Process development and validation of expanded regulatory T cells for prospective applications: an example of manufacturing a personalized advanced therapy medicinal product.

BACKGROUND: A growing number of clinical trials have shown that regulatory T (Treg) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional Treg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for Treg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of Treg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype). RESULTS: The GMP-compliant protocol defined in this work allows at least 4.11 × 109 Treg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit Treg cell immunomodulatory function. CONCLUSIONS: These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the Treg cell number and function, which may slow the progression of certain diseases.

Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function.

BACKGROUND: Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. METHODS: We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. RESULTS: Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. CONCLUSIONS: These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation.

Finding a new therapeutic approach for no-option Parkinsonisms: mesenchymal stromal cells for progressive supranuclear palsy.

BACKGROUND: The trophic, anti-apoptotic and regenerative effects of bone marrow mesenchymal stromal cells (MSC) may reduce neuronal cell loss in neurodegenerative disorders. METHODS: We used MSC as a novel candidate therapeutic tool in a pilot phase-I study for patients affected by progressive supranuclear palsy (PSP), a rare, severe and no-option form of Parkinsonism. Five patients received the cells by infusion into the cerebral arteries. Effects were assessed using the best available motor function rating scales (UPDRS, Hoehn and Yahr, PSP rating scale), as well as neuropsychological assessments, gait analysis and brain imaging before and after cell administration. RESULTS: One year after cell infusion, all treated patients were alive, except one, who died 9 months after the infusion for reasons not related to cell administration or to disease progression (accidental fall). In all treated patients motor function rating scales remained stable for at least six-months during the one-year follow-up. CONCLUSIONS: We have demonstrated for the first time that MSC administration is feasible in subjects with PSP. In these patients, in whom deterioration of motor function is invariably rapid, we recorded clinical stabilization for at least 6 months. These encouraging results pave the way to the next randomized, placebo-controlled phase-II study that will definitively provide information on the efficacy of this innovative approach. Trial registration ClinicalTrials.gov NCT01824121.

Defining the identity of human adipose-derived mesenchymal stem cells.

Adipose-derived mesenchymal stem cells (ADMSCs) are an ideal population for regenerative medical application. Both the isolation procedure and the culturing conditions are crucial steps, since low yield can limit further cell therapies, especially when minimal adipose tissue harvests are available for cell expansion. To date, a standardized procedure encompassing both isolation sites and expansion methods is missing, thus making the choice of the most appropriate conditions for the preparation of ADMSCs controversial, especially in view of the different applications needed. In this study, we compared the effects of three different commercial media (DMEM, aMEM, and EGM2), routinely used for ADMSCs expansion, and two supplements, FBS and human platelet lysate, recently proven to be an effective alternative to prevent xenogeneic antibody transfer and immune alloresponse in the host. Notably, all the conditions resulted in being safe for ADMSCs isolation and expansion with platelet lysate supplementation giving the highest isolation and proliferation rates, together with a commitment for osteogenic lineage. Then, we proved that the high ADMSC hematopoietic supportive potential is performed through a constant and abundant secretion of both GCSF and SCF. In conclusion, this study further expands the knowledge on ADMSCs, defining their identity definition and offers potential options for in vitro protocols for clinical production, especially related to HSC expansion without use of exogenous cytokines or genetic modifications.

Differential microRNA signature of human mesenchymal stem cells from different sources reveals an "environmental-niche memory" for bone marrow stem cells.

Human mesenchymal stem cells (MSCs) are multipotent cells offering valuable hopes for the treatment of degenerative diseases. MSCs can be found among differentiated cells in many tissues and organs but, unfortunately, their phenotypic similarity hinders a robust cell characterization and discrimination from diverse tissue harvests. MicroRNAs (miRNAs) are crucial managers of gene expression with intriguing and still poorly known roles in stem cell maintenance and differentiation. To identify miRNAs that can discriminate among MSCs, we performed a whole-genome comparative miRNA expression profiling analysis on adipose (AD), bone marrow (BM) and cord blood (CB) derived MSCs, all three considered among the most promising in the field of regenerative medicine. miRNA expression patterns were very similar, meeting their extensive phenotypic and functional overlaps. An in-depth comparison of the few most differentially expressed miRNAs allowed the identification of a highly restricted molecular signature consisting of 5 BMMSC, 11 ADMSC and 11 CBMSC specific miRNAs. Functional analysis of their validated targets allowed the identification of an "environmental-niche memory" for BMMSC and an "epithelial" commitment for ADMSC, providing new insights into the molecular mechanisms discriminating between these MSCs, a crucial element to identify the most appropriate stem cell source for clinical application.

Off-the-Shelf Cord-Blood Mesenchymal Stromal Cells: Production, Quality Control, and Clinical Use.

Background Recently, mesenchymal stromal cells (MSCs) have gained recognition for their clinical utility in transplantation to induce tolerance and to improve/replace pharmacological immunosuppression. Cord blood (CB)-derived MSCs are particularly attractive for their immunological naivety and peculiar anti-inflammatory and anti-apoptotic properties. OBJECTIVES: The objective of this study was to obtain an inventory of CB MSCs able to support large-scale advanced therapy medicinal product (ATMP)-based clinical trials. STUDY DESIGN: We isolated MSCs by plastic adherence in a GMP-compliant culture system. We established a well-characterized master cell bank and expanded a working cell bank to generate batches of finished MSC(CB) products certified for clinical use. The MSC(CB) produced by our facility was used in approved clinical trials or for therapeutic use, following single-patient authorization as an immune-suppressant agent. RESULTS: We show the feasibility of a well-defined MSC manufacturing process and describe the main indications for which the MSCs were employed. We delve into a regulatory framework governing advanced therapy medicinal products (ATMPs), emphasizing the need of stringent quality control and safety assessments. From March 2012 to June 2023, 263 of our Good Manufacturing Practice (GMP)-certified MSC(CB) preparations were administered as ATMPs in 40 subjects affected by Graft-vs.-Host Disease, nephrotic syndrome, or bronco-pulmonary dysplasia of the newborn. There was no infusion-related adverse event. No patient experienced any grade toxicity. Encouraging preliminary outcome results were reported. Clinical response was registered in the majority of patients treated under therapeutic use authorization. CONCLUSIONS: Our 10 years of experience with MSC(CB) described here provides valuable insights into the use of this innovative cell product in immune-mediated diseases.

Human CD34+ cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand target both tumor cells and tumor vasculature.

Adenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 10(5) tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell-derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling-stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.

First clinical application of cord blood mesenchymal stromal cells in children with multi-drug resistant nephrotic syndrome.

BACKGROUND AND OBJECTIVES: Children with multi-drug resistant idiopathic nephrotic syndrome (MDR-INS) usually progress to end-stage kidney disease with a consistent risk of disease recurrence after transplantation. New therapeutic options are needed for these patients. Mesenchymal stromal cells (MSCs) are multipotential non-hematopoietic cells with several immunomodulatory properties and growing clinical applications. Cord blood-derived MSC have peculiar anti-inflammatory and immunosuppressive properties. We aimed at assessing safety and efficacy of cord-blood-derived MSCs (CB-MSCs) in children with MDR-INS. DESIGN, SETTING, PARTICIPANTS: Prospective, open-label, single arm phase I-II pilot study. Pediatric patients with MDR-INS, resistant to at least two lines of therapy, were enrolled. Allogenic CB-MSCs were administered intravenously on days 0, 14, and 21 at a dose of 1.5 × 106 cells/kg. Patients were followed for at least 12 months. The primary outcomes were safety and toxicity. The secondary outcome was remission at 12 months evaluated by urinary protein/urinary creatinine ratio (uPr/uCr). Circulating regulatory T cells (Tregs) were monitored. RESULTS: Eleven pediatric patients with MDR-INS (10 females, median age 13 years) resistant to a median of 3 previous lines of therapy were enrolled. All patients completed the CB-MSC infusion schedule. No patient experienced any infusion-related adverse event or toxicity. Nine patients were assessable for efficacy. At the 12 months follow-up after the treatment, the median uPr/uCr did not change significantly from baseline (8.13 vs. 9.07; p = 0.98), while 3 patients were in partial or complete remission. A lower baseline uPr/uCr was a predictor of remission (2.55 vs. 8.74; p = 0.0238). Tregs count was not associated with CB-MSCs therapy. CONCLUSIONS: CB-MSCs are safe and may have a role in the immunosuppressive therapy of pediatric patients with MDR-INS. This preliminary experience paves the way toward further phase II studies addressing MSC efficacy in immune-mediated kidney diseases.

Chondrogenic and BMP-4 primings confer osteogenesis potential to human cord blood mesenchymal stromal cells delivered with biphasic calcium phosphate ceramics.

Bone marrow mesenchymal stem/stromal cells (BMSCs) show great promise for bone repair, however they are isolated by an invasive bone marrow harvest and their regenerative potential decreases with age. Conversely, cord blood can be collected non-invasively after birth and contains MSCs (CBMSCs) that can be stored for future use. However, whether CBMSCs can replace BMSCs targeting bone repair is unknown. This study evaluates the in vitro osteogenic potential of unprimed, osteogenically primed, or chondrogenically primed CBMSCs and BMSCs and their in vivo bone forming capacity following ectopic implantation on biphasic calcium phosphate ceramics in nude mice. In vitro, alkaline phosphatase (intracellular, extracellular, and gene expression), and secretion of osteogenic cytokines (osteoprotegerin and osteocalcin) was significantly higher in BMSCs compared with CBMSCs, while CBMSCs demonstrated superior chondrogenic differentiation and secretion of interleukins IL-6 and IL-8. BMSCs yielded significantly more cell engraftment and ectopic bone formation compared to CBMSCs. However, priming of CBMSCs with either chondrogenic or BMP-4 supplements led to bone formation by CBMSCs. This study is the first direct quantification of the bone forming abilities of BMSCs and CBMSCs in vivo and, while revealing the innate superiority of BMSCs for bone repair, it provides avenues to induce osteogenesis by CBMSCs.

Safety and Effectiveness of Cell Therapy in Neurodegenerative Diseases: Take-Home Messages From a Pilot Feasibility Phase I Study of Progressive Supranuclear Palsy.

Mesenchymal stromal cells (MSCs) are multipotent cells with anti-inflammatory properties. Here we tested the safety of MSCs in patients with progressive supranuclear palsy (PSP; ClinicalTrials.gov: NCT01824121; Eudract No. 2011-004051-39). Seven patients were treated. To improve the safety, protocol adjustments were made during the performance of the study. The objectives of our work were: (1) to assess the safety of MSCs and (2) to identify critical issues in cell therapies for neurodegenerative diseases. Autologous MSCs from the bone marrow of PSP patients were administered through the internal carotid arteries. 1-year survival and number of severe adverse events were considered as safety endpoints. Clinical rating scales, neuropsychological assessments, gait and posture analysis, single-photon emission computed tomography, positron emission tomography, and brain magnetic resonance (BMR) were performed at different follow-up times. Peripheral blood levels of inflammatory cytokines were measured before and after cell infusion. Six of the seven treated patients were living 1 year after cell infusion. Asymptomatic spotty lesions were observed at BMR after 24 h in six of the seven treated patients. The last patient in the preliminary cohort (Case 5) exhibited transiently symptomatic BMR ischemic alterations. No severe adverse events were recorded in the last two treated patients. Interleukin-8 serum concentrations decreased in three patients (Case 2, 3, and 4). An adaptive study design, appropriate and up-to-date efficacy measures, adequate sample size estimation, and, possibly, the use of a cellular and/or allogeneic cell sources may help in performing phase II trials in the field.

A circular RNA map for human induced pluripotent stem cells of foetal origin.

BACKGROUND: Adult skin fibroblasts represent the most common starting cell type used to generate human induced pluripotent stem cells (F-hiPSC) for clinical studies. Yet, a foetal source would offer unique advantages, primarily the absence of accumulated somatic mutations. Herein, we generated hiPSC from cord blood multipotent mesenchymal stromal cells (MSC-hiPSC) and compared them with F-hiPSC. Assessment of the full activation of the pluripotency gene regulatory network (PGRN) focused on circular RNA (circRNA), recently proposed to participate in the control of pluripotency. METHODS: Reprogramming was achieved by a footprint-free strategy. Self-renewal and pluripotency of cord blood MSC-hiPSC were investigated in vitro and in vivo, compared to parental MSC, to embryonic stem cells and to F-hiPSC. High-throughput array-based approaches and bioinformatics analyses were applied to address the PGRN. FINDINGS: Cord blood MSC-hiPSC successfully acquired a complete pluripotent identity. Functional comparison with F-hiPSC showed no differences in terms of i) generation of mesenchymal-like derivatives, ii) their subsequent adipogenic, osteogenic and chondrogenic commitment, and iii) their hematopoietic support ability. At the transcriptional level, specific subsets of mRNA, miRNA and circRNA (n = 4,429) were evidenced, casting a further layer of complexity on the PGRN regulatory crosstalk. INTERPRETATION: A circRNA map of transcripts associated to naïve and primed pluripotency is provided for hiPSC of clinical-grade foetal origin, offering insights on still unreported regulatory circuits of the PGRN to consider for the optimization and development of efficient differentiation protocols for clinical translation. FUNDING: This research was funded by Ricerca Corrente 2012-2018 by the Italian Ministry of Health.

A computational approach to compare microvessel distributions in tumors following antiangiogenic treatments.

Experimental approaches currently used to quantify the activity of antiangiogenic treatments in cancer therapy do not generally address the importance of spatial distribution of microvessels in target tissues. We report a new computerized method to assess tumor vascularization by quantifying the distribution of functional microvessels as revealed by in vivo staining with sulfosuccinimidyl-6-(biotinamido) hexanoate. Our approach was based on pixel dilation of digital images of blood vessels and addressed the space-filling property of the vessel layouts. This was practically achieved computing the number of dilation cycles (Halo index) needed to permeate a pre-defined amount of each image. Our approach was validated on human tumor xenografts in nonobese diabetic/severe combined immunodeficient mice treated with the antiangiogenic drug sorafenib. For each experimental model, area normalization allowed the unbiased comparison of several hundreds of images showing different amounts of vascular tissue. In two different tumor types, comparison of Halo values showed statistically significant differences between control and sorafenib-treated samples. Conversely, this effect was not observed in samples from an additional xenograft known to resist the antiangiogenic treatment. By separating the analysis of vessel area from the quantification of vessel distributions, our approach can potentially contribute to a better evaluation of the antiangiogenic or vascular-disrupting activity of new drugs or treatments.

IFN-gamma enhances the antimyeloma activity of the fully human anti-human leukocyte antigen-DR monoclonal antibody 1D09C3.

To investigate the therapeutic activity of the fully human anti-HLA-DR antibody 1D09C3 in multiple myeloma (MM), we reevaluated HLA-DR expression on CD138(+) cells, analyzed the capacity of IFN-gamma to up-regulate HLA-DR expression on MM cell lines, and tested the in vitro and in vivo activity of 1D09C3 alone or in combination with IFN-gamma. CD138(+)HLA-DR(+) cells were detected in 31 of 60 patients, with 15 of 60 patients having >/=20% CD138(+)HLA-DR(+) cells (median, 50%; range, 23-100). Because primary plasma cells cannot be efficiently cultured in vitro, we used a panel of MM cell lines with a dim/negative to bright HLA-DR expression to evaluate 1D09C3-induced cell death. Annexin V/propidium iodide (PI) staining showed that 1D09C3-induced cell death correlated with constitutive HLA-DR expression. Induction of HLA-DR by IFN-gamma restored the sensitivity of HLA-DR dim cell lines to 1D09C3. In vivo, the combined IFN-gamma/1D09C3 treatment significantly increased the median survival of nonobese diabetic/severe combined immunodeficient mice xenografted with KMS-11 cell line, compared with controls (147 versus 48 days, P

Targeting TRAIL agonistic receptors for cancer therapy.

Based on preclinical studies demonstrating that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts a potent and cancer cell-specific proapoptotic activity, recombinant TRAIL as well as agonistic anti-TRAIL-R1 and anti-TRAIL-R2 antibodies recently entered clinical trials. Additionally, gene therapy approaches using TRAIL-encoding adenovirus (Ad-TRAIL) are currently being developed to overcome the limitations inherent to TRAIL receptor targeting, i.e., pharmacokinetic of soluble TRAIL, pattern of receptor expression, and tumor cell resistance. To optimize gene therapy approaches, CD34+ cells transduced with Ad-TRAIL (CD34-TRAIL+) have been investigated as cellular vehicles for TRAIL delivery. Transduced cells exhibit a potent tumor killing activity on a variety of tumor cell types both in vitro and in vivo and are also cytotoxic against tumor cells resistant to soluble TRAIL. Studies in tumor-bearing nonobese diabetic/severe combined immunodeficient mice suggest that the antitumor effect of CD34-TRAIL+ cells is mediated by both direct tumor cell killing due to apoptosis and indirect tumor cell killing due to vascular-disrupting mechanisms. The clinical translation of cell and gene therapy approaches represent a challenging strategy that might achieve systemic tumor targeting and increased intratumor delivery of the therapeutic agent.

The anti-human leukocyte antigen-DR monoclonal antibody 1D09C3 activates the mitochondrial cell death pathway and exerts a potent antitumor activity in lymphoma-bearing nonobese diabetic/severe combined immunodeficient mice.

The fully human anti-HLA-DR antibody 1D09C3 has been shown to delay lymphoma cell growth in severe combined immunodeficient (SCID) mice. The present study was aimed at (a) investigating the mechanism(s) of 1D09C3-induced cell death and (b) further exploring the therapeutic efficacy of 1D09C3 in nonobese diabetic (NOD)/SCID mice. The chronic lymphocytic leukemia cell line JVM-2 and the mantle cell lymphoma cell line GRANTA-519 were used. Generation of reactive oxygen species (ROS) and mitochondrial membrane depolarization were measured by flow cytometry following cell incubation with dihydroethidium and TMRE, respectively. Western blot analysis was used to detect c-Jun-NH(2)-kinase (JNK) phosphorylation and apoptosis-inducing factor (AIF). NOD/SCID mice were used to investigate the activity of 1D09C3 in early- or advanced-stage tumor xenografts. In vitro, 1D09C3-induced cell death involves a cascade of events, including ROS increase, JNK activation, mitochondrial membrane depolarization, and AIF release from mitochondria. Inhibition of JNK activity significantly reduced 1D09C3-induced apoptosis, indicating that 1D09C3 activity involves activation of the kinase. In vivo, 1D09C3 induces long-term disease-free survival in a significant proportion of tumor-bearing mice treated at an early stage of disease. Treatment of mice bearing advanced-stage lymphoma results in a highly significant prolongation of survival. These data show that 1D09C3 (a) exerts a potent antitumor effect by activating ROS-dependent, JNK-driven cell death, (b) cures the great majority of mice treated at an early-stage of disease, and (c) significantly prolongs survival of mice with advanced-stage disease.

Highly efficient gene transfer into mobilized CD34+ hematopoietic cells using serotype-5 adenoviral vectors and BoosterExpress Reagent.

OBJECTIVE: To optimize transduction efficiency of mobilized CD34(+) cells with serotype-5 adenoviruses (Ad5s), we investigated the activity of the chemical cocktail BoosterExpress Reagent in enhancing Ad5-mediated gene transfer into CD34(+) cells. METHODS: Enriched CD34(+) cells were transduced with three different Ad5s at increasing multiplicity of infections (MOIs) in the presence and absence of BoosterExpress Reagent. Percentages of transduced cells and levels of transgene expression were quantified by flow cytometry. Propidium iodide staining and colony growth were used to assess the toxicity of the transduction protocol. Expression of adenovirus receptors was investigated by flow cytometry. RESULTS: Irrespective of the Ad5 used, transduction with BoosterExpress Reagent using an MOI of 500 resulted in an average six- to seven-fold increase of transduction efficiencies and 1.5- to 2-fold increase of the levels of transgene expression, which could be detected up to 7 days post-transduction. Although BoosterExpress Reagent alone did not affect the plating efficiency of CD34(+) cells, adenovector transduction plus BoosterExpress Reagent significantly reduced the plating efficiency due to the marked increase of transduced cells. However, adenoviral transduction in the presence of BoosterExpress Reagent failed to significantly reduce the recovery of CD34(+) cells as compared with transduction in the absence of the compound. Coxsackievirus and adenovirus receptor as well as alpha(v)beta(3), alpha(v)beta(5), alpha(5), and beta(1) integrins were upregulated by BoosterExpress Reagent. CONCLUSIONS: BoosterExpress Reagent allows high-levels of durable transgene expression, thus making CD34(+) cells a suitable target for Ad5-mediated gene transfer.

Tips and Tricks for Validation of Quality Control Analytical Methods in Good Manufacturing Practice Mesenchymal Stromal Cell Production.

Mesenchymal stromal cells (MSC) for cellular therapy in European Union are classified as advanced therapy medicinal products (ATMPs), and their production must fulfill the requirements of Good Manufacturing Practice (GMP) rules. Despite their classification as medicinal products is already well recognized, there is still a lack of information and indications to validate methods and to adapt the noncompendial and compendial methods to these peculiar biological products with intrinsic characteristics that differentiate them from classic synthetic or biologic drugs. In the present paper, we present the results of the validation studies performed in the context of MSC development as ATMPs for clinical experimental use. Specifically, we describe the validation policies followed for sterility testing, endotoxins, adventitious viruses, cell count, and immunophenotyping. Our work demonstrates that it is possible to fully validate analytical methods also for ATMPs and that a risk-based approach can fill the gap between the prescription of the available guidelines shaped on traditional medicinal products and the peculiar characteristics of these novel and extremely promising new drugs.

CD52 antigen expressed by malignant plasma cells can be targeted by alemtuzumab in vivo in NOD/SCID mice.

OBJECTIVE: To explore new treatments specifically targeting malignant plasma cells (PCs), we examined CD52 antigen expression on primary PCs as well as multiple myeloma (MM) cell lines, and investigated in vivo the antimyeloma activity of alemtuzumab. MATERIALS AND METHODS: PCs were enriched from the marrow of MM patients (n = 39) according to CD138 expression and then analyzed by 3-color flow cytometry and quantitative PCR. The in vivo activity of alemtuzumab was evaluated in a xenotransplant model of MM in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. RESULTS: CD52 expression revealed a substantial heterogeneity in terms of both percentage of positive cells and fluorescence intensity, with 25/39 (64%) MM patients showing >or=30% CD138(+) PCs expressing the CD52 antigen (mean = 79%; range, 33-100%). Similarly to primary cells, cell lines showed heterogeneous CD52 expression. Expression of CD52 mRNA by quantitative PCR analysis strongly correlated with CD52 antigen detection by flow cytometry. In vivo, alemtuzumab treatment significantly increased the median survival of animals with an early- (64 vs 77 days, p

Antitumor activity of human CD34+ cells expressing membrane-bound tumor necrosis factor-related apoptosis-inducing ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of transformed cells while sparing normal cells. To enhance the therapeutic index of soluble (s)TRAIL, we used CD34+ cells transduced with a replication-deficient adenovirus encoding the human TRAIL gene (CD34-TRAIL+) for the systemic delivery of membrane-bound (m)TRAIL to lymphoid tumors. CD34-TRAIL+ cells were evaluated for their activity in vitro and in vivo in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with sTRAIL-sensitive and -resistant tumors. In vitro, coculturing CD34-TRAIL+ cells with sTRAIL-sensitive or -resistant lymphoma cell lines induced significant levels of caspase-dependent tumor cell death. In vivo, CD34-TRAIL+ cells significantly increased the survival of NOD/SCID mice bearing sTRAIL-sensitive or -resistant lymphoid tumors at an early or advanced stage of disease. No obvious toxicity was observed on administration of CD34-TRAIL+ cells. Histological analysis revealed high-level expression of the agonistic receptor TRAIL-R2 by tumor endothelial cells, and efficient tumor homing of transduced cells. Injection of CD34-TRAIL+ cells resulted in extensive damage of tumor vasculature followed by hemorrhagic necrosis exhibiting a perivascular distribution. These results show that CD34-TRAIL+ cells might be an efficient vehicle for mTRAIL delivery to tumors, where they exert a potent antitumor effect possibly mediated by both direct tumor cell killing and indirect vascular-disrupting mechanisms.

A Chemically Defined Medium-Based Strategy to Efficiently Generate Clinically Relevant Cord Blood Mesenchymal Stromal Colonies.

During the last decade it has been demonstrated that mesenchymal progenitors are present and can be isolated also from cord blood (CB). Recently, we managed to set up a standard protocol allowing the isolation of mesenchymal stromal cells (MSCs) with high proliferative potential and multiple differentiation capabilities, whereas the generation rate of MSC-initiating colonies could still be further improved. Herein, we strikingly succeeded in defining some simple and basic culture conditions based on the use of a chemically defined medium that increased the colony isolation efficiency up to almost 80% of processed CB units. Importantly, this result was achieved irrespective of CB unit white blood cell content and time elapsed from delivery, two limiting parameters involved with processing CB units. Thus, this high efficiency is guaranteed without strict selection of the starting material. In addition, since we are profoundly concerned about how different culture conditions can influence cell behavior, we devoted part of this study to in-depth characterization of the established CB-MSC populations to confirm their stemness features in this novel isolation and culture system. Therefore, an extended study of their immunophenotype, including classical pericytic markers, and a detailed molecular analysis addressing telomere length and also stemness-related microRNA contribution were performed. In summary, we propose a straightforward, extremely efficient, and reliable approach to isolate and expand thoroughly characterized CB-MSCs, even when poor-quality CB units are the only available source, or there is no space for an isolation to fail.