Good manufacturing practice production of CD34+ progenitor-derived NK cells for adoptive immunotherapy in acute myeloid leukemia.

Allogeneic natural killer (NK) cell-based immunotherapy is a promising, well-tolerated adjuvant therapeutic approach for acute myeloid leukemia (AML). For reproducible NK cell immunotherapy, a homogenous, pure and scalable NK cell product is preferred. Therefore, we developed a good manufacturing practice (GMP)-compliant, cytokine-based ex vivo manufacturing process for generating NK cells from CD34+ hematopoietic stem and progenitor cells (HSPC). This manufacturing process combines amongst others IL15 and IL12 and the aryl hydrocarbon receptor antagonist StemRegenin-1 (SR1) to generate a consistent and active NK cell product that fits the requirements for NK cell immunotherapy well. The cell culture protocol was first optimized to generate NK cells with required expansion and differentiation capacity in GMP-compliant closed system cell culture bags. In addition, phenotype, antitumor potency, proliferative and metabolic capacity were evaluated to characterize the HSPC-NK product. Subsequently, seven batches were manufactured for qualification of the process. All seven runs demonstrated consistent results for proliferation, differentiation and antitumor potency, and preliminary specifications for the investigational medicinal product for early clinical phase trials were set. This GMP-compliant manufacturing process for HSPC-NK cells (named RNK001 cells) is used to produce NK cell batches applied in the clinical trial 'Infusion of ex vivo-generated allogeneic natural killer cells in combination with subcutaneous IL2 in patients with acute myeloid leukemia' approved by the Dutch Ethics Committee (EudraCT 2019-001929-27).

IL-15 superagonist N-803 improves IFNγ production and killing of leukemia and ovarian cancer cells by CD34+ progenitor-derived NK cells.

Allogeneic natural killer (NK) cell transfer is a potential immunotherapy to eliminate and control cancer. A promising source are CD34 + hematopoietic progenitor cells (HPCs), since large numbers of cytotoxic NK cells can be generated. Effective boosting of NK cell function can be achieved by interleukin (IL)-15. However, its in vivo half-life is short and potent trans-presentation by IL-15 receptor α (IL-15Rα) is absent. Therefore, ImmunityBio developed IL-15 superagonist N-803, which combines IL-15 with an activating mutation, an IL-15Rα sushi domain for trans-presentation, and IgG1-Fc for increased half-life. Here, we investigated whether and how N-803 improves HPC-NK cell functionality in leukemia and ovarian cancer (OC) models in vitro and in vivo in OC-bearing immunodeficient mice. We used flow cytometry-based assays, enzyme-linked immunosorbent assay, microscopy-based serial killing assays, and bioluminescence imaging, for in vitro and in vivo experiments. N-803 increased HPC-NK cell proliferation and interferon (IFN)γ production. On leukemia cells, co-culture with HPC-NK cells and N-803 increased ICAM-1 expression. Furthermore, N-803 improved HPC-NK cell-mediated (serial) leukemia killing. Treating OC spheroids with HPC-NK cells and N-803 increased IFNγ-induced CXCL10 secretion, and target killing after prolonged exposure. In immunodeficient mice bearing human OC, N-803 supported HPC-NK cell persistence in combination with total human immunoglobulins to prevent Fc-mediated HPC-NK cell depletion. Moreover, this combination treatment decreased tumor growth. In conclusion,  N-803 is a promising IL-15-based compound that boosts HPC-NK cell expansion and functionality in vitro and in vivo. Adding N-803 to HPC-NK cell therapy could improve cancer immunotherapy.

A phase I/II minor histocompatibility antigen-loaded dendritic cell vaccination trial to safely improve the efficacy of donor lymphocyte infusions in myeloma.

Allogeneic stem cell transplantation (allo-SCT) with or without donor lymphocyte infusions (DLI) is the only curative option for several hematological malignancies. Unfortunately, allo-SCT is often associated with GvHD, and patients often relapse. We therefore aim to improve the graft-versus-tumor effect, without increasing the risk of GvHD, by targeting hematopoietic lineage-restricted and tumor-associated minor histocompatibility antigens using peptide-loaded dendritic cell (DC) vaccinations. In the present multicenter study, we report the feasibility, safety and efficacy of this concept. We treated nine multiple myeloma patients with persistent or relapsed disease after allo-SCT and a previous DLI, with donor monocyte-derived mHag-peptide-loaded DC vaccinations combined with a second DLI. Vaccinations were well tolerated and no occurrence of GvHD was observed. In five out of nine patients, we were able to show the induction of mHag-specific CD8+ T cells in peripheral blood. Five out of nine patients, of which four developed mHag-specific T cells, showed stable disease (SD) for 3.5-10 months. This study shows that mHag-based donor monocyte-derived DC vaccination combined with DLI is safe, feasible and capable of inducing objective mHag-specific T-cell responses. Future research should focus on further improvement of the vaccination strategy, toward translating the observed T-cell responses into robust clinical responses.

Long-term follow-up of a retrospective comparison of reduced-intensity conditioning and conventional high-dose conditioning for allogeneic transplantation from matched related donors in myelodysplastic syndromes.

This study shows the long-term updated outcomes of a multicenter retrospective study which analyzed 843 patients with myelodysplastic syndrome (MDS) who underwent transplantation with an HLA-identical sibling donor with either reduced-intensity conditioning (RIC) in 213 patients, or standard myeloablative conditioning (MAC) in 630 patients. In multivariate analysis, the 13-year relapse rate was significantly increased after RIC (31% after MAC vs 48% in RIC; HR, 1.5; 95% CI, 1.1-1.9; P=0.04), but with no differences in overall survival (OS) (30% after MAC vs 27% in RIC; P=0.4) and PFS (29 vs 21%, respectively, P=0.3). Non-relapse mortality was higher in MAC (40 vs 31%; P=0.1), especially in patients older than 50 years (50 vs 33%, P<0.01). In addition, long-term follow-up confirms the importance of other variables on 13-year OS, mainly MDS risk category, disease phase, cytogenetics and receiving a high donor cell dose, irrespective of the conditioning regimen used.

Allogeneic hematopoietic cell transplantation for multiple myeloma in Europe: trends and outcomes over 25 years. A study by the EBMT Chronic Malignancies Working Party.

We describe the use and outcomes of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for multiple myeloma (MM) in Europe between January 1990 and December 2012. We identified 7333 patients, median age at allo-HSCT was 51 years (range: 18-78), of whom 4539 (62%) were males. We distinguished three groups: (1) allo-HSCT upfront (n=1924), (2) tandem auto-allo-HSCT (n=2004) and (3) allo-HSCT as a second line treatment and beyond (n=3405). Overall, there is a steady increase in numbers of allo-HSCT over the years. Upfront allo-HSCT use increased up to year 2000, followed by a decrease thereafter and represented 12% of allo-HSCTs performed in 2012. Tandem auto-allo-HSCT peaked around year 2004 and contributed to 19% of allo-HSCTs in 2012. Allo-HSCT as salvage after one or two or three autografts was steadily increasing over the last years and represented 69% of allo-HSCTs in 2012. Remarkable heterogeneity in using allo-HSCT was observed among the different European countries. The 5-year survival probabilities from time of allo-HSCT for the three groups after year 2004 were 42%, 54% and 32%, respectively. These results show that the use of allo-HSCT is increasing in Europe, especially as second line treatment and beyond. There is an unmet need for well-designed prospective studies investigating allo-HSCT as salvage therapy for MM.

Conditioning intensity in middle-aged patients with AML in first CR: no advantage for myeloablative regimens irrespective of the risk group-an observational analysis by the Acute Leukemia Working Party of the EBMT.

In recipients of allogeneic hematopoietic stem cell transplantation with AML in CR1, reduced intensity (RIC) conditioning regimens are usually given to older patients and myeloablative regimens (MAC) to younger patients. We analyzed whether in middle-aged patients aged 40-60 years, MAC was superior to RIC in cytogenetically higher risk AML. Among 2974 patients, 1638 had MAC and 1336 RIC transplants. Cytogenetics were high risk in 508, intermediate risk in 2297 and low risk in 169. Overall survival (OS) was higher in patients with RIC with low-risk cytogenetics but not in the intermediate- or poor-risk AML. Relapse incidence was lower with MAC in poor- and intermediate-risk AML. Nonrelapse mortality (NRM) was higher in MAC in all cytogenetic risk groups. Multivariate analysis confirmed a significant leukemia-free survival and OS advantage for RIC in low risk but no advantage of MAC in intermediate- and poor-risk leukemia. In patients aged 40-60 years, MAC has no advantage over RIC. We confirm lower relapse but higher NRM risks with MAC. MAC is not superior in patients with higher risk cytogenetics, but is inferior to RIC in the small cohort of AML patients with low-risk cytogenetics.

The impact of circulating suppressor cells in multiple myeloma patients on clinical outcome of DLIs.

Allo-SCT followed by DLIs can establish long-term remissions in multiple myeloma (MM) patients. In many patients, however, the immunotherapeutic graft-versus-tumor (GVT) effect is moderate and not sustained, implying that immune suppression is mediated, among other factors, by regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs). Towards a better understanding and, eventually, manipulation of the immune-regulatory mechanisms in transplanted MM patients, we retrospectively sought a correlation between DLI outcome and circulating CD14(+) MDSCs, CD14(-) MDSCs and Tregs in 53 MM patients before their first DLI. We found significantly elevated frequencies of highly suppressive CD14(+) MDSCs, CD14(-) MDSCs and Tregs in pre-DLI samples from patients. Higher frequencies of Tregs, but not of MDSCs, were significantly associated with non-responsiveness to DLI. Furthermore, a lower frequency of Tregs predicted the development of chronic GVHD, which, in turn, displayed a high association with GVT. Elevated Treg frequencies before DLI were also associated with significantly shorter PFS and OS. Hence, our data reinforce the idea of active suppression of antitumor responses by Tregs in MM patients and therefore suggest that targeting patient Tregs before DLI may improve outcome of DLI.

Comparison of upfront tandem autologous-allogeneic transplantation versus reduced intensity allogeneic transplantation for multiple myeloma.

We performed a retrospective analysis of the European Group for Blood and Marrow Transplantation database comparing the outcomes of multiple myeloma patients who received tandem autologous followed by allogeneic PSCT (auto-allo) with the outcomes of patients who underwent a reduced intensity conditioning allograft (early RIC) without prior autologous transplant. From 1996 to 2013, we identified a total of 690 patients: 517 patients were planned to receive auto-allo and 173 received an early RIC allograft without prior autologous transplant. With a median follow-up of 93 months, 5-year PFS survival was significantly better in the auto-allo group, 34% compared with 22% in the early RIC group (P<0.001). OS was also significantly improved in the auto-allo group with a 5-year rate of 59% vs 42% in the early RIC group (P=0.001). The non-relapse mortality rate was lower in the auto-allo group than in the early RIC group, with 1- and 3-year rates of 8% and 13% vs 20% and 28%, respectively (P<0.001). The relapse/progression rate was similar in the two groups, with 5-year rates of 50% for auto-allo and 46% for early RIC (P=0.42). These data suggest that planned tandem autologous allograft can improve overall survival compared with upfront RIC allograft alone in patients with multiple myeloma.

Use of G-CSF to hasten neutrophil recovery after auto-SCT for AML is not associated with increased relapse incidence: a report from the Acute Leukemia Working Party of the EBMT.

Application of G-CSF in AML is controversial as leukemic blasts may express receptors interacting with the cytokine, which may stimulate leukemia growth. We retrospectively analyzed the impact of G-CSF use to accelerate neutrophil recovery after auto-SCT on outcome. Adults with AML in first CR autografted between 1994 and 2010 were included. Nine hundred and seventy two patients were treated with G-CSF after auto-SCT whereas 1121 were not. BM and PB were used as a source of stem cells in 454 (22%) and 1639 (78%) cases, respectively. The incidence of relapse at 5 years in the BM-auto-SCT group was 38% for patients receiving post-transplant G-CSF and 43% for those not treated with G-CSF, P=0.46. In the PB-auto-SCT cohort, respective probabilities were 48% and 49%, P=0.49. No impact of the use of G-CSF could be demonstrated with respect to the probability of leukemia-free survival: in the BM-auto-SCT group, 51% for G-CSF(+) and 48% for G-CSF(-), P=0.73; in PB-auto-SCT group, 42% for G-CSF(+) and 43% for G-CSF(-), P=0.83. Although G-CSF administration significantly shortened the neutropenic phase, no beneficial effect was observed with regard to non-relapse mortality. In patients with AML, the use of G-CSF after auto-SCT is not associated with increased risk of relapse irrespective of the source of stem cells used.

Allogeneic stem cell transplantation for older advanced MDS patients: improved survival with young unrelated donor in comparison with HLA-identical siblings.

We investigated whether a young human leukocyte antigen (HLA)-matched unrelated donor (MUD) should be preferred as donor to an HLA-identical sibling (MRD) for older patients with myelodysplastic syndrome (MDS) (≥ 50 years) who underwent allogeneic stem cell transplantation (AHSCT). Outcomes of 719 MDS patients with a median age of 58 years (range, 50-73 years) who received AHSCT from related (n=555) or unrelated (n=164) donors between 1999 and 2008 and reported to the European Group for Blood and Marrow Transplantation were analyzed. The median donor age of the MRD was 56 years (range: 35-78), in contrast to 34 years (range: 19-64) for the MUDs. Influence of donor's age on survival was not observed for MRD (hazard ratio (HR): 1.01 (95% confidence interval (CI): 0.99-1.02), P=0.2), but there was a significant impact of MUD's age on outcome (HR: 1.03 (95% CI: 1.01-1.06); P=0.02). Transplantation from younger MUDs (<30 years) had a significant improved 5-year overall survival in comparison with MRD and older MUDs (>30 years): 40% vs 33% vs 24% (P=0.04). In a multivariate analysis, AHSCT from young MUD (<30 years) remained a significant factor for improved survival in comparison with MRD (HR: 0.65 (95% CI: 0.45-0.95), P=0.03), which should be considered in donor selection for older patients.

Donor lymphocyte infusions for the treatment of chronic myeloid leukemia relapse following peripheral blood or bone marrow stem cell transplantation.

Peripheral blood used as a source of stem cells for transplantation (PBSCT) is known to exert stronger immune-mediated effects compared with BM (BMT). We decided to retrospectively analyze the impact of stem cell source on the OS of CML patients who relapsed after either matched related donor PBSCT (N=168) or BMT (N=216) and were treated with donor lymphocyte infusions (DLI). Univariate analysis revealed a lower probability of OS after DLI in patients relapsing after PBSCT vs BMT (66% vs 79% at 5 years, P=0.013). However, a multivariate Cox analysis did not reveal any significant impact of PBSCT as a risk factor for decreased OS for patients transplanted in first chronic phase (CP1; hazard ratio (HR) 1.036, 95% confidence interval (CI) 0.619-1.734). A statistical interaction term suggested that the impact of stem cell source on OS after DLI was different for those transplanted in advanced phases (negative impact of previous PBSCT-HR 2.176, 95% CI 0.930-5.091). In summary, the stem cell source does not affect the OS of CML patients who underwent PBSCT in CP1, relapsed and were treated with DLI. However, when the patients were transplanted in advanced phases, previous PBSCT seems to negatively affect OS after DLI compared with BMT.

Induction of multiple myeloma-reactive T cells during post-transplantation immunotherapy with donor lymphocytes and recipient DCs.

Recently, we demonstrated that reduced intensity conditioning (RIC) followed by partial T-cell-depleted SCT creates a platform for inducing the graft-versus-myeloma effect by adjuvant immunotherapy. Here, we evaluated mHA-specific T-cell responses in a multiple myeloma (MM) patient who was treated with RIC-SCT followed by donor lymphocyte infusion (DLI) and subsequent recipient DC vaccination. We isolated a mHA-specific CTL clone with the capacity to target MM tumor cells from this patient experiencing long-term CR. This CTL clone recognizes an HLA-A3-restricted mHA and mediates killing of both primary MM cells and the MM-cell line U266, while BM-derived fibroblasts are not recognized. CTL-specific T-cell receptor (TCR) transcripts could be detected by quantitative PCR analysis in both peripheral blood and BM during tumor remission. Interestingly, a strong increase of CTL-specific TCR transcripts at the BM tumor site was observed following DLI and recipient DC vaccination, while the TCR signal in peripheral blood decreased. These findings illustrate that the approach of partial T-cell-depleted RIC-SCT followed by post-transplantation immunotherapy induces mHA-specific T-cell responses targeting MM tumor cells.

Adult metachromatic leukodystrophy treated by allo-SCT and a review of the literature.

Five patients with adult-onset metachromatic leukodystrophy (MLD) underwent allo-SCT. Conditioning was reduced in intensity and grafts were obtained from voluntary unrelated donors. All but one graft were depleted of T-lymphocytes. Patient age at transplantation varied from 18 to 29 (median, 27) years. Two patients rejected their graft and MLD progressed. The recipient of the unmanipulated graft converted to complete donor chimerism with normalization of arylsulphatase A (ARSA) levels. Despite ARSA normalization, he deteriorated. Another patient was a mixed chimera. Following escalated doses of donor lymphocyte infusions he converted to complete donor chimerism. His levels of ARSA correlated positively with the percentage of donor cells and MLD was not progressive. The fifth patient died after 35 days from complications associated with GVHD. We conclude that results of allo-SCT in symptomatic MLD patients are poor. However, allo-SCT may stop progression of MLD in selected patients.

Thalidomide and lenalidomide in primary myelofibrosis.

Primary myelofibrosis is a clonal haematopoietic stem cell disease, characterised by marrow stromal fibrosis, extramedullary haematopoiesis, splenomegaly, hepatomegaly and progressive cytopenia. Therapeutic options once cytopenia has developed are limited to supportive care, such as erythrocyte transfusions and growth factors. The aetiology has become more clear, especially since JAK-2 mutations were found, resulting in increased production of cytokines. The immune-modulating drug thalidomide and its derivative lenalidomide have shown to be effective in reducing cytopenia, most probably by inhibiting the cytokine responses. In some patients the bone marrow fibrosis disappears. We describe the experience with these drugs in a cohort of 14 patients for thalidomide and seven for lenalidomide (in six patients lenalidomide was given after thalidomide and one patient received lenalidomide upfront). Thalidomide gave clinical improvement in 6/14 patients, but its use was limited mainly due to toxicity, especially the development of neuropathy. The drug could be given for a median period of 15.5 months in responding patients. Lenalidomide was effective in 4/7 of the patients, in some patients with no response on thalidomide. Due to the more favourable toxicity profile, the median duration of therapy was 19 months, with 3/4 patients on therapy longer than 19 months. These data are discussed in view of the clinical studies published. We conclude that lenalidomide is preferred in myelofibrosis, given a higher response rate and more favourable toxicity profile. If no response the addition of prednisone can be considered. In some patients it can normalise haemoglobin and make them transfusion independent.

CD3+/CD19+-depleted grafts in HLA-matched allogeneic peripheral blood stem cell transplantation lead to early NK cell cytolytic responses and reduced inhibitory activity of NKG2A.

Natural killer (NK) cells have an important function in the anti-tumor response early after stem cell transplantation (SCT). As part of a prospective randomized phase III study, directly comparing the use of CD3(+)/CD19(+)-depleted peripheral blood stem cell (PBSC) harvests with CD34(+)-selected PBSC harvests in allogeneic human leukocyte antigen-matched SCT, we here show that the use of CD3(+)/CD19(+)-depleted PBSC grafts leads to early NK cell repopulation and reconstitution of the CD56(dim) and CD56(bright) NK cell subsets, with concomitant high cytolytic capacity. In the CD34 group, this process took significantly longer. Moreover, in the CD3/19 group after reconstitution, a higher percentage of killer immunoglobulin-like receptor-positive NK cells was found. Although similar percentages of CD94-positive NK cells were found in both groups, in the CD34 group, almost all expressed the inhibitory CD94:NKG2A complex, whereas in the CD3/19 group, the inhibitory CD94:NKG2A and the activating CD94:NKG2C complex were equally distributed. This preferential development of NKG2C-expressing NK cells in the CD3/19 group was paralleled by a loss of NKG2A-mediated inhibition of NK cell degranulation. These results show that the use of CD3(+)/CD19(+)-depleted grafts facilitates strong NK cell cytolytic responses directly after SCT, and the rapid emergence of an NK cell receptor phenotype that is more prone to activation.

NOD2 polymorphisms predict severe acute graft-versus-host and treatment-related mortality in T-cell-depleted haematopoietic stem cell transplantation.

Single nucleotide polymorphisms (SNPs) in the NOD2 gene have significant impact on both treatment-related mortality (TRM) and acute GVHD (aGVHD) in haematopoietic stem cell transplantation (HSCT). The effect of these polymorphisms when using T-cell-depleted grafts has been poorly studied. We retrospectively analysed NOD2 polymorphisms in a cohort of 85 patients and donors who received an HLA-identical sibling partially T-cell-depleted HSCT (0.5 x 10(6) CD3+ T cells per kg) following idarubicin-containing conditioning regimens. NOD2 polymorphisms were present in 14 of 85 (16.5%) of patients and 18 of 85 (21%) of donors. The risk of severe aGVHD (grade III-IV) and the 1-year TRM was significantly higher in the presence of NOD2 polymorphisms (hazard ratio (HR) 6.0, P=0.02 for severe aGVHD and HR 3.3, P=0.02 for TRM, respectively) and was most prominent in cases where patient and donor both had a polymorphism (HR 10.5, P=0.002 and HR 3.9, P=0.002). There was also a trend towards increased risk of bacteraemia due to coagulase-negative staphylococci in patients with an NOD2 polymorphism. We conclude that NOD2 polymorphism screening should be used to optimize donor selection and antimicrobial prophylaxis to reduce the occurrence of aGVHD and TRM following allogeneic HSCT.

Dynamics in chimerism of T cells and dendritic cells in relapsed CML patients and the influence on the induction of alloreactivity following donor lymphocyte infusion.

Donor lymphocyte infusion (DLI) after allogeneic SCT induces complete remissions in approximately 80% of patients with relapsed CML in chronic phase, but some patients do not respond to DLI. We studied absolute numbers of dendritic cell (DC) subsets and chimerism in T cells and two subsets of blood DCs (myeloid DCs (MDCs) and plasmacytoid DCs (PDCs)) in relation to DLI-induced alloreactivity. Based on T cell and DC chimerism, we identified three groups. Four patients were completely donor chimeric in T cells and DC subsets. These patients had an early stage of relapse, and three of the four patients attained complete molecular remission (CMolR) without significant GVHD. Six patients were completely donor in T cells and mixed chimeric in DC subsets. All patients entered CMolR, but this was associated with GVHD in four and cytopenia in three patients. Five patients had mixed chimerism in T cells and complete recipient chimerism in MDC; only two patients entered CMolR. Our data suggest that the combination of donor T cells and mixed chimerism in DC subsets induces a potent graft-versus-leukemia (GVL) effect in association with GVHD. DLI in patients with an early relapse and donor chimerism in both T cells and DC subsets results in GVL reactivity without GVHD.

Multiple myeloma patients receiving pre-emptive donor lymphocyte infusion after partial T-cell-depleted allogeneic stem cell transplantation show a long progression-free survival.

The purpose of this study was to determine the role of pre-emptive donor lymphocyte infusion (pDLI) after partial T-cell-depleted allogeneic SCT in patients with multiple myeloma (MM). A cohort of 24 MM patients was treated with partial T-cell-depleted myeloablative SCT between December 1997 and April 2002. These patients were intended to receive pDLI after SCT. The overall response rate after SCT was 83% (20 of 24 patients) with 10 patients (42%) in complete remission (CR). Transplant-related mortality within 1 year after SCT was 29%. Thirteen patients (54%) received pDLI and four patients in partial remission reached CR. GVHD>grade I after pDLI developed in 4 out of 13 patients (30%). Four patients received therapeutic DLI, without preceding pDLI. Eleven patients (46%) are alive, with a median follow-up of 67 months (range, 48-100 months). Seven of these patients (29%) are in continuous CR (CCR), which was confirmed by a negative patient-specific IgH PCR in four patients. All seven patients in CCR received pDLI. Although myeloablative SCT in MM induces high toxicity, we show that the concept of T-cell depletion followed by pDLI is promising and needs to be investigated in a reduced-intensity conditioning setting.