Management of adults and children receiving CAR T-cell therapy: 2021 best practice recommendations of the European Society for Blood and Marrow Transplantation (EBMT) and the Joint Accreditation Committee of ISCT and EBMT (JACIE) and the European Haematology Association (EHA).

BACKGROUND: Several commercial and academic autologous chimeric antigen receptor T-cell (CAR-T) products targeting CD19 have been approved in Europe for relapsed/refractory B-cell acute lymphoblastic leukemia, high-grade B-cell lymphoma and mantle cell lymphoma. Products for other diseases such as multiple myeloma and follicular lymphoma are likely to be approved by the European Medicines Agency in the near future. DESIGN: The European Society for Blood and Marrow Transplantation (EBMT)-Joint Accreditation Committee of ISCT and EBMT (JACIE) and the European Haematology Association collaborated to draft best practice recommendations based on the current literature to support health care professionals in delivering consistent, high-quality care in this rapidly moving field. RESULTS: Thirty-six CAR-T experts (medical, nursing, pharmacy/laboratory) assembled to draft recommendations to cover all aspects of CAR-T patient care and supply chain management, from patient selection to long-term follow-up, post-authorisation safety surveillance and regulatory issues. CONCLUSIONS: We provide practical, clinically relevant recommendations on the use of these high-cost, logistically complex therapies for haematologists/oncologists, nurses and other stakeholders including pharmacists and health sector administrators involved in the delivery of CAR-T in the clinic.

Superior physical and mental health of healthy volunteers before and five years after mobilized stem cell donation.

BACKGROUND: Safety, tolerability and efficacy of granulocyte colony-stimulating factor (G-CSF) for mobilization of hematopoietic stem and progenitor cells (HSPCs) from healthy donors have been conclusively demonstrated. This explicitly includes, albeit for smaller cohorts and shorter observation periods, biosimilar G-CSFs. HSPC donation is non-remunerated, its sole reward being "warm glow", hence harm to donors must be avoided with maximal certitude. To ascertain, therefore, long-term physical and mental health effects of HSPC donation, a cohort of G-CSF mobilized donors was followed longitudinally. METHODS: We enrolled 245 healthy volunteers in this bi-centric long-term surveillance study. 244 healthy volunteers began mobilization with twice-daily Sandoz biosimilar filgrastim and 242 underwent apheresis after G-CSF mobilization. Physical and mental health were followed up over a period of 5-years using the validated SF-12 health questionnaire. RESULTS: Baseline physical and mental health of HSPC donors was markedly better than in a healthy reference population matched for ethnicity, sex and age. Physical, but not mental health was sharply diminished at the time of apheresis, likely due to side effects of biosimilar G-CSF, however had returned to pre-apheresis values by the next follow-up appointment after 6 months. Physical and mental health slightly deteriorated over time with kinetics reflecting the known effects of aging. Hence, superior physical and mental health compared to the general healthy non-donor population was maintained over time. CONCLUSIONS: HSPC donors are of better overall physical and mental health than the average healthy non-donor. Superior well-being is maintained over time, supporting the favorable risk-benefit assessment of volunteer HSPC donation. Trial registration National Clinical Trial NCT01766934.

Mesenchymal stromal cells for osteonecrosis.

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.

Progressive multifocal leukoencephalopathy in a patient post allo-HCT successfully treated with JC virus specific donor lymphocytes.

BACKGROUND: Progressive multifocal leukoencephalopathy is a demyelinating CNS disorder. Reactivation of John Cunningham virus leads to oligodendrocyte infection with lysis and consequent axonal loss due to demyelination. Patients usually present with confusion and seizures. Late diagnosis and lack of adequate therapy options persistently result in permanent impairment of brain functions. Due to profound T cell depletion, impairment of T-cell function and potent immunosuppressive factors, allogeneic hematopoietic cell transplantation recipients are at high risk for JCV reactivation. To date, PML is almost universally fatal when occurring after allo-HCT. METHODS: To optimize therapy specificity, we enriched JCV specific T-cells out of the donor T-cell repertoire from the HLA-identical, anti-JCV-antibody positive family stem cell donor by unstimulated peripheral apheresis [1]. For this, we selected T cells responsive to five JCV peptide libraries via the Cytokine Capture System technology. It enables the enrichment of JCV specific T cells via identification of stimulus-induced interferon gamma secretion. RESULTS: Despite low frequencies of responsive T cells, we succeeded in generating a product containing 20 000 JCV reactive T cells ready for patient infusion. The adoptive cell transfer was performed without complication. Consequently, the clinical course stabilized and the patient slowly went into remission of PML with JCV negative CSF and containment of PML lesion expansion. CONCLUSION: We report for the first time feasibility of generating T cells with possible anti-JCV activity from a seropositive family donor, a variation of virus specific T-cell therapies suitable for the post allo transplant setting. We also present the unusual case for successful treatment of PML after allo-HCT via virus specific T-cell therapy.

The osteo-inductive activity of bone-marrow-derived mononuclear cells resides within the CD14+ population and is independent of the CD34+ population.

Bone marrow mononuclear cells (BMC) seeded on a scaffold of ß-tricalcium phosphate (ß-TCP) promote bone healing in a critical-size femur defect model. Being BMC a mixed population of predominantly mature haematopoietic cells, which cell type(s) is(are) instrumental for healing remains elusive. Although clinical therapies using BMC are often dubbed as stem cell therapies, whether stem cells are relevant for the therapeutic effects is unclear and, at least in the context of bone repair, seems dubious. Instead, in light of the critical contribution of monocytes and macrophages to tissue development, homeostasis and injury repair, in the current study it was hypothesised that BMC-mediated bone healing derived from the stem cell population. To test this hypothesis, bone remodelling studies were performed in an established athymic rats critical-size femoral defect model, with ß-TCP scaffolds augmented with complete BMC or BMC immunomagnetically depleted of stem cells (CD34+) or monocytes/macrophages (CD14+). Bone healing was assessed 8 weeks after transplantation. Compared to BMC-augmented controls, when CD14- BMC, but not CD34- BMC were transplanted into the bone defect, femora possessed dramatically decreased biomechanical stability and new bone formation was markedly reduced, as measured by histology. The degree of vascularisation did not differ between the two groups. It was concluded that the monocyte fraction within the BMC provided critical osteo-inductive cues during fracture healing. Which factors were responsible at the molecular levels remained elusive. However, this study marked a significant progress towards elucidating the mechanisms by which BMC elicit their therapeutic effects, at least in bone regeneration.

Automated isolation of primary antigen-specific T cells from donor lymphocyte concentrates: results of a feasibility exercise.

BACKGROUND: The safety and clinical efficacy of adoptive transfer of prospectively isolated antigen-specific T cells are well established. Several competing selection methods are available, one of which is based on immunomagnetic enrichment of T cells secreting IFNγ after incubation with the relevant antigen. The proprietary, GMP-conforming selection technology, called 'cytokine capture system' (CCS) is established in many laboratories for the CliniMACS Plus system. It is robust and efficient, but labour-intensive and incompatible with a single-shift working schedule. An automatic immunomagnetic cell processing system, CliniMACS Prodigy ('Prodigy'), including a protocol for fully automatic CCS execution was recently released. MATERIAL AND METHODS: Feasibility of clinical-scale CMV-specific T-cell selection using Prodigy was evaluated using leukoapheresis products from five healthy CMV sero-positive volunteers. Clinical reagents and consumables were used throughout. RESULTS: The process required no operator input beyond set-up and QC-sample collection, that is, feasibility was given. An IFNγ-secreting target T-cell population was detectable after stimulation, and >2 log-scale relative depletion of not CMV-reactive T cells in the target population was achieved. Purity, that is the frequency of CMV-reactive T cells among all CD3(+) cells ranged between 64 and 93%. CONCLUSION: The CCS protocol on Prodigy is unrestrictedly functional. It runs fully automatically beyond set-up and thus markedly reduces labour. The quality of the products generated is similar to products generated with CliniMACS Plus. The automatic system is thus suitable for routine clinical application.

Delayed onset of graft-versus-host disease in immunodeficent human leucocyte antigen-DQ8 transgenic, murine major histocompatibility complex class II-deficient mice repopulated by human peripheral blood mononuclear cells.

Haematopoietic humanization of mice is used frequently to study the human immune system and its reaction upon experimental intervention. Immunocompromised non-obese diabetic (NOD)-Rag1(-/-) mice, additionally deficient for the common gamma chain of cytokine receptors (γc) (NOD-Rag1(-/-) γc(-/-) mice), lack B, T and natural killer (NK) cells and allow for efficient human peripheral mononuclear cell (PBMC) engraftment. However, a major experimental drawback for studies using these mice is the rapid onset of graft-versus-host disease (GVHD). In order to elucidate the contribution of the xenogenic murine major histocompatibility complex (MHC) class II in this context, we generated immunodeficient mice expressing human MHC class II [human leucocyte antigen (HLA)-DQ8] on a mouse class II-deficient background (Aß(-/-) ). We studied repopulation and onset of GVHD in these mouse strains following transplantation of DQ8 haplotype-matched human PBMCs. The presence of HLA class II promoted the repopulation rates significantly in these mice. Virtually all the engrafted cells were CD3(+) T cells. The presence of HLA class II did not advance B cell engraftment, such that humoral immune responses were undetectable. However, the overall survival of DQ8-expressing mice was prolonged significantly compared to mice expressing mouse MHC class II molecules, and correlated with an increased time span until onset of GVHD. Our data thus demonstrate that this new mouse strain is useful to study GVHD, and the prolonged animal survival and engraftment rates make it superior for experimental intervention following PBMC engraftment.

Leucodepletion for hyperleucocytosis--first report on a novel technology featuring electronic interphase management.

BACKGROUND AND OBJECTIVES: Therapeutic leucodepletion plays an established role in the initial treatment of patients with acute myeloid leukaemia (AML) and possibly other leukaemias presenting with leucostasis. Recently, a new leucodepletion technology, Spectra Optia IDL, has become available that differs from its predecessor, COBE Spectra MNC, by a variety of electronic supports, including by electronic adjustment of buffy coat positioning at the collection port. Given the paucity of patients in need of leucodepletions and marked differences in clinical presentation as well as blast properties (e.g. size, density), formal clinical trials comparing leucodepletion technologies have never been executed. MATERIALS AND METHODS: Here, we present aggregate data from eight leucodepletions performed in AML patients with clinical signs of leucostasis between 11/2011 and 07/2012 with the new device and compare the apheresis outcomes with those from fifteen leucodepletions performed with the old technology between 06/2010 and 10/2011. RESULTS: Patients did not differ with respect to epidemiological data. Pre-apheresis leucocyte count (WBC) was significantly higher in Spectra Optia IDL patients. Tolerability was excellent with both devices. Basic apheresis denominators such as duration, processed volume, inlet pump rate, ACD-A consumption and product volume were very similar. A negative correlation between pre-apheresis WBC and collection efficiency was noted. Mean collection efficiency for leucocytes with Spectra Optia IDL (47·3%) was similar to that with COBE Spectra MNC (50·5%). Platelet attrition was similar with both devices, approximately 30%. CONCLUSION: The novel, electronically guided leukapheresis system is suitable for leucodepletion.

Safety and efficacy of healthy volunteer stem cell mobilization with filgrastim G-CSF and mobilized stem cell apheresis: results of a prospective longitudinal 5-year follow-up study.

BACKGROUND AND OBJECTIVES: G-CSF-mobilized peripheral blood stem cells have long replaced marrow as the major source for allogeneic transplants. Conclusive evidence questioning the long-term safety of G-CSF for donors has not been provided, but the cumulative number of followed donors remains insufficient to rule out rare adverse events. A long-term active follow-up study of G-CSF-mobilized healthy volunteer donors was therefore performed. PATIENTS AND METHODS: Two hundred and three successive donors were evaluated pre-apheresis, subjected to G-CSF-mobilization/apheresis, and actively followed for 5 years by the same physicians and laboratories. Follow-up laboratory work included standard biochemical/haematological tests and T-cell phenotyping. RESULTS: Donor epidemiology was typical for reported stem cell donor cohorts. Acute adverse effects of G-CSF and apheresis were mild and transient, consistent with the previous reports. Mean circulating CD34(+) cells after nine doses of G-CSF were 124 per µl. Other biochemical/haematological parameters were also altered, consistent with G-CSF treatment. Spleen enlargement was modest. At first follow-up, all clinical and laboratory parameters had normalized. Leucocyte/lymphocyte counts and CD4/CD8 ratios were the same as during premobilization work-up and remained unchanged throughout. A single severe but likely unrelated adverse event, a case of papillary thyroid carcinoma, was reported. CONCLUSION: The studies add an observation time of almost 500 donor years to the growing body of evidence of the long-term safety of G-CSF for allogeneic donor stem cell mobilization.

Mobilized allogeneic peripheral stem/progenitor cell apheresis with Spectra Optia v.5·0, a novel, automatic interface-controlled apheresis system: results from the first feasibility trial.

BACKGROUND AND OBJECTIVES: G-CSF mobilized peripheral blood stem/progenitor cells are frequently used for allogeneic transplantation. Available manual apheresis systems generate stem cell products of consistently high quality. Short-comings include need for continuous interface monitoring/adjustment, interface instability in donors with inconsistent blood flow, high collection variability, high platelet loss, and failure to electronically document apheresis parameters. MATERIAL AND METHODS: A fundamentally different, novel apheresis system, Spectra Optia v.5·0, featuring optical sensors, which provide real-time automatic interface and product collect line control, and newly developed tubing sets, was designed to address these short-comings. In a prospective validation study, 30 healthy volunteer stem cell donors were subjected to apheresis with Spectra Optia to test feasibility and effectiveness. Results were compared to 608 historic first-day allogeneic aphereses with COBE Spectra MNC. RESULTS: Usability and function of automatic interface control of Spectra Optia were good. Most collection parameters, including collection efficiency and product size, were similar. Cells were viable and provided timely engraftment. Platelet loss with Spectra Optia was 25% less than with COBE Spectra MNC. Products contained fewer erythrocytes, but more granulocytes. CONCLUSION: The automatic apheresis system Spectra Optia is functional and user-friendly. Thus Spectra Optia aphereses are associated with similar, and equally variable, collection efficiencies as COBE Spectra MNC.

[Potential of hematopoietic stem cells as the basis for generation of advanced therapy medicinal products]. / Potenzial hämatopoetischer Stammzellen als Ausgangsmaterial für Arzneimittel für neuartige Therapien.

Individualized, (stem) cell-based therapies of congenital and acquired illnesses are among the most exciting medical challenges of the twenty-first century. Before the full potential of such therapies can be achieved, many basic scientific and biotechnological questions remain to be answered. What is the ideal source for the generation of such cellular drugs is one of those issues. In many respects, hematopoietic stem cells fulfill the requirements for stem cells as starting material for novel cellular therapeutics, including the simple access to large amounts of stem cells, the availability of good phenotypic markers for their prospective isolation, and an extensive body of knowledge about the in vitro manipulation of these cells. This manuscript discusses the general and specific usability of hematopoietic stem cells as starting material for novel cellular therapeutics and presents some examples of hematological and nonhematological therapeutic approaches which are based on hematopoietic stem cells.

Accumulation of soluble inflammatory mediators between blood donation and pre-storage leucocyte depletion.

BACKGROUND AND OBJECTIVES: Leucocyte-derived cytokines accumulate in stored whole blood. Pre-storage leucocyte depletion has reduced cytokine levels and, consequently, febrile non-haemolytic transfusion reactions. As leucocyte filtration and component separation can be performed until 24 h after donation, we hypothesized that within this time, inflammatory cytokines might accumulate. MATERIALS AND METHODS: Serial plasma samples were collected 4, 10 and 20 h after donation and cytokine concentrations were measured. RESULTS: Interleukin-8 increased > 20-fold and soluble CD40 ligand > sixfold during the observation time, less pronounced changes for several other mediators were also observed. CONCLUSION: Leucocyte depletion within 10 h of blood donation will reduce the concentrations of pyrogenic mediators.

Autologous cell-based therapy for treatment of large bone defects: from bench to bedside.

OBJECTIVES: Reconstruction of long segmental bone defects is demanding for patients and surgeons, and associated with long-term treatment periods and substantial complication rates in addition to high costs. While defects up to 4-5 cm length might be filled up with autologous bone graft, heterologous bone from cadavers, or artificial bone graft substitutes, current options to reconstruct bone defects greater than 5 cm consist of either vascularized free bone transfers, the Masquelet technique or the Ilizarov distraction osteogenesis. Alternatively, autologous cell transplantation is an encouraging treatment option for large bone defects as it eliminates problems such as limited autologous bone availability, allogenic bone immunogenicity, and donor-site morbidity, and might be used for stabilizing loose alloplastic implants. METHODS: The authors show different cell therapies without expansion in culture, with ex vivo expansion and cell therapy in local bone defects, bone healing and osteonecrosis. Different kinds of cells and scaffolds investigated in our group as well as in vivo transfer studies and BMC used in clinical phase I and IIa clinical trials of our group are shown. RESULTS: Our research history demonstrated the great potential of various stem cell species to support bone defect healing. It was clearly shown that the combination of different cell types is superior to approaches using single cell types. We further demonstrate that it is feasible to translate preclinically developed protocols from in vitro to in vivo experiments and follow positive convincing results into a clinical setting to use autologous stem cells to support bone healing.

Transfer of minimally manipulated CMV-specific T cells from stem cell or third-party donors to treat CMV infection after allo-HSCT.

Cytomegalovirus (CMV) infection is a common, potentially life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We assessed prospectively the safety and efficacy of stem cell-donor- or third-party-donor-derived CMV-specific T cells for the treatment of persistent CMV infections after allo-HSCT in a phase I/IIa trial. Allo-HSCT patients with drug-refractory CMV infection and lacking virus-specific T cells were treated with a single dose of ex vivo major histocompatibility complex-Streptamer-isolated CMV epitope-specific donor T cells. Forty-four allo-HSCT patients receiving a T-cell-replete (D+ repl; n=28) or T-cell-depleted (D+ depl; n=16) graft from a CMV-seropositive donor were screened for CMV-specific T-cell immunity. Eight D+ depl recipients received adoptive T-cell therapy from their stem cell donor. CMV epitope-specific T cells were well supported and became detectable in all treated patients. Complete and partial virological response rates were 62.5% and 25%, respectively. Owing to longsome third-party donor (TPD) identification, only 8 of the 57 CMV patients transplanted from CMV-seronegative donors (D-) received antigen-specific T cells from partially human leukocyte antigen (HLA)-matched TPDs. In all but one, TPD-derived CMV-specific T cells remained undetectable. In summary, adoptive transfer correlated with functional virus-specific T-cell reconstitution in D+ depl patients. Suboptimal HLA match may counteract expansion of TPD-derived virus-specific T cells in D- patients.

NF-κB-dependent DNA damage-signaling differentially regulates DNA double-strand break repair mechanisms in immature and mature human hematopoietic cells.

Hematopoietic stem and progenitor cells (HSPC), that is, the cell population giving rise not only to all mature hematopoietic lineages but also the presumed target for leukemic transformation, can transmit (adverse) genetic events, such as are acquired from chemotherapy or ionizing radiation. Data on the repair of DNA double-strand-breaks (DSB) and its accuracy in HSPC are scarce, in part contradictory, and mostly obtained in murine models. We explored the activity, quality and molecular components of DSB repair in human HSPC as compared with mature peripheral blood lymphocytes (PBL). To consider chemotherapy/radiation-induced compensatory proliferation, we established cycling HSPC cultures. Comparison of pathway-specific repair activities using reporter systems revealed that HSPC were severely compromised in non-homologous end joining and homologous recombination but not microhomology-mediated end joining. We observed a more pronounced radiation-induced accumulation of nuclear 53BP1 in HSPC relative to PBL, despite evidence for comparable DSB formation from cytogenetic analysis and γH2AX signal quantification, supporting differential pathway usage. Functional screening excluded a major influence of phosphatidylinositol-3-OH-kinase (ATM/ATR/DNA-PK)- and p53-signaling as well as chromatin remodeling. We identified diminished NF-κB signaling as the molecular component underlying the observed differences between HSPC and PBL, limiting the expression of DSB repair genes and bearing the risk of an inaccurate repair.

'Sepsis' and multi-organ failure: predictors of poor outcome after hematopoietic stem cell transplantation in children.

Prognostic scores, such as the PRISM and APACHE II, have been established, predicting with reasonable accuracy the outcome of patients admitted to intensive care units (ICU). In keeping with previous reports, we found, however, that these scores failed to perform in a series of 28 recipients of hematopoietic auto- or allografts (BMT) who required ICU admission for reasons including respiratory (82%) and multi-organ (36%) failure. We therefore retrospectively analyzed the charts of these patients, evaluating predisposing factors and prognostic variables which might confound the validity of these ICU tools which in other clinical scenarios have proven so valuable. Of all the parameters tested, logistic analysis established the following as predictors for poor outcome: increased C-reactive protein (CRP) to > 10 mg/dl (P = 0.04), macroscopic hemorrhage (P = 0.04), hypotension (mean arterial pressure < normal) (P = 0.04) and GVHD > or = III (P = 0.002). Most of these factors are not accounted for by the standard prognostic questionnaires. The development of an 'oncological' or 'post-BMT' risk of mortality score, taking into account these patients' specific clinical problems, might improve the risk assessment for this patient group, and might thus facilitate the timely recognition of those patients most in need of more intensive therapeutic measures.

High interleukin-10 serum levels are associated with fatal outcome in patients after bone marrow transplantation.

IL-10 plays an important role in the control of immune reactions during systemic infection. Here, IL-10 serum levels were investigated in patients after BMT. The IL-10 levels correlated with the clinical course of the patients and with serum levels of C-reactive protein (CRP) and neopterin (NP). A total of 26 patients with AML (7), ALL (12), CML (2), NHL (3) and multifocal Ewing's sarcoma (2) had received autologous (10) or allogeneic (16) BMT from related (9) or unrelated donors (7). Routine serum samples were obtained prior to BMT and at days 46 and 100 after BMT. However, in patients with severe complications additional samples were drawn at individual points in time. Prior to BMT, IL-10 serum levels were not detectable in 24/24 patients. Post-BMT, 11 patients developed elevated IL-10 levels, of these eight died of complications (DOC), whereas only one of 15 patients with undetectable IL-10 died of complications, indicating that high IL-10 levels were significantly correlated with severe life-threatening complications (chi2, P < 0.01). To determine the pathomechanism and role of the increased IL-10 levels, they were correlated to the respective NP and CRP serum concentrations. CRP and NP concentrations were found significantly elevated in patients with detectable IL-10, indicating a severe acute phase reaction associated with macrophage activation. In conclusion, high IL-10 serum levels in patients after BMT were significantly associated with fatal outcome. Since IL-10 is a strong suppressor of T cell immunity, high IL-10 production in patients with severe complications such as septic shock or GVHD > grade II after BMT might lead to functional immunodeficiency contributing to the poor prognosis of these patients.