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Background: Prolonged immunosuppression or delayed T-cell recovery may favor Epstein-Barr virus (EBV) infection or reactivation after allogeneic hematopoietic stem cell transplantation (HSCT), which can lead to post-transplant lymphoproliferative disease (PTLD) and high-grade malignant B-cell lymphoma. Cytokine-induced killer (CIK) cells with dual specific anti-tumor and virus-specific cellular immunity may be applied in this context.
Methods: CIK cells with EBV-specificity were generated from peripheral blood mononuclear cells (PBMCs), expanded in the presence of interferon-γ, anti-CD3, interleukin (IL)-2 and IL-15 and were pulsed twice with EBV consensus peptide pool. CIK cells with EBV-specificity and conventional CIK cells were phenotypically and functionally analyzed. Additionally, CIK cells with EBV-specificity were applied to a patient with EBV-related PTLD rapidly progressing to highly aggressive B-cell lymphoma on a compassionate use basis after approval and agreement by the regulatory authorities.
Results: Pre-clinical analysis showed that generation of CIK cells with EBV-specificity was feasible. In vitro cytotoxicity analyses showed increased lysis of EBV-positive target cells, enhanced proliferative capacity and increased secretion of cytolytic and proinflammatory cytokines in the presence of EBV peptide-displaying target cells. In addition, 1 week after infusion of CIK cells with EBV-specificity, the patient's highly aggressive B-cell lymphoma persistently disappeared. CIK cells with EBV-specificity remained detectable for up to 32 days after infusion and infusion did not result in acute toxicity.
Discussion: The transfer of both anti-cancer potential and T-cell memory against EBV infection provided by EBV peptide-induced CIK cells might be considered a therapy for EBV-related PTLD.
Background: Cytokine-induced-killer (CIK) cells are a promising immunotherapeutic approach for impending relapse following hematopoietic stem cell transplantation (HSCT). However, there is a high risk for treatment failure associated with severe graft versus host disease (GvHD) necessitating pharmaceutical intervention post-transplant. Whether immunosuppression with mycophenolate mofetil (MMF) or Ciclosporin A (CsA) influences the cytotoxic effect of CIK cell immunotherapy is still an open issue.
Methods: CIK cells were generated from PBMC as previously described followed by co-incubation with mycophenolic acid (MPA) or CsA. Proliferation, cytotoxicity and receptor expression were investigated following short- (24 h), intermediate- (3 days) and long-term (7 days) MPA incubation with the intention to simulate the in vivo situation when CIK cells were given to a patient with relevant MPA/CsA plasma levels.
Results: Short-term MPA treatment led to unchanged proliferation capacity and barely had any effect on viability and cytotoxic capability in vitro. The composition of CIK cells with respect to T-, NK-like T- and NK cells remained stable. Intermediate MPA treatment lacked effects on NKG2D, FasL and TRAIL receptor expression, while an influence on proliferation and viability was detectable. Furthermore, long-term treatment significantly impaired proliferation, restricted viability and drastically reduced migration-relevant receptors accompanied by an alteration in the CD4/CD8 ratio. CD3+CD56+ cells upregulated receptors relevant for CIK cell killing and migration, whereas T cells showed the most interference through significant reductions in receptor expression. Interestingly, CsA treatment had no significant influence on CIK cell viability and the cytotoxic potential against K562.
Conclusions: Our data indicate that if immunosuppressant therapy is indispensable, efficacy of CIK cells is maintained at least short-term, although more frequent dosing might be necessary.
Chimeric antigen receptor (CAR) T cells are a novel class of anti-cancer therapy in which autologous or allogeneic T cells are engineered to express a CAR targeting a membrane antigen. In Europe, tisagenlecleucel (Kymriah™) is approved for the treatment of refractory/relapsed acute lymphoblastic leukemia in children and young adults as well as relapsed/refractory diffuse large B-cell lymphoma, while axicabtagene ciloleucel (Yescarta™) is approved for the treatment of relapsed/refractory high-grade B-cell lymphoma and primary mediastinal B-cell lymphoma. Both agents are genetically engineered autologous T cells targeting CD19. These practical recommendations, prepared under the auspices of the European Society of Blood and Marrow Transplantation, relate to patient care and supply chain management under the following headings: patient eligibility, screening laboratory tests and imaging and work-up prior to leukapheresis, how to perform leukapheresis, bridging therapy, lymphodepleting conditioning, product receipt and thawing, infusion of CAR T cells, short-term complications including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome, antibiotic prophylaxis, medium-term complications including cytopenias and B-cell aplasia, nursing and psychological support for patients, long-term follow-up, post-authorization safety surveillance, and regulatory issues. These recommendations are not prescriptive and are intended as guidance in the use of this novel therapeutic class.
Osteonecrosis (ON) is an acquired debilitating skeletal disorder, which is caused by a multitude of traumatic and non-traumatic etiological factors. Vascular damage, mechanical stress and increased intraosseous pressure have been discussed as contributors to ON. The optimal treatment of ON remains to be determined, since the current gold standard, core decompression, is insufficiently effective. Specific properties of mesenchymal stromal cells (MSCs) provide the rationale for their assessment in advanced stages of ON: Osteoinductive potential has been demonstrated and MSC preparations of suitable quality for use as medicinal products have been developed. Here we review the scant information on the use of allogeneic or autologous MSCs in advanced ON as well as potentially supportive data from pre-clinical studies with autologous bone marrow mononuclear cells (auto BM-MNCs), which have been studied quite extensively and the presumed therapeutic effect of which was attributed to the rare MSCs contained in these cell products. Outcomes in clinical trials with MSCs and auto-BM-MNCs remain preliminary and non-definitive, at best promising, with respect to their pharmacological effect. Clearly, though, the application of any of these cell therapies was technically feasible and safe in that it was associated with low complication rates. The heterogeneity of cell type and source, study protocols, cell manufacturing, cell properties, cell doses and surgical techniques might contribute to inconsistent results.
Background: Because of limitations of transportation imposed by the COVID-19 pandemic, current recommendation calls for cryopreservation of allogeneic stem cell transplants before patient conditioning. A single cell therapy laboratory was selected to function as the central cryopreservation hub for all European registry donor transplants intended for the Australian-Pacific region. We examined properties of these transplants to ascertain how quality is maintained.
Methods: We analyzed 100 pandemic-related allogeneic mobilized blood-derived stem cell apheresis products generated at 30 collection sites throughout Europe, shipped to and cryopreserved at our center between April and November of 2020. Products were shipped in the cool, subsequently frozen with DMSO as cryoprotectant. Irrespective of origin, all products were frozen within the prescribed shelf-life of 72 h.
Results: Prior to cryopreservation, viable stem cell and leukocyte count according to the collection site and our reference laboratory were highly concordant (r2 = 0.96 and 0.93, respectively) and viability was > 90% in all instances. Median nominal post-thaw recovery of viable CD34+ cells was 42%. Weakly associated with poorer CD34+ cell recovery was higher leukocyte concentration, but not time lag between apheresis or addition of cryopreservant, respectively, and start of freezing. The correlation between pre- and post-thaw CD34+ cell dose was high (r2 = 0.85), hence predictable. Neutrophil and platelet engraftment were prompt with no evidence of dose dependency within the range of administered cell doses (1.31–15.56 × 106 CD34+ cells/kg).
Conclusions: General cryopreservation of allogeneic stem cell transplants is feasible. While more than half of the CD34+ cell content is lost, the remaining stem cells ensure timely engraftment.
Ionizing radiation generates DNA double-strand breaks (DSB) which, unless faithfully repaired, can generate chromosomal rearrangements in hematopoietic stem and/or progenitor cells (HSPC), potentially priming the cells towards a leukemic phenotype. Using an enhanced green fluorescent protein (EGFP)-based reporter system, we recently identified differences in the removal of enzyme-mediated DSB in human HSPC versus mature peripheral blood lymphocytes (PBL), particularly regarding homologous DSB repair (HR). Assessment of chromosomal breaks via premature chromosome condensation or γH2AX foci indicated similar efficiency and kinetics of radiation-induced DSB formation and rejoining in PBL and HSPC. Prolonged persistence of chromosomal breaks was observed for higher LET charged particles which are known to induce more complex DNA damage compared to X-rays. Consistent with HR deficiency in HSPC observed in our previous study, we noticed here pronounced focal accumulation of 53BP1 after X-ray and carbon ion exposure (intermediate LET) in HSPC versus PBL. For higher LET, 53BP1 foci kinetics was similarly delayed in PBL and HSPC suggesting similar failure to repair complex DNA damage. Data obtained with plasmid reporter systems revealed a dose- and LET-dependent HR increase after X-ray, carbon ion and higher LET exposure, particularly in HR-proficient immortalized and primary lymphocytes, confirming preferential use of conservative HR in PBL for intermediate LET damage repair. HR measured adjacent to the leukemia-associated MLL breakpoint cluster sequence in reporter lines revealed dose dependency of potentially leukemogenic rearrangements underscoring the risk of leukemia-induction by radiation treatment.
Background: Emerging evidence indicates that mesenchymal stromal cells (MSCs) isolated from different tissue sources may be used in vivo as tissue restorative agents. To date, there is no evidence, however, on migration and proliferation ("wound healing") potential of different subsets of MSCs. The main goal of this study was therefore to compare the in vitro "wound healing" capacity of MSCs generated from positively selected CD271+ bone marrow mononuclear cells (CD271-MSCs) and MSCs generated by plastic adherence (PA-MSCs).
Methods: The in vitro model of wound healing (CytoSelect™ 24-Well Wound Healing Assay) was used in order to compare the migration and proliferation potential of CD271-MSCs and PA-MSCs of passage 2 and 4 cultured in presence or absence of growth factors or cytokines.
Results: CD271-MSCs of both passages when compared to PA-MSCs demonstrated a significantly higher potential to close the wound 12 and 24 h after initiation of the wound healing assay (P < 0.003 and P < 0.002, respectively). Noteworthy, the migration capacity of PA-MSCs of second passage was significantly improved after stimulation with FGF-2 (P < 0.02), PDGF-BB (P < 0.006), MCP-1 (P < 0.002) and IL-6 (P < 0.03), whereas only TGF-β enhanced significantly migration process of PA-MSCs of P4 12 h after the treatment (P < 0.02). Interestingly, treatment of CD271-MSCs of both passages with growth factors or cytokines did not affect their migratory potential.
Conclusions: Our in vitro data provide the first evidence that CD271-MSCs are significantly more potent in "wound healing" than their counterparts PA-MSCs.
Leukemia cells reciprocally interact with their surrounding bone marrow microenvironment (BMM), rendering it hospitable to leukemia cell survival, for instance through the release of small extracellular vesicles (sEVs). In contrast, we show here that BMM deficiency of pleckstrin homology domain family M member 1 (PLEKHM1), which serves as a hub between fusion and secretion of intracellular vesicles and is important for vesicular secretion in osteoclasts, accelerates murine BCR-ABL1+ B-cell acute lymphoblastic leukemia (B-ALL) via regulation of the cargo of sEVs released by BMM-derived mesenchymal stromal cells (MSCs). PLEKHM1-deficient MSCs and their sEVs carry increased amounts of syntenin and syndecan-1, resulting in a more immature B-cell phenotype and an increased number/function of leukemia-initiating cells (LICs) via focal adhesion kinase and AKT signaling in B-ALL cells. Ex vivo pretreatment of LICs with sEVs derived from PLEKHM1-deficient MSCs led to a strong trend toward acceleration of murine and human BCR-ABL1+ B-ALL. In turn, inflammatory mediators such as recombinant or B-ALL cell–derived tumor necrosis factor α or interleukin-1β condition murine and human MSCs in vitro, decreasing PLEKHM1, while increasing syntenin and syndecan-1 in MSCs, thereby perpetuating the sEV-associated circuit. Consistently, human trephine biopsies of patients with B-ALL showed a reduced percentage of PLEKHM1+ MSCs. In summary, our data reveal an important role of BMM-derived sEVs for driving specifically BCR-ABL1+ B-ALL, possibly contributing to its worse prognosis compared with BCR-ABL1− B-ALL, and suggest that secretion of inflammatory cytokines by cancer cells in general may similarly modulate the tumor microenvironment.
Background: Blood donation saves lives. Provided they are in good health, male volunteers can donate as often as six times per year from the age of 18 into their late sixties. The burden of blood donation is very unevenly distributed, with a small minority of altruistic individuals providing this critical resource. While the consequences of persistent iron depletion in blood donors have been studied in the context of cancer and coronary heart disease, potential effects of the erythropoietic stress from repetitive large-volume phlebotomy remain unexplored. We sought to investigate if and how repeated blood donations affect the clonal composition of the hematopoietic stem and progenitor cell (HSPC) compartment.
Methods: 105 healthy, male individuals with an extensive blood donation history (median of 120 donations per donor; median age of 66 yrs.) were screened for the presence of clonal hematopoiesis (CH) using a sequencing panel covering 141 genes commonly mutated in human myeloid neoplasms. The control cohort consisted of 103 healthy, male donors with a median of 5 donations per donor and a median age of 63. Donors positive for CH were subsequently studied longitudinally. The pathogenicity of detected variants was compared using established scoring systems. Finally, to assess the functional consequences of blood-donation induced CH, selected CH mutations were introduced by CRISPR-mediated editing into HSPCs from human cord blood (CB) or bone marrow (BM). The effect of these mutations was tested under different stress stimuli using functional ex vivo long-term culture initiating cells (LTC-IC) assays.
Results: Compared to the control cohort, frequent donors were significantly more likely to have mutations in genes encoding for epigenetic modifiers (44.7 vs. 22.3 %), most specifically in the two genes most commonly mutated in CH, DNMT3A and TET2 (35.2 vs. 20.3 %). However, no difference in the variant allele frequency (VAF) of detected mutations was found between the groups. Longitudinal analysis revealed that the majority of the mutations remained at a stable VAF over an observation period of approximately one year. Three DNMT3A variants from the frequent donor cohort were introduced into healthy HSPCs and functionally analyzed: All expanded in response to EPO, but none responded to LPS or IFNγ stimulation. This contrasted with the leukemogenic DNMT3A R882H mutation, which did not expand in the presence of EPO but instead responded strongly to inflammatory stimuli.
Conclusions: Frequent whole blood donation is associated with a higher prevalence of CH driven by mutations in genes encoding for epigenetic modifiers, with DNMT3A and TET2 being the most common. This increased CH prevalence is not associated with a higher pathogenicity of the associated variants and is likely a result of the selection of clones with improved responsiveness to EPO under the condition of bleeding stress. Our data show that even highly frequent blood donations over many years is not increasing the risk for malignant clones further underscoring the safety of repetitive blood donations. To our knowledge, this is the first CH study analyzing a cohort of individuals known for their superior health and survival, able to donate blood until advanced age. Thus, our analysis possibly identified mutations associated with beneficial outcomes, rather than a disease condition, such as mutations in DNMT3A that mediated the improved expansion of HSPCs in EPO enriched environments. Our data support the notion of ongoing Darwinian evolution in humans at the somatic stem cell level and present EPO as one of the environmental factors to which HSPCs with specific mutations may respond with superior fitness.
Specific protocols define eligibility, conditioning, donor selection, graft composition and prophylaxis of graft vs. host disease for children and young adults undergoing hematopoietic stem cell transplant (HSCT). However, international protocols rarely, if ever, detail supportive care, including pharmaceutical infection prophylaxis, physical protection with face masks and cohort isolation or food restrictions. Supportive care suffers from a lack of scientific evidence and implementation of practices in the transplant centers brings extensive restrictions to the child's and family's daily life after HSCT. Therefore, the Board of the Pediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT) held a series of dedicated workshops since 2017 with the aim of initiating the production of a set of minimal recommendations. The present paper describes the consensus reached within the field of infection prophylaxis.
(1) Background: Refractory acute graft-versus-host disease (R-aGvHD) remains a leading cause of death after allogeneic stem cell transplantation. Survival rates of 15% after four years are currently achieved; deaths are only in part due to aGvHD itself, but mostly due to adverse effects of R-aGvHD treatment with immunosuppressive agents as these predispose patients to opportunistic infections and loss of graft-versus-leukemia surveillance resulting in relapse. Mesenchymal stromal cells (MSC) from different tissues and those generated by various protocols have been proposed as a remedy for R-aGvHD but the enthusiasm raised by initial reports has not been ubiquitously reproduced.
(2) Methods: We previously reported on a unique MSC product, which was generated from pooled bone marrow mononuclear cells of multiple third-party donors. The products showed dose-to-dose equipotency and greater immunosuppressive capacity than individually expanded MSCs from the same donors. This product, MSC-FFM, has entered clinical routine in Germany where it is licensed with a national hospital exemption authorization. We previously reported satisfying initial clinical outcomes, which we are now updating. The data were collected in our post-approval pharmacovigilance program, i.e., this is not a clinical study and the data is high-level and non-monitored.
(3) Results: Follow-up for 92 recipients of MSC-FFM was reported, 88 with GvHD ≥°III, one-third only steroid-refractory and two-thirds therapy resistant (refractory to steroids plus ≥2 additional lines of treatment). A median of three doses of MSC-FFM was administered without apparent toxicity. Overall response rates were 82% and 81% at the first and last evaluation, respectively. At six months, the estimated overall survival was 64%, while the cumulative incidence of death from underlying disease was 3%.
(4) Conclusions: MSC-FFM promises to be a safe and efficient treatment for severe R-aGvHD.
Background: MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study.
Methods: The levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied.
Results: The levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles.
Conclusions: Serum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs.
Albumin, the most abundant plasma protein, not only controls osmotic blood pressure, but also serves as a carrier for various small molecules, including pharmaceuticals. Its impact on pharmacological properties of many drugs has been extensively studied over decades. Here, we focus on its interaction with the following mobilizing agents: Granulocyte-colony stimulating factor (G-CSF) and AMD3100, where such analyses are lacking. These compounds are widely used for hematopoietic stem cell mobilization of healthy donors or patients. Using albumin-deficient (Alb−/−) mice, we studied the contribution of albumin to mobilization outcomes. Mobilization with the bicyclam CXCR4 antagonist AMD3100 was attenuated in Alb−/− mice compared to wild-type littermates. By contrast, mobilization with recombinant human G-CSF (rhG-CSF), administered twice daily over a five-day course, was significantly increased in Alb−/− mice. In terms of a mechanism, we show that rhG-CSF bioavailability in the bone marrow is significantly improved in Alb−/− mice, compared to wild-type (WT) littermates, where rhG-CSF levels dramatically drop within a few hours of the injection. These observations likely explain the favorable mobilization outcomes with split-dose versus single-dose administration of rhG-CSF to healthy donors.
Drug resistance is an obstacle in the therapy of acute lymphoblastic leukemia (ALL). Whether the physical properties such as the motility of the cells contribute to the survival of ALL cells after drug treatment has recently been of increasing interest, as they could potentially allow the metastasis of solid tumor cells and the migration of leukemia cells. We hypothesized that chemotherapeutic treatment may alter these physical cellular properties. To investigate the motility of chemotherapeutics-treated B-cell ALL (B-ALL) cells, patient-derived B-ALL cells were treated with chemotherapy for 7 days and left for 12 h without chemotherapeutic treatment. Two parameters of motility were studied, velocity and migration distance, using a time-lapse imaging system. The study revealed that compared to non-chemotherapeutically treated B-ALL cells, B-ALL cells that survived chemotherapy treatment after 7 days showed reduced motility. We had previously shown that Tysabri and P5G10, antibodies against the adhesion molecules integrins α4 and α6, respectively, may overcome drug resistance mediated through leukemia cell adhesion to bone marrow stromal cells. Therefore, we tested the effect of integrin α4 or α6 blockade on the motility of chemotherapeutics-treated ALL cells. Only integrin α4 blockade decreased the motility and velocity of two chemotherapeutics-treated ALL cell lines. Interestingly, integrin α6 blockade did not affect the velocity of chemoresistant ALL cells. This study explores the physical properties of the movements of chemoresistant B-ALL cells and highlights a potential link to integrins. Further studies to investigate the underlying mechanism are warranted.
The decision in September 2011 in the UK to accept blood donations from non-practicing men who have sex with men (MSM) has received significant public attention. Will this rule change substantially boost the number of blood donations or will it make our blood less safe? Clearly, most European countries have a blood procurement problem. Fewer young people are donating, while the population is aging and more invasive therapies are requiring more blood. Yet if that was the reason for allowing non-practicing MSM to donate, clearly re-admission of some other, much larger populations that are currently deferred from donation should likewise be considered. As far as risks for blood safety are concerned, evidence has been provided that the current quality of infectious disease marker testing significantly mitigates against, although does not completely eradicate, risks associated with admission of donors with a high risk of carrying certain blood-transmissible agents. However, it could be argued that more effective recruitment of the non-donor pool, which is substantially larger than the group of currently ineligible donors, would be a better strategy. Recruitment of this group will benefit the availability of blood without jeopardizing the current excellent safety profile of blood.
CXCL12-CXCR4 signaling controls multiple physiological processes and its dysregulation is associated with cancers and inflammatory diseases. To discover as-yet-unknown endogenous ligands of CXCR4, we screened a blood-derived peptide library for inhibitors of CXCR4-tropic HIV-1 strains. This approach identified a 16 amino acid fragment of serum albumin as an effective and highly specific CXCR4 antagonist. The endogenous peptide, termed EPI-X4, is evolutionarily conserved and generated from the highly abundant albumin precursor by pH-regulated proteases. EPI-X4 forms an unusual lasso-like structure and antagonizes CXCL12-induced tumor cell migration, mobilizes stem cells, and suppresses inflammatory responses in mice. Furthermore, the peptide is abundant in the urine of patients with inflammatory kidney diseases and may serve as a biomarker. Our results identify EPI-X4 as a key regulator of CXCR4 signaling and introduce proteolysis of an abundant precursor protein as an alternative concept for chemokine receptor regulation.
Background: Certain disadvantages of the standard hematopoietic stem and progenitor cell (HSPC) mobilizing agent G-CSF fuel the quest for alternatives. We herein report results of a Phase I dose escalation trial comparing mobilization with a peptidic CXCR4 antagonist POL6326 (balixafortide) vs. G-CSF.
Methods: Healthy male volunteer donors with a documented average mobilization response to G-CSF received, following ≥6 weeks wash-out, a 1–2 h infusion of 500–2500 µg/kg of balixafortide. Safety, tolerability, pharmacokinetics and pharmacodynamics were assessed.
Results: Balixafortide was well tolerated and rated favorably over G-CSF by subjects. At all doses tested balixafortide mobilized HSPC. In the dose range between 1500 and 2500 µg/kg mobilization was similar, reaching 38.2 ± 2.8 CD34 + cells/µL (mean ± SEM). Balixafortide caused mixed leukocytosis in the mid-20 K/µL range. B-lymphocytosis was more pronounced, whereas neutrophilia and monocytosis were markedly less accentuated with balixafortide compared to G-CSF. At the 24 h time point, leukocytes had largely normalized.
Conclusions: Balixafortide is safe, well tolerated, and induces efficient mobilization of HSPCs in healthy male volunteers. Based on experience with current apheresis technology, the observed mobilization at doses ≥1500 µg/kg of balixafortide is predicted to yield in a single apheresis a standard dose of 4× 10E6 CD34+ cells/kg from most individuals donating for an approximately weight-matched recipient. Exploration of alternative dosing regimens may provide even higher mobilization responses.
Trial Registration European Medicines Agency (EudraCT-Nr. 2011-003316-23) and clinicaltrials.gov (NCT01841476)
Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.
The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is ∼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34+ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4–8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.