Extended characterization of anti-CD19 CAR T cell products manufactured at the point of care using the CliniMACS Prodigy system: comparison of donor sources and process duration.

BACKGROUND AIMS: The CliniMACS Prodigy closed system is widely used for the manufacturing of chimeric antigen receptor T cells (CAR-T cells). Our study presents an extensive immunophenotypic and functional characterization and comparison of the properties of anti-CD19 CAR-T cell products obtained during long (11 days) and short (7 days) manufacturing cycles using the CliniMACS Prodigy system, as well as cell products manufactured from different donor sources of T lymphocytes: from patients, from patients who underwent HSCT, and from haploidentical donors. We also present the possibility of assessing the efficiency of transduction by an indirect method. METHODS: Seventy-six CD19 CAR-T cell products were manufactured using the CliniMACS Prodigy automated system. Immunophenotypic properties, markers of cell activation and exhaustion, antitumor, anti-CD19 specific activity in vitro of the manufactured cell products were evaluated. As an indirect method for assessing the efficiency of transduction, we used the method of functional assessment of cytokine secretion and expression of the CD107a marker after incubation of CAR-T cells with tumor targets. RESULTS: The CliniMACS Prodigy platform can produce a product of CD19 CAR-T cells with sufficient cell expansion (4.6 × 109 cells-median for long process [LP] and 1.6 × 109-for short process [SP]), transduction efficiency (43.5%-median for LP and 41.0%-for SP), represented mainly by T central memory cell population, with low expression of exhaustion markers, and with high specific antitumor activity in vitro. We did not find significant differences in the properties of the products obtained during the 7- and 11-day manufacturing cycles, which is in favor of reducing the duration of production to 7 days, which may accelerate CAR-T therapy. We have shown that donor sources for CAR-T manufacturing do not significantly affect the composition and functional properties of the cell product. CONCLUSIONS: This study demonstrates the possibility of using the CliniMACS Prodigy system with a shortened 7-day production cycle to produce sufficient amount of functional CAR-T cells. CAR transduction efficiency can be measured indirectly via functional assays.

Automatic generation of alloreactivity-reduced donor lymphocytes and hematopoietic stem cells from the same mobilized apheresis product.

INTRODUCTION: In vitro or in vivo depletion of alloreactive T cells can facilitate haplo-identical hematopoietic stem cell transplantation (HSCT). Very satisfactory transplant outcomes were thus reported for TCRαß/CD19-depleted hematopoietic stem/progenitor cell (HSPC) grafts. The current semi-automatic manufacturing process on the CliniMACS Plus, although robust, still requires a significant amount of manual labor to be completed. Towards advancing and further facilitating large scale cell processing, a new TCRαß/CD19 depletion module combined with the previously described CD45RA depletion module (to serve as allo-reactivity attenuated donor lymphocyte infusion) was established on the CliniMACS Prodigy. METHODS: We evaluated six apheresis products from G-CSF-mobilized volunteer donors which were split automatically by the Prodigy, one portion each depleted of CD45RA+ or of TCRαß+ and CD19+ cells. We investigated critical quality attributes for both products. Products were assessed for recovery of HSPCs and mature subsets, as well as depletion efficiency of targeted cells using flow cytometry. Effects of apheresis and product age post 48 h storage at 2-6 °C as well as freeze-thawing on product viability and recovery of WBC and HPSCs were assessed by flow cytometry. RESULTS: Ten sequential automatic processes were completed with minimal hands-on time beyond tubing set installation. Depletion efficiency of CD45RA+ resp. TCRαß+ and CD19+ cells was equivalent to previous reports, achieving mean depletions of 4 log of targeted cells for both products. HSPC products retained TCRγδ+ and NK cells. 48 h storage of apheresis product was associated with the expected modest loss of HSPCs, but depletions remained efficient. Depleted products were stable until at least 72 h after apheresis with stem cell viabilities > 90%. Freeze-thawing resulted in loss of NK cells; post-thaw recovery of viable CD45+ and HSPCs was > 70% and in line with expectation. CONCLUSION: The closed, GMP-compatible process generates two separate medicinal products from the same mobilized apheresis product. The CD45RA-depleted products contained functional memory T cells, whereas the TCRαß/CD19-depleted products included HSPCs, TCRγδ+ and NK cells. Both products are predicted to be effectively depleted of GVH-reactivity while providing immunological surveillance, in support of haplo-identical HSCT.

Binding Affinity of Trastuzumab and Pertuzumab Monoclonal Antibodies to Extracellular HER2 Domain.

The binding affinity of trastuzumab and pertuzumab to HER2 has been studied using both experimental and in silico methods. The experiments were conducted using the antibodies in their complete IgG form, as used in clinical therapy, and the extracellular domain of the HER2 protein in solution. This approach provides a precise, reproducible, and reliable view of the interaction between them in physicochemical conditions similar to those found in the tumoral environment. Dynamic light scattering and size exclusion chromatography coupled with tetra detection were utilized to characterize the protein complexes, measure their concentrations, and calculate the equilibrium-free binding energy, ΔGbind. In addition, PRODIGY, a QSAR-like model with excellent predictive ability, was employed to obtain in silico ΔGbind estimations. The results obtained indicate that pertuzumab exhibits a slightly higher binding affinity to HER2 than trastuzumab. The difference in binding affinity was explained based on the contribution of the different interfacial contact (IC) descriptors to the ΔGbind value estimated by the PRODIGY model. Furthermore, experiments revealed that the pertuzumab IgG antibody binds preferentially to two HER2 proteins, one per Fab fragment, while trastuzumab mainly forms a monovalent complex. This finding was interpreted based on a geometrical model that identified steric crowding in the trastuzumab-HER2 complex as compared with the pertuzumab-HER2 complex.

Comparison of the Lunar Prodigy and Stratos DR Dual-Energy X-Ray Absorptiometers to Assess Regional Bone Mineral Density.

PURPOSE: The first objective of the study was to assess the agreement between the Stratos DR (DMS) and the GE Prodigy (GE) DXAs in determining femoral neck, total hip and lumbar spine aBMD. The second objective was to assess the potential impact of leg positioning (hip flexed at 90° or not) on lumbar spine aBMD. METHODS: Forty-six individuals (n=42 women, 91.3%), with a mean age of 59.7 ± 13 years and mean BMI of 23.8 ± 4.7 kg/m², were scanned consecutively on the same day using the two devices. In a subgroup (n=30), two consecutive Stratos DR scans (with hip flexed at 90° or not) at the lumbar spine were conducted. Predictive equations for hip and lumbar spine aBMD were derived from linear regression of the data. RESULTS: Correlation coefficients for aBMD measured with the two DXAs were characterised by an R² of 0.76 for the femoral neck, 0.89 for the total hip, and 0.86 for the lumbar spine. However, the derived equations for aBMD determination showed an intercept significantly different from 0 for hip aBMD, and a slope significantly different from 1 for lumbar spine aBMD. These results highlight a bias between the two measurements, thus requiring the determination of specific cross-calibration equations for hip and lumbar spine, femoral neck excepted. When compared with values on the Prodigy, mean aBMD on the Stratos DR was higher at the femoral neck (+4.8%, p<0.001) and total hip (+9.6%, p<0.001) and lower at L2-L4 (-8.8%, p<0.001). The coefficient of variation (CV%) for the two consecutive measures at lumbar spine (with different positioning) with the Stratos DR was 2.9%. CONCLUSIONS: The difference in aBMD measured with the two DXAs illustrates the need to define cross-calibration equations when comparing data across systems in order to avoid erroneous conclusions.

Cross-Calibration of Prodigy and Horizon A Densitometers and Precision of the Horizon A Densitometer.

We performed this study to enable a reliable transition for clinical study participants and patients from a GE Lunar Prodigy to a Hologic Horizon A dual-energy X-ray absorptiometry (DXA) scanner and to assess the reproducibility of measurements made on the new DXA scanner. Forty-five older adults had one spine, hip, and total body scan on a Prodigy dual-energy X-ray absorptiometry (DXA) scanner and 2 spine, hip, and total body scans, with repositioning, on a new Hologic Horizon A DXA scanner. Linear regression models were used to derive cross calibration equations for each measure on the 2 scanners. Precision (group root-mean-square average coefficient of variation) of bone mineral density (BMD) of the total hip, femoral neck, and lumbar spine (L1-L4), and total body fat, bone, and lean mass, appendicular lean mass, and trabecular bone score (TBS) was assessed using the International Society of Clinical Densitometry's (ISCD's) Advanced Precision Calculation Tool. Correlation coefficients for the BMD and body composition measures on the 2 scanners ranged from 0.94 to 0.99 (p<0.001). When compared with values on the Prodigy, mean BMD on the Horizon A was lower at each skeletal site (0.136 g/cm2 lower at the femoral neck and 0.169 g/cm2 lower at the lumbar spine (L1-4)), fat mass was 0.47 kg lower, and lean mass was 4.50 kg higher. Precision of the Horizon A scans was 1.60% for total hip, 1.94% for femoral neck, and 1.25% for spine (L1-4) BMD. Precision of TBS was 1.67%. Precision of total body fat mass was 2.16%, total body lean mass was 1.26%, appendicular lean mass was 1.97%, and total body bone mass was 1.12%. The differences in BMD and body composition values on the 2 scanners illustrate the importance of cross-calibration to account for these differences when transitioning clinical study participants and patients from one scanner to another.

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.

AIM Platform: A Novel Nano Artificial Antigen-Presenting Cell-Based Clinical System Designed to Consistently Produce Multi-Antigen-Specific T-Cell Products with Potent and Durable Anti-Tumor Properties.

Over the last decade, tremendous progress has been made in the field of adoptive cell therapy. The two prevailing modalities include endogenous non-engineered approaches and genetically engineered T-cell approaches. Endogenous non-engineered approaches include dendritic cell-based systems and tumor-infiltrating lymphocytes (TIL) that are used to produce multi-antigen-specific T-cell products. Genetically engineered approaches, such as T-cell receptor engineered cells and chimeric antigen receptor T cells are used to produce single antigen-specific T-cell products. It is noted by the authors that there are alternative methods to sort for antigen-specific T cells such as peptide multimer sorting or cytokine secretion assay-based sorting, both of which are potentially challenging for broad development and commercialization. In this review, we are focusing on a novel nanoparticle technology that generates a non-engineered product from the endogenous T-cell repertoire. The most common approaches for ex vivo activation and expansion of endogenous, non-genetically engineered cell therapy products rely on dendritic cell-based systems or IL-2 expanded TIL. Hurdles remain in developing efficient, consistent, controlled processes; thus, these processes still have limited access to broad patient populations. Here, we describe a novel approach to produce cellular therapies at clinical scale, using proprietary nanoparticles combined with a proprietary manufacturing process to enrich and expand antigen-specific CD8+ T-cell products with consistent purity, identity, and composition required for effective and durable anti-tumor response.

Manufacturing chimeric antigen receptor T cells: issues and challenges.

Clinical trials of adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have delivered unprecedented responses in patients with relapsed refractory B-cell malignancy. These results have prompted Food and Drug Administration (FDA) approval of two CAR T-cell products in this high-risk patient population. The widening range of indications for CAR T-cell therapy and increasing patient numbers present a significant logistical challenge to manufacturers aiming for reproducible delivery systems for high-quality clinical CAR T-cell products. This review discusses current and novel CAR T-cell processing methodologies and the quality control systems needed to meet the increasing clinical demand for these exciting new therapies.

Depletion of αß+ T and B Cells Using the CliniMACS Prodigy: Results of 10 Graft-Processing Procedures from Haploidentical Donors.

BACKGROUND: Depletion of TCRαß+ T cells and B cells with the CliniMACS Plus® has been used for haploidentical hematopoietic stem cell transplantation for a decade. The depletion procedure is time and labour demanding and with variable reported efficiencies. Recently, an automated procedure was launched for the CliniMACS Prodigy® (Miltenyi Biotec) but reported data are scarce. Here, we report the results of the first ten TCRαß+ and B cell depletion procedures for clinical use performed at our centre. MATERIALS AND METHODS: All transplants were from a parent to a child. Collection of peripheral blood stem cells was performed after filgrastim mobilisation by use of the Spectra Optia® (TerumoBCT) set on the MNC program. Because of insufficient hematopoietic stem cell mobilisation, 1 donor received additional plerixafor. RESULTS: We performed ten uncomplicated processes with the CliniMACS Prodigy. We found the results of the depletion procedures satisfactory with a median log reduction of TCRαß+ cells of -4.21 (range -3.98 to -4.74), resulting in a median number of TCRαß+ cells in the depleted product of 28.6 × 103/kg recipient weight (range 14.9-69.7 × 103/kg). The median CD34 recovery was 83% (range 70-100). To achieve a sufficient number of CD34+ cells, we performed an additional CD34+ enrichment procedure using the CliniMACS Plus for 3 patients. The B cell depletion was slightly less efficient with a median log reduction of -3.72 (range -2.83 to -4.20). CONCLUSION: Overall, we found the TCRαß and CD19 depletion procedure on the CliniMACS Prodigy easy to handle and reliable, providing reproducible good results.

Closed-system manufacturing of CD19 and dual-targeted CD20/19 chimeric antigen receptor T cells using the CliniMACS Prodigy device at an academic medical center.

BACKGROUND AIMS: Multiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion. Open processing steps that increase risk of contamination and production failure are required. This complex process requires skilled personnel and costly clean-room facilities and infrastructure. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients. METHODS: The CliniMACS Prodigy with TCT process software and the TS520 tubing set that allows closed-system processing for cell enrichment, transduction, washing and expansion was used. We used MACS-CD4 and CD8-MicroBeads for enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 ζ-cfrag vectors expressing scFv for CD19 or CD20/CD19 antigens for transduction, TexMACS medium-3%-HS-IL2 for culture and phosphate-buffered saline/ethylenediaminetetraacetic acid buffer for washing. Processing time was 13 days. RESULTS: Enrichment (N = 7) resulted in CD4/CD8 purity of 98 ± 4.0%, 55 ± 6% recovery and CD3+ T-cell purity of 89 ± 10%. Vectors at multiplicity of infection 5-10 resulted in transduction averaging 37%. An average 30-fold expansion of 108 CD4/CD8-enriched cells resulted in sufficient transduced T cells for clinical use. CAR-T cells were 82-100% CD3+ with a mix of CD4+ and CD8+ cells that primarily expressed an effector-memory or central-memory phenotype. Functional testing demonstrated recognition of B-cells and for the CAR-20/19 T cells, CD19 and CD20 single transfectants were recognized in cytotoxic T lymphocyte and interferon-γ production assays. DISCUSSION: The CliniMACS Prodigy device, tubing set TS520 and TCT software allow CAR-T cells to be manufactured in a closed system at the treatment site without need for clean-room facilities and related infrastructure.

Generation of alloreactivity-reduced donor lymphocyte products retaining memory function by fully automatic depletion of CD45RA-positive cells.

BACKGROUND AIMS: For patients needing allogeneic stem cell transplantation but lacking a major histocompatibility complex (MHC)-matched donor, haplo-identical (family) donors may be an alternative. Stringent T-cell depletion required in these cases to avoid lethal graft-versus-host disease (GVHD) can delay immune reconstitution, thus impairing defense against virus reactivation and attenuating graft-versus-leukemia (GVL) activity. Several groups reported that GVHD is caused by cells residing within the naive (CD45RA+) T-cell compartment and proposed use of CD45RA-depleted donor lymphocyte infusion (DLI) to accelerate immune reconstitution. We developed and tested the performance of a CD45RA depletion module for the automatic cell-processing device CliniMACS Prodigy and investigated quality attributes of the generated products. METHODS: Unstimulated apheresis products from random volunteer donors were depleted of CD45RA+ cells on CliniMACS Prodigy, using Good Manufacturing Practice (GMP)-compliant reagents and methods throughout. Using phenotypic and functional in vitro assays, we assessed the cellular constitution of CD45RA-depleted products, including T-cell subset analyses, immunological memory function and allo-reactivity. RESULTS: Selections were technically uneventful and proceeded automatically with minimal hands-on time beyond tubing set installation. Products were near-qualitatively CD45RA+ depleted, that is, largely devoid of CD45RA+ T cells but also of almost all B and natural killer cells. Naive and effector as well as γ/δ T cells were greatly reduced. The CD4:CD8 ratio was fivefold increased. Mixed lymphocyte reaction assays of the product against third-party leukocytes revealed reduced allo-reactivity compared to starting material. Anti-pathogen responses were retained. DISCUSSION: The novel, closed, fully GMP-compatible process on Prodigy generates highly CD45RA-depleted cellular products predicted to be clinically meaningfully depleted of GvH reactivity.

BKV-specific T cells in the treatment of severe refractory haemorrhagic cystitis after HLA-haploidentical haematopoietic cell transplantation.

BACKGROUND: Haemorrhagic cystitis caused by BK virus (BKV) is a known complication of allogeneic haematopoietic cell transplantation (HCT) and is relatively common following HLA-haploidentical transplantation. Adoptive immunotransfer of virus-specific T cells from the donor is a promising therapeutic approach, although production of these cells is challenging, particularly when dealing with low-frequency T cells such as BKV-specific T cells. CASE REPORT: Here, we present a patient who, following haploidentical HCT, developed severe BKV haemorrhagic cystitis, resistant to standard therapy. He responded well to adoptive transfer of donor cells enriched in BKV-specific T cells using the new second-generation CliniMACS Prodigy and the Cytokine Capture System from Miltenyi Biotec. Treatment led to full resolution of both the symptoms and viraemia without unwanted complications. CONCLUSION: Our observations suggest that use of products enriched with BKV-specific T cells generated using this system is safe and efficient in HLA-haploidentical HCT where BKV cystitis can be a serious complication.

Cognitive Expertise: An ALE Meta-Analysis.

Expert performance constitutes the endpoint of skill acquisition and is accompanied by widespread neuroplastic changes. To reveal common mechanisms of reorganization associated with long-term expertise in a cognitive domain (mental calculation, chess, language, memory, music without motor involvement), we used activation likelihood estimation meta-analysis and compared brain activation of experts to nonexperts. Twenty-six studies matched inclusion criteria, most of which reported an increase and not a decrease of activation foci in experts. Increased activation occurred in the left rolandic operculum (OP 4) and left primary auditory cortex and in bilateral premotor cortex in studies that used auditory stimulation. In studies with visual stimulation, experts showed enhanced activation in the right inferior parietal cortex (area PGp) and the right lingual gyrus. Experts' brain activation patterns seem to be characterized by enhanced or additional activity in domain-specific primary, association, and motor structures, confirming that learning is localized and very specialized.

Automation of cellular therapy product manufacturing: results of a split validation comparing CD34 selection of peripheral blood stem cell apheresis product with a semi-manual vs. an automatic procedure.

BACKGROUND: Automation of cell therapy manufacturing promises higher productivity of cell factories, more economical use of highly-trained (and costly) manufacturing staff, facilitation of processes requiring manufacturing steps at inconvenient hours, improved consistency of processing steps and other benefits. One of the most broadly disseminated engineered cell therapy products is immunomagnetically selected CD34+ hematopoietic "stem" cells (HSCs). METHODS: As the clinical GMP-compliant automat CliniMACS Prodigy is being programmed to perform ever more complex sequential manufacturing steps, we developed a CD34+ selection module for comparison with the standard semi-automatic CD34 "normal scale" selection process on CliniMACS Plus, applicable for 600 × 10(6) target cells out of 60 × 10(9) total cells. Three split-validation processings with healthy donor G-CSF-mobilized apheresis products were performed; feasibility, time consumption and product quality were assessed. RESULTS: All processes proceeded uneventfully. Prodigy runs took about 1 h longer than CliniMACS Plus runs, albeit with markedly less hands-on operator time and therefore also suitable for less experienced operators. Recovery of target cells was the same for both technologies. Although impurities, specifically T- and B-cells, were 5 ± 1.6-fold and 4 ± 0.4-fold higher in the Prodigy products (p = ns and p = 0.013 for T and B cell depletion, respectively), T cell contents per kg of a virtual recipient receiving 4 × 10(6) CD34+ cells/kg was below 10 × 10(3)/kg even in the worst Prodigy product and thus more than fivefold below the specification of CD34+ selected mismatched-donor stem cell products. The products' theoretical clinical usability is thus confirmed. CONCLUSIONS: This split validation exercise of a relatively short and simple process exemplifies the potential of automatic cell manufacturing. Automation will further gain in attractiveness when applied to more complex processes, requiring frequent interventions or handling at unfavourable working hours, such as re-targeting of T-cells.

Automated manufacturing of chimeric antigen receptor T cells for adoptive immunotherapy using CliniMACS prodigy.

Novel cell therapies derived from human T lymphocytes are exhibiting enormous potential in early-phase clinical trials in patients with hematologic malignancies. Ex vivo modification of T cells is currently limited to a small number of centers with the required infrastructure and expertise. The process requires isolation, activation, transduction, expansion and cryopreservation steps. To simplify procedures and widen applicability for clinical therapies, automation of these procedures is being developed. The CliniMACS Prodigy (Miltenyi Biotec) has recently been adapted for lentiviral transduction of T cells and here we analyse the feasibility of a clinically compliant T-cell engineering process for the manufacture of T cells encoding chimeric antigen receptors (CAR) for CD19 (CAR19), a widely targeted antigen in B-cell malignancies. Using a closed, single-use tubing set we processed mononuclear cells from fresh or frozen leukapheresis harvests collected from healthy volunteer donors. Cells were phenotyped and subjected to automated processing and activation using TransAct, a polymeric nanomatrix activation reagent incorporating CD3/CD28-specific antibodies. Cells were then transduced and expanded in the CentriCult-Unit of the tubing set, under stabilized culture conditions with automated feeding and media exchange. The process was continuously monitored to determine kinetics of expansion, transduction efficiency and phenotype of the engineered cells in comparison with small-scale transductions run in parallel. We found that transduction efficiencies, phenotype and function of CAR19 T cells were comparable with existing procedures and overall T-cell yields sufficient for anticipated therapeutic dosing. The automation of closed-system T-cell engineering should improve dissemination of emerging immunotherapies and greatly widen applicability.

Fully automated expansion and activation of clinical-grade natural killer cells for adoptive immunotherapy.

BACKGROUND AIMS: Ex vivo expansion of natural killer (NK) cells is a strategy to produce large numbers of these effector cells for immunotherapy. However, the transfer of bench-top expansion protocols to clinically applicable methods is challenging for NK cell-based therapy because of regulatory aspects and scale-up issues. Therefore, we developed an automated, large-scale NK cell expansion process. METHODS: Enriched NK cells were expanded with interleukin-2 and irradiated clinical-grade Epstein-Barr virus-transformed lymphoblastoid feeder cells with the use of an automated system in comparison to manual expansion, and the cells were investigated for their functionality, phenotype and gene expression. RESULTS: Automated expansion resulted in a mean 850-fold expansion of NK cells by day 14, yielding 1.3 (± 0.9) × 10(9) activated NK cells. Automatically and manually produced NK cells were comparable in target cell lysis, degranulation and production of interferon-γ and tumor necrosis factor-α and had similar high levels of antibody-dependent cellular cytotoxicity against rituximab-treated leukemic cells. NK cells after automated or manual expansion showed similar gene expression and marker profiles. However, expanded NK cells differed significantly from primary NK cells including upregulation of the functional relevant molecules TRAIL and FasL and NK cell-activating receptors NKp30, NKG2D and DNAM-1. Neither automatically nor manually expanded NK cells showed reduced telomere length indicative of a conserved proliferative potential. CONCLUSIONS: We established an automated method to expand high numbers of clinical-grade NK cells with properties similar to their manually produced counterparts. This automated process represents a highly efficient tool to standardize NK cell processing for therapeutic applications.

Automated CD34+ cell isolation of peripheral blood stem cell apheresis product.

BACKGROUND AIMS: Immunomagnetic enrichment of CD34+ hematopoietic "stem" cells (HSCs) using paramagnetic nanobead coupled CD34 antibody and immunomagnetic extraction with the CliniMACS plus system is the standard approach to generating T-cell-depleted stem cell grafts. Their clinical beneficence in selected indications is established. Even though CD34+ selected grafts are typically given in the context of a severely immunosuppressive conditioning with anti-thymocyte globulin or similar, the degree of T-cell depletion appears to affect clinical outcomes and thus in addition to CD34 cell recovery, the degree of T-cell depletion critically describes process quality. An automatic immunomagnetic cell processing system, CliniMACS Prodigy, including a protocol for fully automatic CD34+ cell selection from apheresis products, was recently developed. We performed a formal process validation to support submission of the protocol for CE release, a prerequisite for clinical use of Prodigy CD34+ products. METHODS: Granulocyte-colony stimulating factor-mobilized healthy-donor apheresis products were subjected to CD34+ cell selection using Prodigy with clinical reagents and consumables and advanced beta versions of the CD34 selection software. Target and non-target cells were enumerated using sensitive flow cytometry platforms. RESULTS: Nine successful clinical-scale CD34+ cell selections were performed. Beyond setup, no operator intervention was required. Prodigy recovered 74 ± 13% of target cells with a viability of 99.9 ± 0.05%. Per 5 × 10E6 CD34+ cells, which we consider a per-kilogram dose of HSCs, products contained 17 ± 3 × 10E3 T cells and 78 ± 22 × 10E3 B cells. CONCLUSIONS: The process for CD34 selection with Prodigy is robust and labor-saving but not time-saving. Compared with clinical CD34+ selected products concurrently generated with the predecessor technology, product properties, importantly including CD34+ cell recovery and T-cell contents, were not significantly different. The automatic system is suitable for routine clinical application.

Cross-calibration of a GE iDXA and Prodigy for total and regional body bone parameters: the importance of using cross-calibration equations for longitudinal monitoring after a system upgrade.

We aimed to determine if cross-calibration equations could be applied to convert GE Lunar Prodigy total and regional bone measurements to the GE iDXA model to support longitudinal monitoring of subjects. The cross-calibration group comprised 63 adults (age 45.1 [12.8] yr; body mass index: 25.6 [3.7] kg/m(2)) and the validation group comprised 25 adults (age 40.5 [11.5] yr; body mass index: 25.7 [3.5] kg/m(2)). The parameters reported were total and regional bone mineral density (BMD), bone mineral content, and bone area. There were significant differences between densitometers for all anatomical regions and reported bone parameters (p < 0.0001); iDXA reported lower BMD than the Prodigy apart from the ribs. Linear regression indicated good agreement for all measurements. Bland-Altman analyses indicated significant bias for all measurements and that cross-calibration equations were required. The derived cross-calibration equations were effective in reducing differences between predicted and measured results for each parameter and at each region apart from leg BMD, where the difference remained significant (0.013 g/cm(2); p < 0.05). Our results indicate that cross-calibration is important to maintain comparability of total body-derived regional bone measurements between the Lunar Prodigy and iDXA.

Effective virus-specific T-cell therapy for high-risk SARS-CoV-2 infections in hematopoietic stem cell transplant recipients: initial case studies and literature review.

The COVID-19 pandemic has exacerbated mortality rates among immunocompromised patients, accentuating the need for novel, targeted therapies. Transplant recipients, with their inherent immune vulnerabilities, represent a subgroup at significantly heightened risk. Current conventional therapies often demonstrate limited effectiveness in these patients, calling for innovative treatment approaches. In immunocompromised transplant recipients, several viral infections have been successfully treated by adoptive transfer of virus-specific T-cells (VST). This paper details the successful application of SARS-CoV-2-specific memory T-cell therapy, produced by an interferon-γ cytokine capture system (CliniMACS® Prodigy device), in three stem cell transplant recipients diagnosed with COVID-19 (case 1: alpha variant, cases 2 and 3: delta variants). These patients exhibited persistent SARS-CoV-2 PCR positivity accompanied by bilateral pulmonary infiltrates and demonstrated only partial response to standard treatments. Remarkably, all three patients recovered and achieved viral clearance within 3 to 9 weeks post-VST treatment. Laboratory follow-up investigations identified an increase in SARS-CoV-2-specific T-cells in two of the cases. A robust anti-SARS-CoV-2 S (S1/S2) IgG serological response was also recorded, albeit with varying titers. The induction of memory T-cells within the CD4 + compartment was confirmed, and previously elevated interleukin-6 (IL-6) and IL-8 levels normalized post-VST therapy. The treatment was well tolerated with no observed adverse effects. While the need for specialized equipment and costs associated with VST therapy present potential challenges, the limited treatment options currently available for COVID-19 within the allogeneic stem cell transplant population, combined with the risk posed by emerging SARS-CoV-2 mutations, underscore the potential of VST therapy in future clinical practice. This therapeutic approach may be particularly beneficial for elderly patients with multiple comorbidities and weakened immune systems.

Cognitive computing technological trends and future research directions in healthcare - A systematic literature review.

BACKGROUND AND AIM: Cognitive Computing systems are the intelligent systems that thinks, understands and augments the capabilities of human brain by blending the technologies of Artificial Intelligence, Machine Learning and Natural Language Processing. In recent days, maintenance or enhancement of health by preclusion, prognosis, and analysis of diseases has become a challenging task. The increasing diseases and its causes becomes a big question before humanity. Limited risk analysis, meticulous training process, and automated critical decision-making are some of the issues of cognitive computing. To overcome this issue, cognitive computing in healthcare works like a medical prodigy which anticipates the disease or illness of the human being and helps the doctors with technological facts to take the timely action. The main aim of this survey article is to explore the present and futuristic technological trends of cognitive computing in healthcare. In this work, different cognitive computing applications are reviewed, and the best application is recommended to the clinicians. Based on this recommendation, the clinicians are able to monitor and analyze the physical health of patients. METHODS: This article presents the systematic literature on the different aspects of cognitive computing in healthcare. Nearly seven online databases such as SCOPUS, IEEE Xplore, Google Scholar, DBLP, Web of Science, Springer and PubMed were screened and the published articles related to cognitive computing in healthcare is collected from 2014 to 2021. In total, 75 articles were selected, examined and their pros and cons are analyzed. The analysis is done with respect to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. RESULTS: The basic findings of this review article and their significance for theory and practice are mindmaps portraying the cognitive computing platforms, cognitive applications in healthcare, and use cases of cognitive computing in healthcare. A detailed discussion section highlighting the present issues, future research directions and recent applications of cognitive computing in healthcare. Accuracy analysis of different cognitive systems conclude that the Medical Sieve achieves 0.95 and Watson For Oncology (WFO) achieves 0.93 and hence proves to be the prominent computing systems for healthcare. CONCLUSIONS: Cognitive computing, an evolving technology in healthcare augments the clinical thought process and enable the doctors to make the right diagnosis and preserve the patient's health in good condition. These systems provides timely care, optimal and cost-effective treatment. This article provides an extensive survey of the importance of cognitive computing in the health sector by highlighting the platforms, techniques, tools, algorithms, applications, and use cases. This survey also explores about the works in the literature on present issues and proposes the future research directions of applying cognitive systems in healthcare.