Artículos de revistas: "GMP-Clinical Grade Processing"

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Wright, Adrienne, Orman L. Snyder, Lane K. Christenson, Hong He y Mark L. Weiss. "Effect of Pre-Processing Storage Condition of Cell Culture-Conditioned Medium on Extracellular Vesicles Derived from Human Umbilical Cord-Derived Mesenchymal Stromal Cells". International Journal of Molecular Sciences 23, n.º 14 (13 de julio de 2022): 7716. http://dx.doi.org/10.3390/ijms23147716.

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EVs can be isolated from a conditioned medium derived from mesenchymal stromal cells (MSCs), yet the effect of the pre-processing storage condition of the cell culture-conditioned medium prior to EV isolation is not well-understood. Since MSCs are already in clinical trials, the GMP-grade of the medium which is derived from their manufacturing might have the utility for preclinical testing, and perhaps, for clinical translation, so the impact of pre-processing storage condition on EV isolation is a barrier for utilization of this MSC manufacturing by-product. To address this problem, the effects of the pre-processing storage conditions on EV isolation, characterization, and function were assessed using a conditioned medium (CM) derived from human umbilical cord-derived MSCs (HUC-MSCs). Hypothesis: The comparison of three different pre-processing storage conditions of CM immediately processed for EV isolation would reveal differences in EVs, and thus, suggest an optimal pre-processing storage condition. The results showed that EVs derived from a CM stored at room temperature, 4 °C, −20 °C, and −80 °C for at least one week were not grossly different from EVs isolated from the CM immediately after collection. EVs derived from an in pre-processing −80 °C storage condition had a significantly reduced polydispersity index, and significantly enhanced dot blot staining, but their zeta potential, hydrodynamic size, morphology and size in transmission electron microscopy were not significantly different from EVs derived from the CM immediately processed for isolation. There was no impact of pre-processing storage condition on the proliferation of sarcoma cell lines exposed to EVs. These data suggest that the CM produced during GMP-manufacturing of MSCs for clinical applications might be stored at −80 °C prior to EV isolation, and this may enable production scale-up, and thus, and enable preclinical and clinical testing, and EV lot qualification.

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Assenmacher, Mario, Nadine Mockel-Tenbrinck, Alexander Scheffold, Georg Rauser, Hermann Bohnenkamp, Jürgen Schmitz, Uwe Odenthal, Bergendahl Veit, Ulrike Kolrep y Melanie Fahrendorff. "New GMP-Grade, Xeno-Component Free Medium for the Activation and Expansion of T Cells". Blood 118, n.º 21 (18 de noviembre de 2011): 4316. http://dx.doi.org/10.1182/blood.v118.21.4316.4316.

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Abstract Abstract 4316 Therapeutic applications of T cells in immunotherapy have recently gained momentum with the promising results in adoptive transfer of antigen-specific T cells for infectious complications after allogeneic stem cell or solid organ transplantation or for immunotherapy of malignant diseases. Activation and expansion of these cells for clinical application under controlled conditions require GMP-grade reagents including appropriate antibodies, cytokines and media. For standardized, reproducible cell cultivation and ex vivo differentiation procedures, a new serum and xeno-component free, GMP-grade medium for clinical use has been developed: High lot-to-lot consistency has been achieved by eliminating protein components not relevant for T cell expansion leaving human serum albumin as the only protein component. The expansion of T cells in this medium upon polyclonal activation using biotinylated antibodies against CD2, CD3 and CD28 loaded on anti-Biotin coated beads resulted in expansion rates similar to other commercially available serum-free media. Using soluble antibodies against CD3 and CD28, more than 30%-higher expansion rates of viable and functional T cells after 6 days of expansion have been achieved with the new xeno-component free medium compared with other serum-fee media. Transferring the same protocol to a high density cell culture system such as a gas permeable rapid expansion device, high densities of T cells with more than 1.5×107 cells/ mL were reached. The generation of antigen-specific T cells using the Cytokine Capture System (IFN-gamma) and the serum and xeno-component free T cell medium showed similar results regarding purity, recovery and background stimulation compared to the use of a standard basal medium supplemented with 10% human AB serum. For the automation of such complex procedures, a new cell processing device was developed. All steps for the antigen-specific T cell processing, i.e. antigen-specific re-stimulation, magnetic enrichment, and in vitro expansion with this T cell medium are performed in this fully automated device, in a closed system under sterile conditions. In conclusion, the newly developed GMP-grade, serum and xeno-component free T cell medium demonstrated high lot-to-lot consistency and was superior in its performance to other commercially available serum-free media in high density cell culture systems. The new medium can be used to replace human AB serum supplementation for the clinical manufacturing of T cells resulting in easier handling and higher consistency. Disclosures: Assenmacher: Miltenyi Biotec GmbH: Employment. Mockel-Tenbrinck:Miltenyi Biotec GmbH: Employment. Scheffold:Miltenyi Biotec GmbH: Employment. Rauser:Miltenyi Biotec GmbH: Employment. Bohnenkamp:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment. Odenthal:Miltenyi Biotec GmbH: Employment. Veit:Miltenyi Biotec GmbH: Employment. Kolrep:Miltenyi Biotec GmbH: Employment. Fahrendorff:Miltenyi Biotec GmbH: Employment.

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Radke, Teja F., Anja Buchheiser, Aurélie Lefort, Mahtab Maleki, Peter Wernet y Gesine Kögler. "GMP-Conform Generation and Cultivation of Unrestricted Somatic Stems Cells (USSC) from Cord Blood Using the SEPAX©-Separation Method a Closed Culture System Applying Cell Stacks." Blood 110, n.º 11 (16 de noviembre de 2007): 1211. http://dx.doi.org/10.1182/blood.v110.11.1211.1211.

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Abstract Generation and characterization of unrestricted somatic stem cells (USSC) from cord blood (CB) was described by our group and has been well established under laboratory conditions [Koegler et al 2004, 2005 and 2006; Sensken et al 2007]. Due to their proliferative and differentiation capacity, USSCs are an interesting candidate for the future development of cellular therapy for tissue repair and tissue regeneration as well as a supportive cell layer to support hematopoietic reconstitution. Since generation and expansion under GMP-grade conditions is mandatory for use in clinical application, the automated cell processing system Sepax (BIOSAFE) with the CS900 separation kit was used for mononuclear cell separation and the subsequent generation of the USSC colonies in the presence of 30% GMP-grade fetal calf serum (Perbio), low-glucose DMEM-medium/10-7M dexamethasone. Expansion of USSC was performed in a closed system (Macopharma) applying cell stacks (Costar Corning). Results achieved so far indicate that the generation frequency and quality of generated USSC under GMP conditions are equal or even superior (45%) to manual generation under laboratory conditions (43%). 20 cord-blood units (mean volume 88,5 +− 15,8 ml; mean number of mononuclear cells 3,1 +−0,6 *108 MNC) have been processed, resulting in 9 USSC-colony formations and lines within 14–28 days. Growth kinetics is equal to the previously established USSC-lines (∼36–48 h / population doubling). Analysis of the immunophenotype as well as the differentiation potential towards the mesenchymal, neural and endodermal lineages also showed no difference to those lines generated manually using Ficoll-separation and normal cell culture flasks (Costar Corning T225). The closed system applied here is perfectly suitable to ensure safe and easy handling of the USSC, including seeding, trypsination and harvesting. In combination with the cell stack system (1, 2, 5 and 10 layers), cell amounts of more than 1.0×109 USSC can be achieved within 4 passages. These USSC products were temperature controlled cryopreserved in the presence of 10% DMSO, HSA and dextran. USSC can be thawed and further expanded in clinical grade quality. On the basis of their pluripotency and expansion under GMP-conditions into large quantities, these USSC from cord blood, when pretested for infectious agents and matched for the major transplantation antigens, may serve as a universal allogeneic stem cell source for tissue repair and tissue regeneration.

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Gaja, Vijay, Jacqueline Cawthray, Clarence R. Geyer y Humphrey Fonge. "Production and Semi-Automated Processing of 89Zr Using a Commercially Available TRASIS MiniAiO Module". Molecules 25, n.º 11 (5 de junio de 2020): 2626. http://dx.doi.org/10.3390/molecules25112626.

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The increased interest in 89Zr-labelled immunoPET imaging probes for use in preclinical and clinical studies has led to a rising demand for the isotope. The highly penetrating 511 and 909 keV photons emitted by 89Zr deliver an undesirably high radiation dose, which makes it difficult to produce large amounts manually. Additionally, there is a growing demand for Good Manufacturing Practices (GMP)-grade radionuclides for clinical applications. In this study, we have adopted the commercially available TRASIS mini AllinOne (miniAiO) automated synthesis unit to achieve efficient and reproducible batches of 89Zr. This automated module is used for the target dissolution and separation of 89Zr from the yttrium target material. The 89Zr is eluted with a very small volume of oxalic acid (1.5 mL) directly over the sterile filter into the final vial. Using this sophisticated automated purification method, we obtained satisfactory amount of 89Zr in high radionuclidic and radiochemical purities in excess of 99.99%. The specific activity of three production batches were calculated and was found to be in the range of 1351–2323 MBq/µmol. ICP-MS analysis of final solutions showed impurity levels always below 1 ppm.

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Van der Loo, Johannes C. M., William Swaney, Diana Nordling, Axel Schambach, Christopher Baum, David A. Williams, Lilith Reeves y Punam Malik. "Production of High Titer cGMP-Grade SIN Gamma-Retroviral Vectors by Transfection in a Closed System Bioreactor". Blood 112, n.º 11 (16 de noviembre de 2008): 3539. http://dx.doi.org/10.1182/blood.v112.11.3539.3539.

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Abstract The need for gamma-retroviral vectors with self-inactivating (SIN) long terminal repeats for clinical trials has prompted a shift in the method with which large scale GMP-grade vectors are produced, from the use of stable producer lines to transient transfection-based techniques. The main challenge of instituting this methodology was to develop SIN retrovirus vectors that produced high amounts of genomic vector RNA in packaging cells, and to design scalable processes for closed system culture, transfection and virus harvest. Using improved expression plasmids, the Vector Production Facility, an academic GMP manufacturing laboratory that is part of the Translational Cores at Cincinnati Children’s Hospital, has developed such a method based on the Wave Bioreactor® production platform. In brief, cells from a certified 293T master cell bank are expanded, mixed with transfection reagents, and pumped into a 2, 10 or 20 Liter Wave Cell Bag containing FibraCel® discs. Cells are cultured in DMEM with GlutaMax® and 10% FBS at 37°C, 5% CO2 at a rocking speed of 22 rpm and 6° angle. At 16–20 hrs post-transfection, the media is changed; virus is harvested at 12-hour intervals, filtered through a leukocyte reduction filter, aliquoted into Cryocyte freezing containers, and frozen at or below −70°C. Several processing parameters, including the confluency of cells harvested prior to transfection, the timing of transfection, the amount of plasmid DNA, exposure of cells to PBS/TrypLESelect, and the timing of the media change post-transfection affected vector titer. Mixing cells with plasmid and transfection mixture prior to seeding onto FibraCel, as compared to transfecting cells 1-day post-seeding (as is standard when using tissue culture plastic) increased the titer from 104 to 4 × 105 IU/mL. Similarly, increasing the amount of plasmid DNA per mL from 4.6 to 9.2 μg doubled the titer in the Wave, while it reduced titer by 20–40% in tissue culture flasks (Fig. 1). Using an optimized protocol, six cGMP-grade SIN gamma-retroviral vectors have now been produced in support of the FDA’s National Toxicology Program (NTP), with unconcentrated vector titers ranging from 1 × 106 to as high as 4 × 107 IU/mL. Using similar processing, we have produced a large scale SIN gamma-retroviral vector (GALV pseudotyped) for an international X-linked SCID trial with average unconcentrated titers of 106 IU/mL in all viral harvests. In summary, the process developed at the Cincinnati Children’s Hospital Vector Production Facility allows for large scale closed-system production of high-titer retroviral vectors for clinical trials using transient transfection. Figure Figure

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Parchment, Ralph E., Robert J. Kinders, Jiuping Jay Ji, Apurva K. Srivastava, Katherine V. Ferry-Galow, Joseph E. Tomaszewski y James H. Doroshow. "Creating clinical target validation groups via quality assured transfer of robust clinical pharmacodynamic (PD) assays from the NCI: Clinical implementation of a PAR immunoassay in tumor biopsies." Journal of Clinical Oncology 31, n.º 15_suppl (20 de mayo de 2013): e22080-e22080. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e22080.

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e22080 Background: Early phase clinical trials of investigational agents benefit from laboratory assays that quantify intended mechanism of action on molecular target (1° PD effects), desired changes in mechanistic biomarkers (2° PD effects), and 3° PD effects on cell survival. Robust PD assay results are valuable for informing go/no‐go decisions about continued development of new agents and for identifying combinations of targeted agents that cover the multiple molecular defects underlying many malignancies. Methods: Readying PD assays for clinical use involves validating analytical performance, identifying and qualifying critical reagents, demonstrating fit‐for‐purpose for the clinical protocol, and finalizing companion SOPs specifying specimen handling and processing. Because clinical PD questions often demand assay performance that meets or even exceeds clinical diagnostic assay standards, but key assay reagents are usually R and D‐rather than GMP‐grade, stringent reagent Quality Control is critical for preventing assay failures due to lot-to-lot variability. Results: The poly(ADP-ribose) (PAR) Immunoassay was the first qualified assay developed by NCI for implementation in early phase clinical trials. Development of stringent production and internal Quality Control specifications for accepting/rejecting new lots of critical reagents and a defined Quality Assurance Plan allowed a network of users to achieve consistent results and quality using R&D-grade source materials. Conclusions: The NCI’s Division of Cancer Treatment and Diagnosis is developing a clinical PD assay portfolio capable of quantifying 1°, 2°, and 3° PD effects. Proven clinical assays are formally transferred from the NCI to requesting sites in academia and industry via laboratory‐based certification and training, centralized access to SOPs, assistance with assay transfer, and participation in the assay’s Quality Assurance Plan. Funded by NCI Contract No HHSN261200800001E.

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Becker, Fabienne, Rigveda Bhave, Soraia Martins, Melanie Hühne, Boris Greber y Gesine Kogler. "Abstract 12: Cord Blood Banking, Technical and Clinical grade GMP- Development of Advanced Therapy Products as HLA-Homozygous iPSC-derived Cardiomyocytes". Stem Cells Translational Medicine 13, Supplement_1 (21 de agosto de 2024): S14. http://dx.doi.org/10.1093/stcltm/szae062.012.

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Abstract Introduction The Cord Blood Bank (CBB) in Düsseldorf stores 21477 active cryopreserved cord blood units (CBUs) licensed by the Paul-Ehrlich Institute, with 1517 transplants delivered worldwide. We have previously documented a stable storage time of 29 years in liquid nitrogen (Liedtke et al. 2024). Since establishing confirmatory typing and high-resolution HLA sequencing for CBUs and segments in 2016, the CBB progressed into advanced cellular therapies with high-qualified source material and applicable grants (BMBF-161B0760B and HEAL-101056712). Production permission for isolation and short-term expansion of CD34+ cells from licensed CBUs was granted, allowing the GMP reprogramming of 17 HLA-homozygous lines in collaboration with CATALENT (Terheyden-Keighley et al. 2024, accepted in SCTM). Objectives As part of the HEAL project, CD34+ HSC-derived iPSCs were differentiated into cardiomyocytes (CM) using suspension cultures to produce CM-aggregates instead of single cells, as studies in non-human primates indicate better engraftment of aggregates than single cells (Gruh et al. 2024). An optimized cryopreservation process for CM-aggregates is under development applying techniques available to CBU banking. Methods iPSCs were differentiated into CM-aggregates via an improved WNT pathway modulation protocol in suspension culture. In addition to the WNT inhibitor CHIR99021, BMP, Activin A and FGF-2 were added to target accessory pathways and achieve robust scalability across different platforms. Following differentiation, the CM-aggregates were cryopreserved in 10% DMSO with HSA or KO-SR supplementation using a controlled-rate freezer adapted to tissue freezing to ensure uniform freezing processes. Results The improved protocol allowed for straightforward upscaling of CM-aggregate differentiation from 6-well plates to Erlenmeyer flasks of different volumes, leading to a 1-2 fold yield of CM-aggregates with a purity of up to 95 % cardiac troponin T (cTnT) and sarcomeric actinin (sAct). Post-cryopreservation recovery of aggregates was between 60-90% with a viability of ~90%. Although CM culture after thawing remains problematic (<60% CM recovery after 5 days), the aggregates restored their morphology and function. Discussion The CBU banking, processing and storage should meet quality and release criteria, including genetic testing, allowing both allogeneic and related CBU selection to provide the best possible starting material. These advanced therapy products can be utilized e.g. for cardiomyocyte production for potential clinical applications. Overview of a GMP-process development for advanced therapy products from selected HLA-homozygous Cord Blood Units

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Zandvliet, Maarten L., J. H. Frederik Falkenburg, Inge Jedema, Roelof Willemze, Henk-Jan Guchelaar y Pauline Meij. "Generation of GMP-Grade CMV pp65-Specific CD8+ and CD4+ Donor T Cell Lines for Treatment of CMV Reactivation after Transplantation." Blood 108, n.º 11 (16 de noviembre de 2006): 2931. http://dx.doi.org/10.1182/blood.v108.11.2931.2931.

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Abstract Reactivation of CMV remains a major cause of morbidity and mortality in immunocompromised recipients of allogeneic stem cell transplantation. Antiviral pharmacotherapy may not be sufficient due to significant toxicity and moderate efficacy. It has been shown that adoptive transfer of donor-derived CMV-specific T cells may be an effective strategy to control established CMV infection. For a persistent function in vivo the presence of both virus-specific CD8+ and CD4+ T cells is essential. Therefore, we developed an optimized protocol for the generation of CMV pp65-specific CD8+ and CD4+ T cell lines which is fully compliable with Good Manufacturing Practice (GMP) conditions. Enrichment for CMV-specific T cells followed by only a short culture period is likely to retain maximal in vivo potential. PBMCs from 7 CMV seropositive donors were stimulated with recombinant pp65 protein (7–70 μg/ml) and/or HLA-A*0201/HLA-B*0702 restricted immunodominant pp65 peptides (NLV/TPR). Peptides used were clinical grade, and recombinant protein was gamma-irradiated (50 kGy, −80 C°) to eliminate possible microbiological contamination. High dose gamma-irradiation of pp65 protein resulted in partial degradation, but antigenic presentation was maintained. IFNγ producing cells were enriched using the IFNγ secretion assay (Miltenyi Biotec) at day 1 after stimulation, and cultured with autologous feeders (10x) and IL-2 (10 or 50 IU IL-2/ml) with or without CD3/28 expansion beads. Addition of high concentrations of protein during initial stimulation had a negative effect on enrichment probably due to non-specific stimulation of cells. Addition of immunodominant pp65 peptides promoted isolation efficiency and proliferation of epitope-specific CD8+ T cells in some donors. Cell lines were analyzed at different time points (day 4–15) using peptide-MHC tetramer and phenotypic markers. In addition, pp65-specificity was evaluated by intracellular IFNγ staining after restimulation with a pp65 protein-spanning pool of 15-mer peptides. CMV-specific lysis was tested in a 51-Cr release assay on pp65-transduced target cells. Enrichment of IFNγ producing cells after pp65 protein stimulation resulted in pp65-specific cell lines consisting of both CD8+ and CD4+ T cells. The T cell subset distribution directly after enrichment did not change during culture and was reproducible for each donor. Moreover, the composition of T cell lines reflected the pp65-specific response in donor PBMC starting material. The CD8+ compartment contained the known immunodominant tetramer staining cells (range 5–100%). The majority of both CD8+ and CD4+ T cells produced IFNγ upon restimulation with the pp65 peptide-pool, and showed CMV-specific lysis of target cells. The phenotype of pp65-specific T cells was predominant CD28+/CD45RO+ and CD45RA−/CCR7−/CD62L−, although CCR7 and CD62L were transiently expressed at day 4 and 7 after stimulation. Cryopreservation did not affect the composition or functionality of T cell lines. In conclusion, this procedure yields GMP-grade T cell lines comprising both CD8+ and CD4+ CMV-specific T cells. Processing and presentation of CMV protein by donor antigen-presenting cells enables selection of the full pp65-specific donor repertoire, without restrictions related to HLA or known epitopes. The choice for a moderate or more vigorous expansion after enrichment remains arbitrary and needs to be evaluated in clinical trials.

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Barz Leahy, Allison, Jennifer Brogdon, Lucy Cain, Amanda M. Dinofia, Joseph A. Fraietta, Richard Hanna, Stephan Kadauke et al. "Cost-Effective Manufacture and Promising Initial Efficacy of huCART19 Cells Manufactured Using the Clinimacs Prodigy Platform". Blood 144, Supplement 1 (5 de noviembre de 2024): 3470. https://doi.org/10.1182/blood-2024-204300.

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Introduction: The manufacturing process for CD19-directed CAR T cells, a life-saving therapy for B-ALL, remains first generation: non-automated, labor-intensive, and requiring expensive clean room facilities for GMP cell manufacture. This approach impacts both patient (pt) safety and access, producing constraints on availability of therapy and manufacturing turnaround. Use of the CliniMACS Prodigy platform allows for GMP, rapid, semi-automated, clinical-scale processing of humanized (hu)CART19 cell products in a point-of-care system and addresses many of these constraints. Based on preclinical data that demonstrated superior efficacy of huCART19 T cells manufactured on the Prodigy platform, we launched a Phase 1/2 study (NCT 05480449) of huCART19 for pediatric B-ALL, with complete Phase 1 and interim Phase 2 results presented here. Methods: Phase 1 included only children and young adults previously treated with CAR T cells. Phase 2 included both CAR-naïve and CAR-exposed patients (pts). After fludarabine/cyclophosphamide lymphodepletion, pts were infused with Prodigy-manufactured huCART19 cells. Results: From 11/2022-05/2024, 17 pts were enrolled. Of 16 pts infused (median age 12 years, range 3-26), 11 were CAR-exposed and 5 were CAR-naïve. Among CAR-exposed pts, 6 had relapsed after CAR therapy, and 5 had developed early B cell recovery. Of CAR-naïve pts, 2 had primary refractory and 3 had multiply-relapsed (all post hematopoietic stem cell transplant, HSCT) disease. Ten pts had evidence of disease at infusion: morphologic (n=6), non-CNS extramedullary (n=1), measurable residual disease by flow cytometry (flow-MRD, n=1), or by high-throughput sequencing (NGS-MRD, n=2). 100% of pts had cells successfully manufactured at the protocol dose in 7-9 days. The median infused cell dose was 4.6x106 huCART19 cells/kg (range, 2-5x106). For all but one patient, 3 surplus doses were also manufactured. Marginal manufacturing cost per patient was $28,427. Dose optimization occurred without a dose limiting toxicity (DLT), and the recommended phase 2 dose (RP2D) was achieved at 5x106 huCART19 cells/kg. Cytokine release syndrome (CRS) was observed in 11 pts (grade 1=8, grade 2=2, grade 3=1) and immune effector cell-associated neurotoxicity syndrome (ICANS) in 2 pts (grade 3=1, grade 4=1). In the Phase 2 retreatment setting, 1 pt with a prior history of seizure disorder and severe neurotoxicity after prior CAR therapy developed fatal cerebral edema in the setting of grade 4 ICANS on day 7. Among the 15 pts evaluable for response at day 28, the overall response rate was 100% (complete morphologic remission, n=14; partial response, n=1 [patient with non-CNS extramedullary disease]), 14 of whom (93%) were flow-MRD negative. There were 13 pts evaluable for NGS (Clonoseq)-MRD at day 28, of which 10 (77%) had undetectable NGS-MRD. One additional pt cleared NGS-MRD at month 3 without intervention. In CAR-naïve pts, 5/5 pts (100%) achieved flow MRD-negative CR; although, 1 patient remained with detectable NGS-MRD. In CAR-exposed pts evaluated at day 28, flow MRD-negative CR was achieved in 8/10 (80%) with one pt with detectable NGS-MRD at day 28 that cleared by month 3 without intervention. With a median follow-up of 4.7 months, event-free survival at 6 months was 94% (95% CI 83%-100%) and at 12 months was 78% (95% CI 53%-100%); pts were censored at alternate treatment or HSCT (n=2) and study withdrawal (n=1). There have been no additional patient deaths from disease or toxicity. Conclusion: Prodigy-manufactured huCART19 T cells yielded a 100% overall response rate in evaluable pts, with a 100% MRD-negative CR rate in CAR-naïve patients, and an 80% flow MRD-negative CR rate in patients with a poor response to prior CAR. This trial shows feasibility of manufacturing 4-1BB cells on the Prodigy cost-effectively, with successful manufacture for each pt and all products meeting release criteria. Investigation will continue into the significance of NGS-MRD positivity following Prodigy-manufactured huCART19 therapy, durability of remission, and potentially faster automated GMP manufacturing.

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Calmeiro, João, Mylène Carrascal, Luís Mendes, Iola F. Duarte, Célia Gomes, João Serra, Amilcar Falcão, Maria Teresa Cruz y Bruno Miguel Neves. "Development of a novel dendritic cell-based immunotherapy targeting cancer stem cells." Journal of Clinical Oncology 37, n.º 15_suppl (20 de mayo de 2019): e14009-e14009. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e14009.

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e14009 Background: Dendritic cells (DCs) are one of the central tools in cellular anti-tumor immunotherapy, being characterized by their capacity for acquiring and processing antigens and ability to produce strong antitumor immune responses. The production of clinical grade ex-vivo monocyte-derived DCs (Mo-DCs) is the most frequent approach for antitumor vaccines production. Recently, therapeutic resistance to radio/chemotherapy and disease recurrence was shown to be in part due to a small cancer stem cell (CSCs) population present in tumors. Methods: Here, we aim to target and eradicate CSCs by developing a novel DC-based immunotherapy vaccine for pancreatic and non-small cells lung cancer (NSCLC), comparing the loading of CSCs vs. classical tumor lysates. Results: CSCs from PANC-1 (pancreatic cancer) and A549 (NSCLC) cell lines were successfully isolated and characterized, overexpressing stem-like markers: NANOG, OCT4, SOX2 and CD133. CSCs resistance to Gemcitabine was also assessed. Before comparing the 2 types of vaccine loading, we also analyzed the impact of 3 GMP free-serum culture media on the phenotype and functional abilities of Mo-DCs. DCs cultured in X-VIVO 15 and AIM-V media show enhanced production of IL-12 and are able to induce a superior stimulation of T cells, mainly CTLs and Th1 subsets. By contrast, DCs cultured in DendriMACS are more prone to induce Treg polarization. Conclusions: Overall, our data demonstrate that blood monocytic precursors present considerable plasticity allowing a tailored differentiation of DCs just by changing the nutritive support. We also highlight the need of critically defining the culture medium to be used in DC cancer immunotherapy in order to attain desired cell characteristics and by consequent robust responses. Finally, our preliminary results indicate that loading DCs with CSCs antigens may be an effective strategy to target and destroy this resilient cancer cell population.

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Dobrila, Ludy, Tracy Zhu, Dan Zamfir, Tao Wang, Michal J. Tarnawski, Rodica Ciubotariu, Maria S. Albano, Andromachi Scaradavou y Pablo Rubinstein. "Increasing Post-Processing Total Nucleated Cell (TNC) Recovery in Cord Blood Banking: Hespan Addition in cGMP Environment". Blood 124, n.º 21 (6 de diciembre de 2014): 1126. http://dx.doi.org/10.1182/blood.v124.21.1126.1126.

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Abstract Purpose. Evaluate the effects of HESPAN (HES) addition on indices of cord blood unit (CBU) potency, stability and safety after automated volume reduction (reduction of erythrocyte bulk and plasma volume) using the AXP AutoXpressTM (AXP) processing system. Background and Methods. TNC recovery varies significantly and unpredictably after volume reduction during CBU processing. However, prompt engraftment of CBU allotransplants correlates with their TNC including hematopoietic stem/progenitor cells (HPC). As a result, TNC is important in selecting CBU for transplantation: for example, 75% of the National Cord Blood Program (NCBP) CBUs shipped during the period 2010-2013 had TNC >120 x 107. Therefore, minimizing TNC losses during processing improves the chances that a CBU will be useful for clinical transplantation. The first method to increase post-processing TNC recovery was the addition of HES to the centrifugal method for CB volume reduction [Rubinstein et al, PNAS (USA), 92:10119-10122, 1995]. Volume reduction consists of concentrating the buffy-coat (containing leukocytes and HPC) by centrifugal stratification and removal of bulk red cells and platelet-containing plasma. NCBP processed manually over 30,000 CBUs using HES during the period 1995-2006. In 2006, the AXP, a closed, automated and FDA-approved processing method, without HES, was implemented. AXP was designed to enhance the MNC and CD34+ cell recoveries but not those of granulocytes. This resulted in lower post- processing TNC counts. Additionally, CBUs with larger volume and TNC content have somewhat higher TNC losses during automated processing. Using the AXP method, NCBP’s HEMACORD® obtained FDA License approval on November 10, 2011. However, since TNC (not MNC or CD34+ count) remains the most commonly used indicator of CBU potency and engraftment ability, we describe here implementation of HES in AXP processing to augment TNC recovery, by adding HES to the CBU to a 1-2% final concentration. Results.The results of manual CBU processing showed that HES addition improves yield, without changes in cell viability after cryopreservation, freezing at -196°C and thawing, after 20 years (from NCBP continuing stability study). HES addition also preserved CFU numbers and CD34+ cell counts after thawing. 1. Comparison of TNC recoveries without and with HES addition in the same CBU: A total of 25 CBUs were initially processed with the AXP platform without HES, as per routine procedure, and the TNC recoveries were calculated. Each CBU was then reconstituted after its initial processing into a new AXP bag set. HES was added aseptically to the reconstituted product and each CBU was processed in the same conditions as first time (same AXP device, centrifuge, etc.). The TNC recoveries after the second volume reduction process (AXP with HES) were ~20% higher on average than after the first (AXP without HES). 2. Comparison of TNC recoveries in different cohorts of CBUs: Thirty clinical-grade CBUs, with volumes 80-156 mL and TNC counts 111-290 x 107, were processed with HES in the AXP system and the results were compared with those of AXP-processed CBUs without HES over an earlier six month period. TNC and CD45+ cell recoveries improved by 16 - 20% maintaining mean post-processing hematocrit at 30.6% (SD ± 2). CD34+ and CD45+ cell viabilities were unchanged: 99% (SD ± 0.7) and 96% (SD ± 2.7), respectively, while their mean recoveries were 95% (SD ± 18) and 94% (SD ± 6). In addition, the same consistent post-processing volume was obtained (mean 20.8 mL, SD ± 0.1). CFU - CD34+ correlation (R2) after processing with HES was 0.788 (not different than what was observed in the CBUs without HES). Six CBUs AXP-processed with HES, were thawed and tested, with very minor losses in cell count and viability, similar to results of thawed AXP-processed CBU without HES. Finally, HES addition did not result in microbial contamination in any of these AXP-processed CBUs. Conclusion. Adding HES to CBUs before automated AXP processing increased substantially the TNC and CD45+ recoveries without loss of viability, while the CD34+ recoveries remained basically unchanged, with a mean of 95%. The post-processing hematocrit was consistent and low. AXP-automated CB processing with HES addition can be performed in the GMP environment, results in higher post-processing TNC and therefore, increases the CB bank’s ability to store larger CBUs that are most useful to patients. Disclosures No relevant conflicts of interest to declare.

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Adair, Jennifer E., Kevin G. Haworth, Guy Sauvageau, Shelly Heimfeld, Jonah D. Hocum, Grant D. Trobridge y Hans-Peter Kiem. "A Point-of-Care Platform for Hematopoietic Stem Cell Gene Therapy". Blood 126, n.º 23 (3 de diciembre de 2015): 4416. http://dx.doi.org/10.1182/blood.v126.23.4416.4416.

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Abstract Hematopoietic stem and progenitor cell (HSPC) gene therapy holds great promise but requires highly technical and dedicated facilities. Current state-of-the-art requires ex vivo HSPC gene transfer in dedicated Good Manufacturing Practices (GMP) facility infrastructure, limiting treatment to highly developed countries. A patient point-of-care strategy would therefore make HSPC gene therapy available to patients worldwide. We developed a short, semi-automated, mostly-closed platform for ex vivo isolation and lentivirus (LV) gene modification of CD34+ HSPCs from either bone marrow or mobilized peripheral blood sources using the CliniMACS Prodigy® (Figure 1). Experiments were performed with a biosafety cabinet and personal protective equipment to simulate anticipated conditions in clinical facilities of underdeveloped countries. A total of 7 custom programs were developed for bone marrow or mobilized peripheral blood CD34+ cell isolation and LV transduction. Addition of a pyrimidoindole derivative, UM 729, permitted efficient transduction of CD34+ HSPCs in <18 hours. Complete semi-automated production took <34 hours from collection to infusion of the gene modified cell product, requiring very little operator hands-on time. Autologous, LV gene-modified CD34+ HSPCs from two nonhuman primates produced using this platform engrafted and supported multilineage hematopoietic repopulation after myeloablative total body irradiation at 1020 cGy. Total cell doses achieved were 27 x 106 and 5.4 x 106 CD34+ cells/kg body weight, respectively. We observed stable, persistent and polyclonal gene marking in peripheral blood granulocytes up to 40% and lymphocytes up to 15% within 4 months after infusion. Neither animal displayed evidence for increased toxicity, including potential contamination from the cell products. We then validated processing of human bone marrow and mobilized apheresis products from healthy adult donors. We demonstrate efficient isolation of human CD34+ HSPCs and up to 60% transduction efficiency with a clinical-grade LV currently being tested in a phase I clinical trial for treatment of HIV-associated lymphoma. Products tested met current FDA approved specifications for infusion. Xenotransplantation of these products into immunodeficient mice resulted in polyclonal engraftment over 12 weeks. These data demonstrate preclinical safety and feasibility of a patient point-of-care strategy for ex vivo LV gene transfer into HSPCs. This platform represents a major advance in global portability of LV mediated HSPC gene therapy. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

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Srivastava, Sandeep K., Sandhya R. Panch, Jianjian Jin, Haneen Shalabi, Nirali N. Shah, Steven L. Highfill y David F. Stroncek. "Abbreviated T-Cell Activation on the Automated Clinimacs Prodigy Device Enhances Bispecific CD19/22 Chimeric Antigen Receptor T-Cell Viability and Fold Expansion, Reducing Total Culture Duration". Blood 132, Supplement 1 (29 de noviembre de 2018): 4551. http://dx.doi.org/10.1182/blood-2018-99-116846.

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Abstract Introduction: As clinical applications for Chimeric Antigen Receptor (CAR) T-cell therapy expand, cell manufacturing incorporating closed-system, automated instruments are supplanting traditional open-system, labor-intensive culture methods. At our institution and others, the CliniMACS Prodigy (Miltenyi Biotec), a closed-system automated device, has demonstrated success in the production of CAR T-cells from T-cell enrichment, activation, viral transduction, and expansion to downstream harvest for cryopreservation/fresh infusion. However, the duration of T-cell activation/viral transduction, and total T-cell culture duration are variable across centers (2-5 days and 7-13 days) and merit evaluation prior to routine use. Methods: Following the opening of our clinical protocol (Clinicaltrials.gov NCT03448393), CD19/CD22 Bispecific CAR T-cell products were manufactured on the Prodigy for 4 patients (Original Method, OM) but CAR T cell manufacturing was felt to be suboptimal. Consequently, we investigated a modified processing method (Modified Method, MM), for the manufacture of clinical grade products using the Prodigy. Specifically, TransAct CD3/CD28 reagent mediated T-cell activation/stimulation and lentiviral transduction (MSCV-CAR1922-WPRE; Lentigen Inc.) was terminated with a wash step at Day 3 (instead of the wash step at Day 5, as in the OM). Overstimulation of the relatively more sensitive patient cells was proposed as a likely cause of suboptimal cell viability and expansion in the OM runs. Final cell harvest was planned between culture days 7-12. A total of 4 apheresis products were evaluated using this MM and compared with the 4 prior runs using the OM. All products were obtained from live or deceased patients with disease profiles similar to patients on the clinical trial. Other process parameters (enrichment for CD4/CD8 subsets, in-process media changes with GMP-TexMACS Medium supplemented with human IL-2 (200IU/mL) and 3% human AB serum) were kept unchanged across the 2 methods. Transduction by Protein L expression, viability and cell phenotype (CD3, CD4/CD8) were measured by flow cytometry. Results: From ~0.1x109 CD4/8 enriched T-cells placed into the Prodigy culture chamber on day 0, the mean viable Total Nucleated Cells (TNC) obtained in the final product was 1.93x109 ± 0.27x109 in the 4 MM runs. This cell dose was accomplished by culture Day 7. In contrast, in the OM runs, the mean viable TNC obtained in the final products between Days 9 and 12 was 0.8x109 ± 0.7x109 (Figure 1a). Viable CAR transduced CD3+ fold increase was calculated for days 0-7 of the MM cultures and 0-9 and 0-12 of the OM cultures depending on day of harvest and for the 4 MM products the average fold increase was 15.3 ± 4.2 by Day 7 (Figure 1b) which was ~3 fold greater than OM products harvested on day 9 or 12. Viability of transduced cells was >80% throughout MM culture. In contrast, viability was about 31% during manufacturing of one of the OM products (Figure 1c). On the day of harvest, >99% of the cells were CD3+ T-cells for all 4 MM products (Figure 1d) with no remaining CD19+CD22+ cells. The CD4/CD8 ratio was as expected and favored CD4 T-cells over CD8 T-cells (Figure 1e). Transduction efficiency based on Protein L binding was >70% for the MM products and passed clinical release criteria (Figure 1f). A head-to-head comparison of the 2 methods from the same starting fraction in one patient product (Figure 1, 3A, 3B) also confirmed all findings above. Conclusions: Our data demonstrate that the modified CD19/CD22 Bispecific CAR T-cell manufacturing method (MM) which terminated T-cell activation/transduction by culture Day 3, resulted in reproducible and robust CAR T cell production, even in the relatively more sensitive patient cells. Viability, Viable TNC recovery, CD3% and Protein L expression were consistently higher with the MM compared to the OM. All final products in the MM met product release criteria. In addition, final product dose requirements were consistently met by culture Day 7 when using the MM, augmenting process efficiency. Consequently, we have adopted the MM for the manufacture of clinical CD19/CD22 Bispecific CAR T cells. However, to determine if this change effects CAR T cell potency, studies have been initiated to compare differences in T-cell subsets, activation/exhaustion/senescence and differentiation markers, and the metabolic activity of cells manufactured by the 2 methods. Disclosures No relevant conflicts of interest to declare.

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Lichtenegger, Felix S., Katrin Deiser, Maurine Rothe, Frauke M. Schnorfeil, Christina Krupka, Christian Augsberger, Thomas Köhnke et al. "Induction of Antigen-Specific T-Cell Responses through Dendritic Cell Vaccination in AML: Results of a Phase I/II Trial and Ex Vivo Enhancement By Checkpoint Blockade". Blood 128, n.º 22 (2 de diciembre de 2016): 764. http://dx.doi.org/10.1182/blood.v128.22.764.764.

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Abstract Postremission therapy is critical for successful elimination of minimal residual disease (MRD) in acute myeloid leukemia (AML). Innovative treatment options are needed for patients that are not eligible for allogeneic stem cell transplantation. As the intrinsic immune response against leukemia-associated antigens (LAAs) is generally low, the clinical application of checkpoint inhibitors as monotherapy is less promising in AML compared to other hemato-oncological diseases. Therapeutic vaccination with autologous dendritic cells (DCs) loaded with LAAs is a promising treatment strategy to induce anti-leukemic immune responses. We have conducted a phase I/II proof-of-concept study using monocyte-derived next-generation DCs as postremission therapy of AML patients with a non-favorable risk profile in CR/CRi after intensive induction therapy (NCT01734304). These DCs are generated using a GMP-compliant 3-day protocol including a TLR7/8 agonist, loaded with RNA encoding the LAAs WT1 and PRAME as well as CMVpp65 as adjuvant/surrogate antigen, and are applied intradermally up to 10 times within 26 weeks. The primary endpoint of the trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control. After the safety and toxicity profile was evaluated within phase I (n=6), the patient cohort was expanded to a total of 13 patients. DCs of sufficient number and quality could be generated from leukapheresis in 11/12 cases. DCs exhibited an immune-stimulatory profile based on high costimulatory molecule expression, IL-12p70 secretion, migration towards a chemokine gradient and processing and presentation of antigen. In 9/9 patients that are currently evaluable, we observed delayed-type hypersensitivity (DTH) responses at the vaccination site, but no grade III/IV toxicities. TCR repertoire analysis by next-generation sequencing revealed an enrichment of particular clonotypes at DTH sites. In the peripheral blood, we detected vaccination-specific T cell responses by multimer staining and by ELISPOT analysis: 7/7 patients showed responses to CMVpp65, including both boosting of pre-existing T cells in CMV+ patients and induction of a primary T cell response in CMV- patients. 2/7 patients exhibited responses to PRAME and WT each. 7/10 vaccinated patients are still alive, and 5/10 are in CR, with an observation period of up to 840 days. In vitro, DC-activated T cells showed an upregulation of PD-1 and LAG-3, while the DCs expressed the respective ligands PD-L1 and HLA-DR. Therefore, we studied the capacity of checkpoint blocking antibodies to further enhance T-cell activation by DCs. We found that blockade of PD-1 and particularly of LAG-3 was highly effective in enhancing both IFN-γ secretion and proliferation of T cells. Both pathways seem to target different T-cell subsets, as PD-1 blockade resulted in increased IFN-γ secretion by TN- and TEM-subsets, while blockade of LAG-3 significantly affected TN- and TCM-subsets. We conclude that vaccination with next-generation LAA-expressing DCs in AML is feasible, safe, and induces anti-leukemic immune responses in vivo. Our in vitro data supports the hypothesis that T-cell activation by means of the vaccine could be further enhanced by blocking PD-1 and/or LAG-3. Disclosures Subklewe: AMGEN Research Munich: Research Funding.

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Deiser, Katrin, Felix S. Lichtenegger, Frauke Schnorfeil, Thomas Koehnke, Torben Altmann, Veit Buecklein, Andreas Moosmann et al. "Next-Generation Dendritic Cell Vaccination in Postremission Therapy of AML: Results of a Clinical Phase I Trial". Blood 126, n.º 23 (3 de diciembre de 2015): 3805. http://dx.doi.org/10.1182/blood.v126.23.3805.3805.

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Abstract Postremission therapy for acute myeloid leukemia (AML) is critical for elimination of minimal residual disease (MRD). In patients not eligible for allogeneic stem cell transplantation, alternative treatment options are needed. Therapeutic vaccination with autologous dendritic cells (DCs) loaded with leukemia-associated antigens (LAAs) is a promising treatment strategy to induce anti-leukemic immune responses and to eradicate chemorefractory cells. We have developed a GMP-compliant 3-day protocol including a TLR7/8 agonist to differentiate monocytes of intensively pretreated AML patients into next-generation DCs. A phase I/II proof-of-concept study has been initiated using next-generation DCs as postremission therapy of AML patients with a non-favorable genetic risk profile in CR after intensive induction therapy (NCT01734304). DCs are loaded with in vitro transcribed RNA encoding the LAAs WT1 and PRAME as well as CMVpp65 as adjuvant and surrogate antigen. Patients are vaccinated intradermally with 5x106 DCs of each antigen species up to 10 times within 26 weeks. The primary endpoint of the phase I/II trial is feasibility and safety of the vaccination. Secondary endpoints are immunological responses and disease control. Based on the safety and toxicity profile of the phase I trial (n=6), phase II has been initiated. In total, 10 patients have been enrolled into the study. DCs of sufficient number and quality were generated from leukapheresis in 8/9 cases. DCs exhibited an immune-stimulatory profile based on high surface expression of positive costimulatory molecules, the capacity to secrete IL-12p70, the migration towards a chemokine gradient and processing and presentation of antigen. 5 patients have completed the vaccination schedule; the 6th and 7th patient have received 7/10 and 4/10 vaccinations, respectively. We observed delayed-type hypersensitivity (DTH) responses at the vaccination site in 6/6 patients, accompanied by slight erythema and indurations at the injection site, but no grade III/IV toxicities. TCR repertoire analysis by next-generation sequencing revealed an enrichment of particular clonotypes at DTH sites. Limited by HLA restriction, we have so far analyzed 4 patients by multimer staining. All of them mounted DC vaccination-specific T cell responses: We detected an increase of WT1-specific T cells in one patient and strong expansion/induction of CMVpp65-specific T cells in one CMV-seropositive and two CMV-seronegative patients. In an individual treatment attempt, an enrolled patient with impending relapse was treated with a combination of DC vaccination and 5-azacytidine, resulting in MRD conversion. Long-term disease control and immunological responses are studied in the ongoing phase II trial. We conclude that vaccination with next-generation LAA-expressing DCs in AML is feasible, safe and induces anti-leukemia-specific immune responses in vivo. Disclosures Subklewe: AMGEN Research (Munich): Research Funding.

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Santra, Mithun, Moira L. Geary, Elizabeth Rubin, Michael Y. S. Hsu, Martha L. Funderburgh, Christine Chandran, Yiqin Du, Deepinder K. Dhaliwal, Vishal Jhanji y Gary Hin-Fai Yam. "Good manufacturing practice production of human corneal limbus-derived stromal stem cells and in vitro quality screening for therapeutic inhibition of corneal scarring". Stem Cell Research & Therapy 15, n.º 1 (8 de enero de 2024). http://dx.doi.org/10.1186/s13287-023-03626-8.

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Abstract Background Mesenchymal stem cells in the adult corneal stroma (named corneal stromal stem cells, CSSCs) inhibit corneal inflammation and scarring and restore corneal clarity in pre-clinical corneal injury models. This cell therapy could alleviate the heavy reliance on donor materials for corneal transplantation to treat corneal opacities. Herein, we established Good Manufacturing Practice (GMP) protocols for CSSC isolation, propagation, and cryostorage, and developed in vitro quality control (QC) metric for in vivo anti-scarring potency of CSSCs in treating corneal opacities. Methods A total of 24 donor corneal rims with informed consent were used—18 were processed for the GMP optimization of CSSC culture and QC assay development, while CSSCs from the remaining 6 were raised under GMP-optimized conditions and used for QC validation. The cell viability, growth, substrate adhesion, stem cell phenotypes, and differentiation into stromal keratocytes were assayed by monitoring the electric impedance changes using xCELLigence real-time cell analyzer, quantitative PCR, and immunofluorescence. CSSC’s conditioned media were tested for the anti-inflammatory activity using an osteoclastogenesis assay with mouse macrophage RAW264.7 cells. In vivo scar inhibitory outcomes were verified using a mouse model of anterior stromal injury caused by mechanical ablation using an Algerbrush burring. Results By comparatively assessing various GMP-compliant reagents with the corresponding non-GMP research-grade chemicals used in the laboratory-based protocols, we finalized GMP protocols covering donor limbal stromal tissue processing, enzymatic digestion, primary CSSC culture, and cryopreservation. In establishing the in vitro QC metric, two parameters—stemness stability of ABCG2 and nestin and anti-inflammatory ability (rate of inflammation)—were factored into a novel formula to calculate a Scarring Index (SI) for each CSSC batch. Correlating with the in vivo scar inhibitory outcomes, the CSSC batches with SI < 10 had a predicted 50% scar reduction potency, whereas cells with SI > 10 were ineffective to inhibit scarring. Conclusions We established a full GMP-compliant protocol for donor CSSC cultivation, which is essential toward clinical-grade cell manufacturing. A novel in vitro QC–in vivo potency correlation was developed to predict the anti-scarring efficacy of donor CSSCs in treating corneal opacities. This method is applicable to other cell-based therapies and pharmacological treatments.

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Schmid, Florian A., Jenny A. Prange, Marko Kozomara, Cornelia Betschart, Rosa A. Sousa, Nicolas Steinke, Manuela Hunziker et al. "Transurethral injection of autologous muscle precursor cells for treatment of female stress urinary incontinence: a prospective phase I clinical trial". International Urogynecology Journal, 12 de abril de 2023. http://dx.doi.org/10.1007/s00192-023-05514-4.

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Abstract Introduction and hypothesis The purpose was to investigate the safety and feasibility of transurethral injections of autologous muscle precursor cells (MPCs) into the external urinary sphincter (EUS) to treat stress urinary incontinence (SUI) in female patients. Methods Prospective and randomised phase I clinical trial. Standardised 1-h pad test, International Consultation on Incontinence Questionnaire-Urinary Incontinence Short Form (ICIQ-UI-SF), urodynamic study, and MRI of the pelvis were performed at baseline and 6 months after treatment. MPCs gained through open muscle biopsy were transported to a GMP facility for processing and cell expansion. The final product was injected into the EUS via a transurethral ultrasound-guided route. Primary outcomes were defined as any adverse events (AEs) during follow-up. Secondary outcomes were functional, questionnaire, and radiological results. Results Ten female patients with SUI grades I–II were included in the study and 9 received treatment. Out of 8 AEs, 3 (37.5%) were potentially related to treatment and treated conservatively: 1 urinary tract infection healed with antibiotics treatment, 1 dysuria and 1 discomfort at biopsy site. Functional urethral length under stress was 25 mm at baseline compared with 30 mm at 6 months’ follow-up (p=0.009). ICIQ-UI-SF scores improved from 7 points at baseline to 4 points at follow-up (p=0.035). MRI of the pelvis revealed no evidence of tumour or necrosis, whereas the diameter of the EUS muscle increased from 1.8 mm at baseline to 1.9 mm at follow-up (p=0.009). Conclusion Transurethral injections of autologous MPCs into the EUS for treatment of SUI in female patients can be regarded as safe and feasible. Only a minimal number of expected and easily treatable AEs were documented.

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