Nicolaas Schaap

Clinical-Grade Generation of Active NK Cells from Cord Blood Hematopoietic Progenitor Cells for Immunotherapy Using a Closed-System Culture Process

Natural killer (NK) cell-based adoptive immunotherapy is a promising treatment approach for many cancers. However, development of protocols that provide large numbers of functional NK cells produced under GMP conditions are required to facilitate clinical studies. In this study, we translated our cytokine-based culture protocol for ex vivo expansion of NK cells from umbilical cord blood (UCB) hematopoietic stem cells into a fully closed, large-scale, cell culture bioprocess. We optimized enrichment of CD34+ cells from cryopreserved UCB units using the CliniMACS system followed by efficient expansion for 14 days in gas-permeable cell culture bags. Thereafter, expanded CD34+ UCB cells could be reproducibly amplified and differentiated into CD56+CD3− NK cell products using bioreactors with a mean expansion of more than 2,000 fold and a purity of >90%. Moreover, expansion in the bioreactor yielded a clinically relevant dose of NK cells (mean: 2×109 NK cells), which display high expression of activating NK receptors and cytolytic activity against K562. Finally, we established a versatile closed washing procedure resulting in optimal reduction of medium, serum and cytokines used in the cell culture process without changes in phenotype and cytotoxic activity. These results demonstrate that large numbers of UCB stem cell-derived NK cell products for adoptive immunotherapy can be produced in closed, large-scale bioreactors for the use in clinical trials.

High Log-Scale Expansion of Functional Human Natural Killer Cells from Umbilical Cord Blood CD34-Positive Cells for Adoptive Cancer Immunotherapy

Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56+CD3− NK cell products could be routinely generated from freshly selected CD34+ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34+ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56+ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34+ cells for cancer immunotherapy.

Donor versus no-donor comparison of newly diagnosed myeloma patients included in the HOVON-50 multiple myeloma study.

To prospectively evaluate allogeneic stem cell transplantation (allo-SCT) for myeloma as part of first-line therapy, a donor versus no-donor analysis was performed of patients treated in the HOVON-50 study, a study that was originally designed to examine thalidomide combined with intensive therapy. Two hundred sixty patients having received an autologous-SCT fulfilled the criteria to be included, 138 patients without an HLA-identical sibling donor and 122 patients with a donor. After a median follow-up of 77 months, complete remission, progression-free survival (PFS), and overall survival were not significantly different between the 2 groups. PFS at 6 years was 28% for patients with a donor versus 22% for patients without a donor (P = .19) and overall survival at 6 years from high-dose melphalan was 55%, irrespective of having a donor (P = .68). Cumulative incidence of nonrelapse mortality at 6 years after autologous-SCT was 16% in the donor group versus 3% in the no-donor group (P < .001). However, PFS was significantly prolonged in the 99 patients who actually proceeded to allo-SCT compared with the 115 patients who continued maintenance or received a second high-dose melphalan, but the difference did not translate into a prolonged survival benefit. These results do not support a general application of allo-SCT in all myeloma patients as part of first-line therapy.

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.

Inhibition of Akt signaling promotes the generation of superior tumor-reactive T cells for adoptive immunotherapy

Effective T-cell therapy against cancer is dependent on the formation of long-lived, stem cell-like T cells with the ability to self-renew and differentiate into potent effector cells. Here, we investigated the in vivo existence of stem cell-like antigen-specific T cells in allogeneic stem cell transplantation (allo-SCT) patients and their ex vivo generation for additive treatment posttransplant. Early after allo-SCT, CD8+ stem cell memory T cells targeting minor histocompatibility antigens (MiHAs) expressed by recipient tumor cells were not detectable, emphasizing the need for improved additive MiHA-specific T-cell therapy. Importantly, MiHA-specific CD8+ T cells with an early CCR7+CD62L+CD45RO+CD27+CD28+CD95+ memory-like phenotype and gene signature could be expanded from naive precursors by inhibiting Akt signaling during ex vivo priming and expansion. This resulted in a MiHA-specific CD8+ T-cell population containing a high proportion of stem cell-like T cells compared with terminal differentiated effector T cells in control cultures. Importantly, these Akt-inhibited MiHA-specific CD8+ T cells showed a superior expansion capacity in vitro and in immunodeficient mice and induced a superior antitumor effect in intrafemural multiple myeloma-bearing mice. These findings provide a rationale for clinical exploitation of ex vivo-generated Akt-inhibited MiHA-specific CD8+ T cells in additive immunotherapy to prevent or treat relapse in allo-SCT patients.

Intestinal Damage Determines the Inflammatory Response and Early Complications in Patients Receiving Conditioning for a Stem Cell Transplantation

Background Stem cell transplantation (SCT) is still complicated by the occurrence of fever and inflammatory complications attributed to neutropenia and subsequent infectious complications. The role of mucosal barrier injury (MBI) of the intestinal tract therein has received little attention. Methods We performed a retrospective analysis in 163 SCT recipients of which data had been collected prospectively on intestinal damage (citrulline), inflammation (C-reactive protein), and neutrophil count. Six different conditioning regimens were studied; 5 myeloablative (MA) and 1 non-myeloablative (NMA). Linear mixed model multivariate and AUC analyses were used to define the role of intestinal damage in post-SCT inflammation. We also studied the relationship between the degree of intestinal damage and the occurrence of early post-SCT complications. Results In the 5 MA regimen there was a striking pattern of inflammatory response that coincided with the occurrence of severe intestinal damage. This contrasted with a modest inflammatory response seen in the NMA regimen in which intestinal damage was limited. With linear mixed model analysis the degree of intestinal damage was shown the most important determinant of the inflammatory response, and both neutropenia and bacteremia had only a minor impact. AUC analysis revealed a strong correlation between citrulline and CRP (Pearson correlation r = 0.96). Intestinal damage was associated with the occurrence of bacteremia and acute lung injury, and influenced the kinetics of acute graft-versus-host disease. Conclusion The degree of intestinal damage after myeloablative conditioning appeared to be the most important determined the inflammatory response following SCT, and was associated with inflammatory complications. Studies should explore ways to ameliorate cytotoxic therapy-induced intestinal damage in order to reduce complications associated with myeloablative conditioning therapy.

Natural Killer Cells Generated from Cord Blood Hematopoietic Progenitor Cells Efficiently Target Bone Marrow-Residing Human Leukemia Cells in NOD/SCID/IL2Rgnull Mice

Natural killer (NK) cell-based adoptive immunotherapy is an attractive adjuvant treatment option for patients with acute myeloid leukemia. Recently, we reported a clinical-grade, cytokine-based culture method for the generation of NK cells from umbilical cord blood (UCB) CD34+ hematopoietic progenitor cells with high yield, purity and in vitro functionality. The present study was designed to evaluate the in vivo anti-leukemic potential of UCB-NK cells generated with our GMP-compliant culture system in terms of biodistribution, survival and cytolytic activity following adoptive transfer in immunodeficient NOD/SCID/IL2Rgnull mice. Using single photon emission computed tomography, we first demonstrated active migration of UCB-NK cells to bone marrow, spleen and liver within 24 h after infusion. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Thereafter, we showed that low dose IL-15 mediates efficient survival, expansion and maturation of UCB-NK cells in vivo. Most importantly, we demonstrate that a single UCB-NK cell infusion combined with supportive IL-15 administration efficiently inhibited growth of human leukemia cells implanted in the femur of mice, resulting in significant prolongation of mice survival. These preclinical studies strongly support the therapeutic potential of ex vivo-generated UCB-NK cells in the treatment of myeloid leukemia after immunosuppressive chemotherapy.

Human secondary lymphoid organs typically contain polyclonally-activated proliferating regulatory T cells.

Immunomodulating regulatory T-cell (Treg) therapy is a promising strategy in autoimmunity and transplantation. However, to achieve full clinical efficacy, better understanding of in vivo human Treg biology is warranted. Here, we demonstrate that in contrast to blood and bone marrow Tregs, which showed a resting phenotype, the majority of CD4(pos)CD25(pos)CD127(neg)FoxP3(pos) Tregs in secondary lymphoid organs were proliferating activated CD69(pos)CD45RA(neg) cells with a hyperdemethylated FOXP3 gene and a broad T-cell receptor-Vβ repertoire, implying polyclonal activation. Activated CD69(pos) Tregs were distributed over both T-cell and B-cell areas, distant from Aire(pos) and CD11c(pos) cells. In contrast to the anergic peripheral blood Tregs, lymphoid organ Tregs had significant ex vivo proliferative capacity and produced cytokines like interleukin-2, while revealing similar suppressive potential. Also, next to Treg-expressing chemokine receptors important for a prolonged stay in lymphoid organs, a significant part of the cells expressed peripheral tissue-associated, functional homing markers. In conclusion, our data suggest that human secondary lymphoid organs aid in the maintenance and regulation of Treg function and homeostasis. This knowledge may be exploited for further optimization of Treg immunotherapy, for example, by ex vivo selection of Tregs with capacity to migrate to lymphoid organs providing an in vivo platform for further Treg expansion.

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.

Efficient nontoxic delivery of PD-L1 and PD-L2 siRNA into dendritic cell vaccines using the cationic lipid SAINT-18.

Dendritic cell (DC)-based vaccination is an appealing strategy to boost graft-versus-tumor immunity after allogeneic stem cell transplantation (allo-SCT), and thereby prevent or counteract tumor recurrence. By exploiting minor histocompatibility antigens (MiHA) presented on hematopoietic cells, donor CD8+ T-cell immunity can be selectively targeted to patient’s hematological tumor cells without the risk of inducing graft-versus-host disease. Previously, we demonstrated that silencing RNA (siRNA) of programmed death-ligand 1 (PD-L1) and PD-L2 on DCs markedly augments the expansion and function of MiHA-specific CD8+ T cells. However, previously applied methods based on electroporation or lipid nanoparticles were either incompatible with target antigen mRNA delivery or required complex manufacturing compliant to Good Manufacturing Practice. Here, we investigated whether transfection using lipoplexes composed of PD-L1 and PD-L2 siRNAs plus SAINT-18:DOPE (ie, SAINT-RED) is an effective and feasible clinical-grade method in DC vaccine manufacturing. We observed that a single siRNA/SAINT-RED transfection resulted in efficient and long-term knockdown of the PD-1 ligands without affecting DC maturation or viability. Furthermore, we demonstrated that SAINT-RED can be heat sterilized without loss of function, facilitating its use in aseptic DC vaccine production. Finally, we showed that the established transfection method can be combined with target antigen mRNA or peptide loading to efficiently stimulate MiHA-specific T-cell expansion and cytokine production. Together, these findings indicate that the developed PD-L siRNA/SAINT-RED transfection protocol in combination with MiHA mRNA or peptide loading can be applied in the generation of clinical-grade DC vaccines to boost antitumor immunity after allo-SCT.