection was omitted to prevent the stimulation of regulatory T cells; instead, it was used for the ex vivo activation of NK cells. Using this protocol, 6 of 16 patients achieved CR or partial response (PR) to therapy, with five becoming eligible for HSCT, and three patients remained disease-free at the 3-year follow-up. In responding patients, clones with high-risk mutations became undetectable after treatment with NK cells. Overall, this study shows that NK cell therapy can induce a response in high-risk AML/MDS patients and suggests the use of haploidentical NK cell infusions as a bridge to HSCT in these patients [17].

In this phase I clinical trial, 11 patients with relapsed/refractory non-M3 AML received a FLAG conditioning regimen followed by three escalating doses of allogeneic CD56 + cells (1 × 10⁶, 3 × 10⁶, and 5 × 10⁶ cells/kg) with five-day intervals. This study evaluated the safety and efficacy of this approach, with follow-up continued until the subsequent transplantation or chemotherapy. Using this protocol, 5 out of the 11 patients studied achieved complete remission. Among these patients, one experienced relapse after 2 months, and one achieved a negative MRD status. Four patients underwent hematopoietic cell transplantation. The 1-year overall survival rate was 36.4% (4/11 patients), as assessed from the date of CD56 + cell therapy.

Methods

Trial design, ethics, and registration

This phase I, nonrandomized, open-label, single-center clinical trial was conducted at the Hematopoietic Stem Cell Transplantation and Cell Therapy Research Center, Taleghani Hospital, affiliated with Shahid Beheshti University of Medical Sciences. The study was approved by the institutional ethics committee (IR.SBMU.RETECH.REC.1402.153) and registered at the Iranian Registry of Clinical Trials (IRCT20230801058996N3) prior to patient recruitment. Written informed consent was obtained from all donors and recipients, or their legal guardians, as required by the Declaration of Helsinki.

Patient eligibility, inclusion, and exclusion criteria

Adult patients (aged over 18 years) with relapsed/refractory non-M3 AML who were not eligible for allo-HSCT were enrolled based on predefined inclusion and exclusion criteria (supplementaryTable 1). Relapsed or refractory disease is defined as either (i) disease recurrence (marrow blasts more than 5%) following an initial CR or (ii) failure to achieve CR after at least two courses of induction chemotherapy [18]. All enrolled patients had a Karnofsky performance scale index ≥ 70% and demonstrated adequate organ function at the time of enrollment (supplementaryTable 1). All patients included in this study failed two or more prior chemotherapies and provided written informed consent for both treatment and data collection. None of the patients included in this study had previously undergone HSCT.

Donor selection

Donor eligibility Immediate relatives (parents, siblings, and offspring) aged 18 to 50 years were systematically screened to identify an appropriate allogeneic donor with a killer-cell immunoglobulin-like receptor (KIR) ligand mismatch in the graft-versus-host direction. In the presence of multiple potential donors, younger individuals were preferred.

Inclusion and exclusion criteria Donor selection was based on the following requirements: (1) body weight greater than 40 kg and a peripheral blood white blood cell (WBC) count exceeding 5,000/µL; (2) negative serologic screening for active infections, including hepatitis B surface antigen (HBsAg) and antibody (HBsAb), hepatitis C virus antibody (HCV Ab), HIV antigen/antibody, cytomegalovirus (CMV Ab), Epstein‒Barr virus (EBV Ab), syphilis (VDRL), varicella-zoster virus (VZV Ab), herpes simplex virus (HSV Ab), and toxoplasma antibody; and (3) the absence of clinical symptoms and confirmation of general health based on comprehensive biochemical testing, including fasting blood sugar (FBS), urea, creatinine, serum transaminases (SGOT, SGPT), total and direct bilirubin, and alkaline phosphatase levels. Ultimately, eligible donors were selected for CD56⁺ cell donation.

HLA and KIR typing Patient and donor HLA typing for the C1/C2 groups and Bw4 epitopes, along with donor KIR genotyping, was performed via PCR-based methods. Donors are considered to mediate NK cell alloreactivity when their recipients lack one or more of the donor’s inhibitory KIR ligands (HLA-C1, HLA-C2, or HLA-Bw4) (Table 1).

Table 1.

Demographics and baseline disease characteristics of the patients

Patient	Sex	Age	Diagnosis (FAB)	Karyotype	Genotype	Recipient HLA I typing	Donor KIR(mismtach)	HLA typing (mismatch)	Disease status before CD56+ cell infusion
1	F	64	M2	N/A	N/A	C1/C2 , Bw6/Bw6	3DL1	-BW4	relapsed
2	F	31	M4	46XX, t(6:7), t(7:21)	FLT3-IDT (neg), NPM (neg)	C2/C2 , Bw4/Bw6	2DL2	-C group 1	refractory
3	F	41	M4	46XX	N/A	C1/C1 , Bw4/Bw4	2DL1	-C group 2	relapsed
4	F	28	M4	45XX,-7, t(3:3)	FLT3-IDT (neg), IDH(1&2) (neg), NPM1(neg), KIT(D816V) (pos), CEBPA(neg)	C1/C1 , Bw6/Bw6	2DL1	-C group 2	refractory
5	M	55	M1	N/A	JAK2 (neg), FLT3-IDT (neg), NPM1(neg), CEBPA (pos)	C1/C2 , Bw6/Bw6	3DL1	-BW4	relapsed
6	M	44	M4	46XY,	NPM (neg), FLT3-IDH (neg)	C1/C1 , Bw6/Bw6	2DL1	-C group 2	relapsed
7	F	57	M0/M1	N/A	N/A	C1/C2 , Bw6/Bw6	3DL1	-BW4	relapsed
8	M	44	M4	46XY	BCR/ABL (neg), CEBPA (neg), NPM1exon12 (neg), FLT3(neg)	C2/C2 , Bw4/Bw6	2DL2/3	-C group 1	refractory
9	M	29	M1	46XY	BCR/ABL (neg), CEBPA (neg), NPM1exon12 (neg), FLT3 (neg)	C1/C2 , Bw6/Bw6	3DL1	-BW4	relapsed
10	M	22	M2	46XY, t(8:21)	CEBPA(neg), NPM1exon12 (neg), FLT3 (neg), RUNX1-RUNX1T1(pos)	C2/C2 , Bw4/Bw4	2DL2/3	-C group 1	relapsed
11	M	22	M0/M1	46XY	C-Kit (neg), ABL/BCR (neg)	C1/C2 , Bw6/Bw6	3DL1	-BW4	relapsed

M: male, F: female, FAB: French-American-British, N/A: not available, FLT3-ITD: FMS-like tyrosine kinase 3-internal tandem duplication, NPM1: Nucleophosmin 1, IDH2: isocitrate dehydrogenase, CEBPA: CCAAT/enhancer-binding protein alpha, AK2: Janus kinase 2, HLA: human leukocyte antigen, KIR: killer-cell immunoglobulin-like receptor, neg: negative, pos: positive

Preparation of CD56⁺ cell-enriched products

CD56⁺ cell enrichment Approximately two days prior to the first cell infusion, donors underwent a 4 h leukapheresis session using the Spectra Optia apheresis system (Terumo BCT, USA). No adverse events or complications were reported during the collection process. The collected products were promptly transferred to the cell processing facility, where CD56⁺ cells were enriched via a single-step magnetic selection protocol with a Miltenyi Biotec CliniMACS ^Plus device and antihuman CD56 microbeads and reagents (CliniMACS® CD56 Reagent, Miltenyi Biotec, Cat. No. 200–070-206) under good manufacturing practice (GMP)-compliant conditions. Following enrichment, the CD56⁺ cells were either fresh or cryopreserved for future administration.

Quality control assessments All final CD56 + cell products were tested for sterility, mycoplasma contamination, and endotoxin levels prior to cryopreservation. Briefly, microbial contamination, including aerobic and anaerobic bacteria, yeast, and fungi, was assessed via the BACTEC 9120 microbial detection system, mycoplasma was detected via PCR using the Mycoplasma Detection Kit (PrimerDesign genesig Kit, UK, Cat. No. z-path-mycoplasma), and endotoxin levels were measured via the gel-clot limulus amoebocyte lysate (LAL) assay (Endosafe LAL kit, Charles River Endosafe, USA, Cat. No. R13012). Total nucleated cell counts were calculated using the Sysmex KX-21 hematology analyzer (Sysmex Corporation, Japan).

Mononuclear cells were labeled with fluorochrome-conjugated antibodies (all from Miltenyi Biotec, Germany) against CD16 (RRID: AB_2733101), CD56 (RRID: AB_2726090), and CD3 (RRID: AB_2725963) in apheresis and enrichment products, and against CD14 (RRID: AB_2655049) and CD19 (RRID: AB_2726195) exclusively in the enrichment products, according to the manufacturer’s protocols. The samples were run on a MACSQuant 10 flow cytometer (Miltenyi Biotec, Germany), and the data were analyzed with FlowJo software version 10.

Cell viability was assessed using 7-AAD (Miltenyi Biotec, Germany, Cat. No. 130–111-568) cytofluorometric staining methods for freshly isolated cells or the trypan blue dye exclusion assay for thawed cells.

Release criteria The release criteria for the CD56⁺ cell product were as follows: cell viability > 90% for freshly infused cells or > 70% post-thaw infusions, CD56⁺ cell content ≥ 90%, contaminating CD19⁺ B cells and CD14⁺ monocytes each < 5%, CD3⁺CD56⁻ T cells < 1%, endotoxin < 0.125 EU/ml, the absence of mycoplasma, and negative microbiological test results. The products were distributed in sterile syringes or infusion bags under GMP conditions.

Treatment protocol

Conditioning regimen Seven days prior to CD56⁺ cell infusion (day -7 to day -2), patients received a five-day FLAG regimen to promote the immunosuppression and lymphodepletion necessary for the homeostatic proliferation of the infused allogeneic CD56⁺ cells. The regimen consisted of intravenous fludarabine (25 mg/m²/day), high-dose cytarabine (3 g/m²/day), and subcutaneous granulocyte colony-stimulating factor (G-CSF, 5 μg/kg/day). Fludarabine and cytarabine were administered from day -7 to day -3, whereas G-CSF was given from day -6 to day -2 (Fig. 1). Throughout the chemotherapy period, all patients received prophylactic antimicrobial therapy in accordance with the institutional protocols of Taleghani Hospital.

Fig. 1.

Schematic presentation of the treatment protocol. Approximately 200–300 mL of leukapheresis production was collected from related donors, and NK and NKT cells were isolated using a single-step positive selection process for CD56 + cells. Patients underwent standard FLAG chemotherapy at least 7 days prior to the first infusion of CD56 + cells. The chemotherapy regimen consisted of daily intravenous administration of fludarabine (25 mg/m²) and cytarabine (3 g/m²) from day –7 to day –3. G-CSF was administered subcutaneously at a dose of 5 µg/kg/day starting on day –6 and continued through day –2. Following a rest period of approximately two days, CD56 + cell infusions were initiated on day 0 and administered in three doses over two weeks. The first dose was infused fresh, while subsequent doses were cryopreserved and thawed before administration. Treatment response was evaluated at two time points: day 28 and between days 60 and 90. In selected patients who were candidates for hematopoietic stem cell transplantation, additional CD56 + cell infusions were administered in two doses prior to transplantation to reduce the risk of relapse

CD56 + cell infusion Two days after the final chemotherapy dose, on day 0, the patient received alloreactive CD56 + cells (1 × 10⁶ cells/kg), followed by two additional escalating doses of CD56 + cells at five-day intervals, ranging from 3 × 10⁶ cells/kg to 5 × 10⁶ cells/kg (Fig. 1). In the first infusion, freshly purified CD56 + cells were administered, while subsequent infusions used cryopreserved aliquots (− 195 °C) that were thawed and washed before infusion. Ten minutes prior to each infusion, either 10 mg of chlorphenamine or 100 mg of hydrocortisone was given intravenously to prevent allergic reactions. The patients did not receive exogenous cytokines or hematopoietic growth factors.

Endpoints

Safety The primary objectives of the study were to evaluate the feasibility and safety of administering escalating doses of allogeneic CD56⁺ cells following a FLAG conditioning regimen in adult patients with relapsed/refractory AML. Adverse events (AEs) were monitored and graded according to the Common Terminology Criteria for Adverse Events (CTCAE), version 4.03. The AE assessment was conducted at the start of the conditioning regimen and continued for 30 days after the final infusion of CD56 + cells. Given that cytopenia is an anticipated consequence of the lymphodepleting regimen, a hematologic toxicity was defined as the persistence of severe cytopenia (absolute neutrophil count ≤ 500/μL, platelet count ≤ 20,000/μL, and hemoglobin levels < 7 g/dL) for 28 days following the initiation of the FLAG regimen.

Safety monitoring criteria required the temporary suspension of enrollment if more than two participants experienced grade 4 or higher toxicities considered possibly, probably, or definitely related to the infused CD56 + cell product, including severe cardiopulmonary, hepatic, neurological, renal, or infectious complications.

Any death attributed to CD56 + cells within 30 days of injection also required trial suspension for safety assessment, while deaths occurring thereafter only warranted suspension if they were definitely related to cell therapy (19).

Efficacy The secondary endpoints focused on evaluating the therapeutic efficacy of the infused CD56⁺ cells. Clinical responses were defined according to the revised International Working Group (IWG) criteria for AML [20], including CR, CR with incomplete hematologic recovery (CRi), PR, stable disease (SD), and progressive disease (PD). Bone marrow (BM) examinations were performed on day + 28 and again between days 60 and 90. MRD was assessed using flow cytometry via AML-specific antibody panels. Overall survival (OS) was defined as the time from CD56⁺ cell infusion to death from any cause, and event-free survival (EFS) was defined as the time to relapse, progression, or death.

Statistical analyses

Descriptive statistics were employed to summarize the demographic characteristics, clinical features, and laboratory findings. All the statistical analyses were conducted via SPSS software version 25. Survival analysis was performed using the Kaplan–Meier method, with estimates reported alongside 95% confidence intervals (CIs) calculated using the Clopper–Pearson method. Comparisons between groups (e.g., responders versus non-responders and HSCT versus no HSCT) were performed using Fisher’s exact test. A P value < 0.05 was considered a descriptive difference.

Results

Patient characteristics

Between September 2022 and November 2023, 15 patients consented and were screened for eligibility, and 12 patients were enrolled in the present phase I trial. Among these patients, one individual developed a pulmonary infection prior to the initiation of CD56 + cell infusion and was consequently withdrawn from the study (Fig. 2). This sample size is consistent with several similar phase I clinical trials that were primarily designed to evaluate safety, tolerability, and feasibility rather than statistical efficacy (17, 19, 21–23). The median age of the patients was 41.5 years (range 22–64 years), with six male participants (Table 1). Eleven patients, three with refractory AML and eight with relapsed disease, proceeded to receive investigational therapy (Table 1). Patients had undergone a median of three lines of chemotherapy (range 2–5) for AML before receiving CD56⁺ cell therapy (Table 2). No patients had received prior allogenic stem cell transplantation. At enrollment, all patients who were in the active phase of the disease with BM demonstrated 7 to 91% (median 18%) blasts and were deemed ineligible for allogeneic HSCT due to progressive disease (Table 2).

Fig. 2.

Study enrollment summary

Table 2.

Baseline characteristics, chemotherapy history, cell infusion details, and clinical outcomes of the 11 study subjects who received CD56⁺ T-cell infusion

Patients	BM Blasts at diagnosis(%)	Prior chemotherapy lines	BM Blasts changes after chemotherapy lines (%)	BM Blasts before CD56+cells infusion(%)	CD56+ cell infusion doses(× 106/kg)					BM Blasts at 1st follow up (%)	MRD at 1st follow up (%)	Disease status	BM Blasts at 2nd follow up(%)	MRD at 2nd follow up(%)	Disease status at 2nd follow-up	HCT after CD56+ cell infusion	One year follow-up outcome
					1st	2nd	3rd	4th	5th
1	N/A	3 + 7, HIDAC, VEN + VID	N/A	18	1	3	5	N/A	NA	25	25	PD	N/A	N/A	N/A	N	Dead
2	30	5 + 2, HIDAC	30 → 10 → 7	7	1	3	5	N/A	N/A	3	3	CR	4	4	CR	Y	Alive in CR
3	80	3 + 7, HIDAC, MEC	80 → 7 → 3 → 10	10	1	3	5	N/A	N/A	1	< 1	CRi	80	-	Relapsed	N	Dead
4	80	3 + 7, 5 + 2, FLAG, MEC,VEN + VID	80 → 12 → 76 → 10 → 6 → 23	23	1	3	5	N/A	N/A	49	49	PD	N/A	N/A	N/A	N	Dead
5	70	3 + 7, 3 + 7, HIDAC	70 → 20 → 5 → 2	21	1	3	5	5	5	2	1.7	CR	1	0.5	CR	Y	Alive in CR
6	85	3 + 7, HiDAC, azacitidin	85 → 1 → 2	90	1	3	5	N/A	N/A	80	80	SD	N/A	N/A	N/A	N	Dead
7	80	3 + 7, HIDAC, VEN + VID	80 → 40 → 2	80	1	3	5	N/A	N/A	60	60	SD	N/A	N/A	N/A	N	Dead
8	21	3 + 7, HIDAC	21 → 6 → 9	9	1	3	5	N/A	N/A	30	30	PD	N/A	N/A	N/A	N	Dead
9	81	3 + 7, HIDAC	81 → 9 → 1	15	1	3	5	5	5	1	0	CR with MRDneg	HCT	-	HCT	Y	Alive in CR
10	45	3 + 7, HIDAC	45 → 6 → 12	12	1	3	5	N/A	N/A	2	0.1	CR	HCT	-	HCT	Y	Alive in CR
11	90	3 + 7, HIDAC, FLAG, MEC	90 → 1 → 6 → 1 → 30 → 91	91	1	3	5	N/A	N/A	60	60	SD	N/A	N/A	N/A	N	Dead

BM: Bone Marrow, MRD: Minimal Residual Disease, HCT: Hematopoietic Cell Transplantation, 3 + 7: 3 days of an anthracycline + 7 days of cytarabine, HIDAC: High-Dose Cytarabine, VEN + VID: Venetoclax + Vidaza, 5 + 2: 5 days of cytarabine + 2 days of an anthracycline, FLAG: Fludarabine + Cytarabine (Ara-C) + G-CSF, MEC: Mitoxantrone + Etoposide + Cytarabine, CR: Complete Remission, CRI: Complete Remission with Incomplete hematologic recovery, CR with MRD-: Complete Remission with undetectable Minimal Residual Disease, PD: Progressive Disease, SD: Stable Disease, NA: Not Available/Not Applicable, Y/N: Yes/No,

Three patients displayed cytogenetic abnormalities: One of whom had a favorable karyotype but still experienced disease relapse, while the remaining two had high-risk cytogenetic profiles. From the perspective of molecular characteristics, most patients had wild-type NPM1 exon 12, FLT3, or CEBPA mutations. One patient was positive for the KIT (D816V) mutation, and another patient was confirmed to be CEBPA-positive.

CD56⁺ cell products

The characteristics of the CD56⁺ cell products of the 11 patients included in this study are summarized in (Supplementary Tables 2 and 3). All the manufacturing procedures were successful, with no failures reported during CD56⁺ cell production. The median leukapheresis volume obtained was 242 mL (199–318 mL). Before enrichment, the median number of mononuclear cells recovered from the leukapheresis product was 23 × 10⁹ (19.8–30.4 × 10⁹), of which 120.1 × 10⁸ (43.4–155.3 × 10⁸) were CD56⁻CD3⁺ T cells, 5.5 × 10⁸ (6.3–44.8 × 10⁸) were CD56⁺CD3⁻ NK cells, and 7.0 × 10⁸ (3.0–18.9 × 10⁸) were CD56⁺CD3⁺ NKT-like cells. Following immunomagnetic enrichment of CD56⁺ cells, the median number of mononuclear cells in the target bag decreased to 1.57 × 10⁹ (1.09–2.93 × 10⁹), with a median of 9.2 × 10⁶ (5.0–15.8 × 10⁶) CD56⁻CD3⁺ T cells, 8.5 × 10⁸ (5.2–24.5 × 10⁸) CD56⁺CD3⁻ NK cells, and 6.5 × 10⁸ (1.1–10.7 × 10⁸) CD56⁺CD3⁺ NKT-like cells (Fig. 3). The mean purity of the final CD56⁺ product was 96.9% (90.6%–99.1%), with a CD56⁺ cell recovery rate of 59.3% (51.3%–77.0%). T cell depletion achieved a median log reduction of -3.11 (-2.68 to -3.48). The median viability of the freshly infused CD56⁺ cell products was 96.5% (94.8%–99.2%). Post-thaw analysis of cryopreserved products revealed excellent viability and recovery rates (median viability: 86%, median recovery: 82%).

Fig. 3.

Representative dot plot of flow cytometry expression of CD3, CD56, and CD16 (A) before and (B) after CD56 + cells selection

Safety

Toxicity profiles were assessed in all treated patients and are summarized in (Supplementary Table 2). No dose-limiting toxicity (DLT) was observed; therefore, the highest administered dose, 5 × 10⁶ CD56⁺ cells/kg, was considered safe and well tolerated. Further dose escalation was not planned, as 5 × 10⁶ cells/kg represented the technical limit of production from a single-step CD56⁺ cell selection approach. The infusions were well tolerated, with no grade 3–5 toxicities observed during CD56⁺ cell infusion or during the 30-day postinfusion monitoring period. Six patients experienced transient, grade 1 or 2 symptoms, including nausea, chills, headache, vomiting, and bone pain, which were considered to have a possible or imaginable causal relationship with CD56⁺ cell infusion (Table 3). We did not observe any symptoms or signs of graft-versus-host disease (GVHD), CRS, or neurotoxicity in any of the subjects.

Table 3.

Overall summary of nonhematologic adverse events related to study therapy

Adverse events Probably related To cell therapy	Total number(%) (N=11)	Grade 1	Grade 2	Grade 3	Grade 4	Grade 5
Nausea	3(27.2%)	2	1	0	0	0
Chills	1(9.0%)	1	0	0	0	0
Headache	2(18.1%)	2	0	0	0	0
Vomiting	2(18.1%)	1	1	0	0	0
Sinus tachycardia	0(00%)	0	0	0	0	0
Bone pain	1(9%)	1	0	0	0	0
Pain in the extremity	0(00%)	0	0	0	0	0
Rash	0(00%)	0	0	0	0	0
Petechiae	0(00%)	0	0	0	0	0
dyspnea	0(00%)	0	0	0	0	0

Adverse events Related to FLAG Regimen chemotherapy	Total number(%) (N=12)	Grade 1	Grade 2	Grade 3	Grade 4	Grade 5
Neutropenia	12(100%)	0	6	4	2	0
Thrombocytopenia	10(83.3%)	3	4	3	0	0
Anemia	7(58.3%)	4	2	1	0	0
Febrile neutropenia/infection	3(25.0%)	-	-	2	1	0
Neurological events	4(33.3%)	2	2	0	0	0
Elevation of liver enzymes	2(16.6%)	2	0	0	0	0
Nausea/vomiting	5(41.6%)	4	1	0	0	0
Mucositis/stomatitis	2(16.6%)	1	1	0	0	0
Cardiotoxicity	1(8.3%)	0	1	0	0	0
Fatigue	4(33.3%)	3	1	0	0	0

All patients in this study experienced at least one chemotherapy-related AE (Table 3). The most common hematologic toxicities were neutropenia (100%), thrombocytopenia (83.3%), and anemia (58.3%). Specifically, neutropenia was observed in all patients, with 50% experiencing grade 2, while the remaining patients suffered from grade 3–4 neutropenia. Thrombocytopenia was reported in 83.3% of patients, predominantly grade 1–2 (58.3%), with fewer cases progressing to grade 3 (25%). Anemia affected 58.3% of patients, with the majority experiencing mild-to-moderate symptoms (grades 1–2), and one patient reporting grade 3 anemia. Febrile neutropenia occurred in 25% of patients, primarily grade 3, with one case suffering grade 4. It is important to note that this patient developed a lung infection and was subsequently withdrawn from the study. Among non-hematologic AEs, neurological events were reported in 33.3% of patients, nausea/vomiting in 41.6%, and mucositis/stomatitis in 16.6%. One patient (8.3%) experienced grade 2 cardiotoxicity, and fatigue affected 33.3% of patients. No grade 5 toxicity or treatment-related death occurred.

Clinical outcomes

Following the administration of the FLAG chemotherapy regimen and CD56 + cell infusion, hematopoietic recovery was similar to what is typically observed after a standard chemotherapy cycle. The median time to absolute neutrophil count recovery (neutrophil count > 0.5 × 10⁶/µL for three consecutive days) was 23 days (range 17–42), and the median time to platelet recovery (platelet count > 20 × 10⁶/µL for three consecutive days) was 21 days (range 18–39).

All patients underwent three planned infusions of CD56 + cells. On day 28 following cell infusion, five patients responded to therapy, four patients achieved CR (4/11, 36.3%), and one patient achieved CR with incomplete hematologic recovery (1/11, 9%). Among them, one patient (P.3) relapsed after two months and succumbed to PD. Additionally, one patient (P.9) achieved negative MRD accompanied by a reduction in blasts from 15% before CD56 + cell infusion to 1% after CD56 + cell infusion. Among the other six patients, three (27.2%) reached SD with a slight reduction in blast percentage, and three (27.2%) experienced PD with an increase in blasts. All six non-remission patients underwent further chemotherapy (Fig. 4).

Fig. 4.

Treatment Outcomes. (A) Swimmer plot showing treatment responses. (B) Kaplan–Meier analysis of overall survival among all patients receiving CD56⁺ cell therapy. Comparison of event-free survival (EFS) between patients who achieved complete remission (CR/CRi) and those who did not respond. Comparison of EFS between patients who underwent allogeneic hematopoietic stem cell transplantation (AHSCT) and those who did not. Both overall survival and EFS were calculated from the start date of the first CD56⁺ cell infusion

Four patients who achieved CR were deemed eligible for HSCT and had suitable stem cell donors. Among them, two patients (P.5 and P.9) received two additional doses of CD56 + cells (5 × 10⁶ cells/kg) as maintenance therapy before undergoing HSCT. These four patients (4/11, 36.3%) proceeded to HLA-matched allogeneic HSCT in sustained remission at days 57, 63, 128, and 92 (median: 85 days) post-CD56 + cell therapy and remained alive with their disease in CR. It is important to note that prior to transplantation, a prophylactic regimen consisting of methotrexate (10 mg/m²) and cyclosporine (3 mg/kg) was administered to prevent GVHD.

With a median follow-up of 6.5 months (range 2–19), the one-year OS and event-free survival (EFS) rates for the entire cohort were both 36.4% (95% CI: 10.9–69.2). Patients who achieved CR/CRi had a numerically higher EFS of 80.0% (95% CI: 28.4–99.5) compared with 0% (95% CI: 0–45.9) in non-responders (p = 0.015). Furthermore, all patients who responded and underwent HSCT remained event-free at one year (100%, 95% CI: 39.8–100), whereas non-responders had an EFS of 0% (p = 0.005). Overall, this phase I study demonstrated the feasibility and acceptable safety of combining the FLAG regimen with CD56⁺ cell infusion. Although not powered to assess efficacy, exploratory findings suggest potential clinical activity and feasibility as a bridging approach to HSCT, warranting further investigation in future phase II studies.

Discussion

Although chemotherapy remains the standard first-line treatment for AML, approximately 10–40% of newly diagnosed patients do not respond to induction therapy and fail to achieve CR, a condition referred to as primary refractory AML. Additionally, among those who initially respond to chemotherapy, nearly half experience disease relapse. Together, chemotherapy resistance and relapse represent the major causes of mortality in AML patients [24]. A variety of salvage regimens, ranging from non-intensive to intensive, have been utilized in refractory/relapsed AML patients to bridge patients to the only potentially curative option, allogeneic HSCT. However, most of these approaches are not promising due to the lack of sufficient efficacy or high toxicity [25, 26]. Cell therapy is a promising alternative to intensive chemotherapy and radiation therapy.

Immunotherapy based on adoptive CD56 + cells (including NK and NKT-like cells) has recently gained attention as a therapeutic strategy. The alloreactivity of NK cells due to donor and recipient KIR and HLA class I mismatch leads to GVL reactions, facilitating engraftment, and protecting against GVHD. Moreover, rapid NK cell recovery after HSCT is associated with improved outcomes, whereas impaired NK cell function is linked to disease progression [27].

In the present study, we report the results of a phase I trial evaluating the feasibility and safety of KIR ligand-mismatched CD56⁺ cells in 11 adult patients with refractory/relapsed non-M3 AML. Our findings, in line with previous studies, showed that administering CD56 + cells was safe, with no infusion-related serious adverse events, including CRS, neurotoxicity, or GVHD [19, 21, 28–30]. Dose escalation was well tolerated, with a high dose of 5 × 10⁶ cells/kg, which represents the maximum yield routinely achievable from single-donor leukapheresis. Our results indicated that five patients achieved CR/CRi and became eligible for allogenic HSCT, and four of them proceeded to subsequent allogenic HSCT. At the censoring date, four patients were alive and in CR following transplantation. These clinical results suggest that this type of cell therapy may convert refractory or relapsed AML patients into transplant candidates, ultimately leading to a possible cure. It is worth noting that patients who did not achieve complete remission after CD56 + cell infusion were subsequently managed with additional chemotherapy regimens rather than remaining in a chemotherapy-free state. These patients received more active therapy, which may have contributed to the observed survival duration of up to 10 months. In our study, the median OS of non-responders was 5.6 months, which is comparable to previously reported OS durations of 3 months in refractory/relapsed AML patients who were treated with the Mito-FLAG chemotherapy regimen without HSCT [31], 5.3 months following first relapse [32] and 2.17 months in refractory/relapsed AML patients who failed to respond to FLAG or FLAG-Ida chemotherapy [33]. This indicates that NK cell infusion did not compromise subsequent treatment opportunities and that survival outcomes in this subgroup were consistent with historical expectations.

Numerous clinical trials have investigated the adoptive transfer of NK cells following HSCT, either as a strategy to treat disease relapse or as a prophylactic (preemptive) approach to inhibit disease progression [34–37]. In addition, clinical trials using allogeneic NK cells outside the transplantation setting to induce remission or as a consolidation therapy to extend CR have been conducted, and various manufacturing strategies and clinical protocols have been developed. Fehniger et al. [28] reported a phase I dose-escalation study in which 12 high-risk AML patients received cyclophosphamide and fludarabine followed by haploidentical NK cell infusions (CD3-CD56 + cells) primed with CTV-1 leukemia cell lysate in their first CR, showing no dose-limiting toxicities; notably, three patients achieved remarkably durable CR lasting 33–48 months after therapy. Björklund et al. [17] explored the safety and efficacy of IL-2-activated haploidentical NK cells in combination with lymphodepleting chemotherapy consisting of fludarabine, cyclophosphamide, and total lymphoid irradiation in 16 patients with refractory or relapsed AML/MDS who were ineligible for HSCT. In this trial, 37.5% of patients (n = 6) responded to NK cell therapy, 31.25% achieved CR, and five patients subsequently underwent allogeneic HSCT. Ciurea et al. [19] performed a phase I study in which 12 relapsed/refractory AML patients received FLAG chemotherapy plus six infusions of haploidentical NK cells expanded with engineered K562 feeder cells, achieving a CR rate of 58.3%, with a favorable safety profile, and five patients bridged to transplantation via the same donor. Conversely, a phase I study evaluating allogeneic expanded NK cell infusion on days 0 and + 7 following a conditioning regimen of fludarabine and endoxan in nine relapsed/refractory AML patients reported no CR achievement [29]. Based on the aforementioned studies, to the best of our knowledge, we are conducting the first clinical trial to evaluate the safety and feasibility of infusing CD56 + cells, comprising a mixture of NK and NKT-like cells following FLAG chemotherapy, with an exploratory assessment of remission inauguration and the potential to act as a bridge to transplantation in adult patients with relapsed or refractory AML. Although these findings are encouraging, they should be interpreted with caution. The small sample size limits the statistical strength of the analysis and precludes firm conclusions regarding the direct antileukemic contribution of the infused CD56⁺ cells. Moreover, the concurrent effect of FLAG chemotherapy must be considered, as historical studies have reported CR/CRi rates of approximately 20–60% with FLAG-based salvage regimens in relapsed/refractory AML [31, 38, 39]; hence, it is plausible that part of the observed responses in our study may be derived from the chemotherapy itself.

It is interesting to note that the CD56 + cells given in our study were more effective in patients with pre-infusion lower tumor burden. The increased clinical efficacy of NK cell alloreactivity in clearing low tumor burden has also been described in previous studies [40, 41]. High tumor burden not only adversely reduces the effector-to-target (E: T) ratio but also creates an immunosuppressive microenvironment that constrains the cytotoxic capacity of NK cells (13). Therefore, strategies that either modify tumor burden or enhance the potency and frequency of infused NK cells are justifiable for improving therapeutic responses in patients with higher tumor burden.

Pre-infusion disease reduction represents one such strategy. In our cohort, patients received the FLAG lymphodepletion, but several alternative combinations were used in conjunction with NK cell therapy, including venetoclax-based therapy alone [42, 43], azacitidine + venetoclax [44], GCLAC [45], and hypomethylating agents [46], may have the potential to reduce disease burden while maintaining an immunologic environment that prevents NK cell suppression. Moreover, additional antineoplastic agents with immunomodulatory properties may be employed to enhance NK cell activity against AML. Specifically, the immunomodulatory drugs lenalidomide and pomalidomide exert antileukemic effects both directly and indirectly by augmenting NK cell-mediated immune responses, accompanied by downregulation of HLA class I expression on AML blasts [47].

Enhancing NK cell potency represents the second major avenue. Short-term activation of NK cells with the combination of IL-12/15/18 results in increased antileukemic activity, improved metabolic adaptability, and greater persistence of NK cells in patients with relapsed/refractory AML (13). Moreover, activation with IL-15 constitutes a straightforward approach supported by prior studies and is commonly used to promote priming, cytotoxic potential, and proliferation of NK cells in both ex vivo and in vivo patients [48, 49]. Another strategy to enhance therapeutic efficacy involves ex vivo NK cell expansion, which increases the number of available effectors. Both feeder-based and feeder-free platforms can generate substantially higher NK cell doses, enabling meaningful dose escalation and supporting repeated or sequential infusions [19]. Notably, higher doses of donor alloreactive NK cells correlate with better clinical responses, highlighting the importance of dose-boosting strategies [50, 51].

Peripheral blood-derived NK cells, which have been widely utilized in clinical trials, are typically obtained from leukapheresis products as the initial source. To enrich the NK cell fraction, T cells can be depleted using anti-CD3 microbeads; however, this approach still yields a mixed population of other cell subsets, including residual B cells and monocytes. To further reduce B cell contamination, anti-CD19 beads are subsequently applied. By sequential depletion of T cells followed by CD56 + cell enrichment, a highly purified NK cell population can be obtained, but this approach remains time-consuming and costly [52]. An alternative and more cost-effective approach is the single-step selection of CD56 + cells, which yields a final cellular product containing a mixed population of NK and NKT-like cells [45, 53].

Both NK (CD3⁻CD56⁺) and NKT-like (CD3⁺CD56⁺) cells belong to the innate lymphocyte population. NKT-like cells constitute only a small fraction (0.2–8%) of total peripheral lymphocytes and are believed to play essential roles in various immune-related diseases, particularly cancer [54]. Previous studies have shown a statistically significant correlation between the number of peripheral blood CD3⁺CD56⁺ NKT-like cells and a favorable prognosis in various types of cancer, including leukemia [54]. Key cytotoxic molecules, such as perforin and granzyme levels, are significantly lower in patients with acute leukemia than in healthy individuals, indicating a CD3 + CD56 + cell functional impairment that may compromise their ability to eliminate leukemic clones [55].

In preclinical models, CD8-expressing CD3 + CD56 + NKT cells have been shown to maintain GVL activity without exacerbating GVHD [56]. Recent trial studies have provided evidence supporting the beneficial immunological effects of NKT-like cells in the setting of allogeneic allo-HSCT. Jaiswal et al. [53] demonstrated that the adoptive transfer of CD56-enriched donor lymphocytes, following post-transplant cyclophosphamide-based haploidentical HSCT, promotes robust reconstitution of NK cells and reduces the incidence of aGVHD. Kulkarni et al. [45] evaluated the therapeutic potential of a single dose of haploidentical CD56⁺ cell infusions (median total dose: 41.6 × 10⁶/kg, comprising 26.47 × 10⁶ CD3⁻CD56⁺ cells/kg and 13.36 × 10⁶ CD3⁺CD56⁺ cells/kg) as an adjunct to sequential transplantation in 14 patients with relapsed/refractory AML, aiming to selectively target and eradicate pretransplant MRD and thereby improve post-HSCT outcomes. In terms of efficacy, although definitive efficacy could not be demonstrated due to the small sample size and high non-relapse mortality, the mean blast percentage decreased from 15.87% to 11.92% following cell infusion, and patients with greater MRD reduction appeared to achieve better disease control post-HSCT. These findings suggest that the transfer of NKT-like cells along with NK cells may represent a promising therapeutic strategy to enhance antileukemic immunity and improve the outcomes in AML patients.

Interleukin-15 (IL-15) plays a crucial role in the differentiation, expansion, and survival of NK cells [57]. Although Miller et al. [16] demonstrated that high-dose lymphodepleting regimens markedly increase endogenous IL-15 production, resulting in promoting the in vivo proliferation of NK cells, but more recent data from Dr. Rezvani’s group indicated that serum IL-15 levels remain largely unchanged following lymphodepleting or NK cell infusion [19]. Notably, even at the lowest doses of infused NK cells, in vivo persistence and expansion of donor-derived NK cells were observed during the first week after the final infusion [19]. Moreover, expansion of regulatory T cells following lymphodepleting therapy in the absence of IL-2 administration has also been reported, which may limit the engraftment and in vivo efficacy of donor-derived NK cells [17]. One limitation of the current study is the absence of longitudinal tracking of donor CD56⁺ cell engraftment kinetics, cytokine profiles, and changes in regulatory T cells following chemotherapy. Thus, future studies are recommended to identify better factors influencing in vivo CD56 + cell expansion and functional activity.

This study is limited by a small patient cohort, a relatively short follow-up period, and a single-center design. To further validate our findings, a randomized phase 2 trial with a larger patient population and extended follow-up is planned to further confirm our observations in the present study. Nonetheless, reporting these results is valuable as it may provide a foundation for developing a rapid and reliable method for manufacturing CD56 + cells applicable to adaptive cell therapies in clinical practice.

Finally, this study was not designed to characterize the features of CD56 + cell products before infusion. This limitation will be addressed in a planned phase 2 clinical trial through a comprehensive analysis of activating and inhibitory receptor expression on CD56 + cells.

Conclusion

The present study showed that multiple infusions of KIR ligand-mismatched CD56 + cells, intended to reduce disease burden and prepare patients with relapsed or refractory AML for subsequent allogeneic HSCT, were feasible and safe. Although tumor response was not the primary objective of this study, 4 out of 11 patients achieved remission and subsequently underwent transplantation, and all of them were alive in remission at the time of the last follow-up. However, despite these encouraging preliminary results, the findings should be interpreted with caution due to the limited sample size, which restricts the ability to draw definitive conclusions regarding clinical efficacy.

Author contributors

E.R.: Conceptualization, Methodology, Data Analysis, and Validation. A.SG. and A.I.: Investigation. A.H. and A.G.: Supervision and Validation A.I., M.S-A., and M.BD.: CD56⁺ cell collection and processing, Flow Cytometry, Molecular assays. M.BD. and M.SA.: Data Analysis. S.P., A.S., and H.RS.: Project Administration. M.BD., S.Y., and A.SG.: Writing – Review & Editing. All authors reviewed and approved the final version of the manuscript.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

E.R.: Conceptualization, Methodology, Data Analysis, and Validation. A.SG. and A.I.: Investigation. A.H. and A.G.: Supervision and Validation A.I., M.S-A., and M.BD.: CD56⁺ cell collection and processing, Flow Cytometry, Molecular assays. M.BD. and M.SA.: Data Analysis. S.P., A.S., and H.RS.: Project Administration. M.BD., S.Y., and A.SG.: Writing – Review & Editing. All authors reviewed and approved the final version of the manuscript.

Funding

The authors received no financial support for the research, authorship, and publication of this article.

Data availability

All the data generated or analyzed during this study have been included in this published article.

Declarations

Conflicts of interest

The authors declare no competing interests.

Consent to participate

All patients and donors provided written informed consent in accordance with the principles outlined in the Declaration of Helsinki.