GMP-compliant Conditions Research Articles

Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions

The unlimited expansion of human progenitor cells in vitro could unlock many prospects for regenerative medicine. However, it remains an important challenge as it requires the decoupling of the mechanisms supporting progenitor self-renewal and expansion from those mechanisms promoting their differentiation. This study focuses on the expansion of human pluripotent stem (hPS) cell-derived pancreatic progenitors (PP) to advance novel therapies for diabetes. We obtained mechanistic insights into PP expansion requirements and identified conditions for the robust and unlimited expansion of hPS cell-derived PP cells under GMP-compliant conditions through a hypothesis-driven iterative approach. We show that the combined stimulation of specific mitogenic pathways, suppression of retinoic acid signaling, and inhibition of selected branches of the TGFβ and Wnt signaling pathways are necessary for the effective decoupling of PP proliferation from differentiation. This enabled the reproducible, 2000-fold, over 10 passages and 40–45 d, expansion of PDX1+/SOX9+/NKX6-1+ PP cells. Transcriptome analyses confirmed the stabilization of PP identity and the effective suppression of differentiation. Using these conditions, PDX1+/SOX9+/NKX6-1+ PP cells, derived from different, both XY and XX, hPS cell lines, were enriched to nearly 90% homogeneity and expanded with very similar kinetics and efficiency. Furthermore, non-expanded and expanded PP cells, from different hPS cell lines, were differentiated in microwells into homogeneous islet-like clusters (SC-islets) with very similar efficiency. These clusters contained abundant β-cells of comparable functionality as assessed by glucose-stimulated insulin secretion assays. These findings established the signaling requirements to decouple PP proliferation from differentiation and allowed the consistent expansion of hPS cell-derived PP cells. They will enable the establishment of large banks of GMP-produced PP cells derived from diverse hPS cell lines. This approach will streamline SC-islet production for further development of the differentiation process, diabetes research, personalized medicine, and cell therapies.

Regulation of multiple signaling pathways promotes the consistent expansion of human pancreatic progenitors in defined conditions.

The unlimited expansion of human progenitor cells in vitro could unlock many prospects for regenerative medicine. However, it remains an important challenge as it requires the decoupling of the mechanisms supporting progenitor self-renewal and expansion from those mechanisms promoting their differentiation. This study focuses on the expansion of human pluripotent stem (hPS) cell-derived pancreatic progenitors (PP) to advance novel therapies for diabetes. We obtained mechanistic insights into PP expansion requirements and identified conditions for the robust and unlimited expansion of hPS cell-derived PP cells under GMP-compliant conditions through a hypothesis-driven iterative approach. We show that the combined stimulation of specific mitogenic pathways, suppression of retinoic acid signaling, and inhibition of selected branches of the TGFβ and Wnt signaling pathways are necessary for the effective decoupling of PP proliferation from differentiation. This enabled the reproducible, 2000-fold, over 10 passages and 40-45 d, expansion of PDX1+/SOX9+/NKX6-1+ PP cells. Transcriptome analyses confirmed the stabilization of PP identity and the effective suppression of differentiation. Using these conditions, PDX1+/SOX9+/NKX6-1+ PP cells, derived from different, both XY and XX, hPS cell lines, were enriched to nearly 90% homogeneity and expanded with very similar kinetics and efficiency. Furthermore, non-expanded and expanded PP cells, from different hPS cell lines, were differentiated in microwells into homogeneous islet-like clusters (SC-islets) with very similar efficiency. These clusters contained abundant β-cells of comparable functionality as assessed by glucose-stimulated insulin secretion assays. These findings established the signaling requirements to decouple PP proliferation from differentiation and allowed the consistent expansion of hPS cell-derived PP cells. They will enable the establishment of large banks of GMP-produced PP cells derived from diverse hPS cell lines. This approach will streamline SC-islet production for further development of the differentiation process, diabetes research, personalized medicine, and cell therapies.

A clinically relevant large-scale biomanufacturing workflow to produce natural killer cells and natural killer cell-derived extracellular vesicles for cancer immunotherapy.

Natural killer cell-derived extracellular vesicles (NK-EVs) have shown promising potential as biotherapeutics for cancer due to their unique attributes as cytotoxic nanovesicles against cancer cells and immune-modulatory activity towards immune cells. However, a biomanufacturing workflow is needed to produce clinical-grade NK-EVs for pre-clinical and clinical applications. This study established a novel biomanufacturing workflow using a closed-loop hollow-fibre bioreactor to continuously produce NK-EVs from the clinically relevant NK92-MI cell line under serum-free, Xeno-free and feeder-free conditions following GMP-compliant conditions. The NK92 cells grown in the bioreactor for three continuous production lots resulted in large quantities of both NK cell and NK-EV biotherapeutics at the end of each production lot (over 109 viable cells and 1013 EVs), while retaining their cytotoxic payload (granzyme B and perforin), pro-inflammatory cytokine (interferon-gamma) content and cytotoxicity against the human leukemic cell line K562 with limited off-target toxicity against healthy human fibroblast cells. This scalable biomanufacturing workflow has the potential to facilitate the clinical translation of adoptive NK cell-based and NK-EV-based immunotherapies for cancer with GMP considerations.

402.1: Towards a GMP-Compliant Protocol for the Differentiation of Human Pluripotent Stem Cells to Beta-like Cells for the Treatment of Type 1 Diabetes

Background: β-cell replacement therapy by allogeneic pancreas or islet transplantation is a promising approach for patients with type 1 diabetes. However, there is a scarcity of organ donors. The generation of insulin-producing pancreatic β-cells from human pluripotent stem cells (hPSC) in vitro would provide an unlimited cell source for drug discovery and cell replacement therapy for diabetes. Here we aim to generate β-cells under GMP (Good Manufacturing Practice)-compliant conditions that can be used for clinical application. Methods: We applied a modified seven-stage (30-day) differentiation protocol to generate hPSC-derived insulin-producing β-like cells in a 3D microwell culture system. Next, in order to scale up the production of hPSC-derived β-cells, we adapted the protocol to spinner flask culture. Differentiation was assessed by qPCR, immunocytochemistry and flowcytometry for stage-specific marker expression. β-cell function was evaluated in vitro by GSIS (Glucose-Stimulated Insulin Secretion), and in vivo by intraperitoneal glucose tolerance tests after transplantation of stage-7 cell clusters in immunodeficient mice and human C-peptide secretion was determined by ELISA. Results: In the microwell set-up, cells acquired a definitive endoderm phenotype at stage 1 (day 3), characterized by expression of SOX17 (87.2% ± 4.6; n=5), FOXA2 (82.8% ± 4.6; n=3), co-expression of c-KIT and CXCR4 (93.3% ± 5.6; n=4), and silencing of pluripotency marker OCT4 (2.8% ± 6; n=5). At the end of stage 4 (day 12), pancreatic progenitor cells were identified by the co-expression of PDX1/NKX6.1 (46.7% ± 3.5; n=3). Finally, at the end of stage 7 (day 30), C-peptide-positive β-like cells (49.5% ± 10.5; n=3) and glucagon-positive α-like cells (18.4%± 6.1; n=3) were present. The stimulation index upon exposure to glucose was 4.4 ± 3.1(n=5). Following transplantation of day-30 clusters into mice, stimulated human C-peptide levels reached 46 pmol/L (n=21), 70.5 pmol/L (n=19) and 327.4 pmol/L (n=11) at day 14, 28 and 60 respectively, indicating further maturation of the cells in vivo after transplantation. Next, we scaled up the cell culture in disposable spinner flasks, and obtained comparable differentiation efficiency. Conclusion: We can generate functional hPSCs-derived β-like cells in GMP-compliant conditions in vitro. We will apply these protocols on a clinical-grade PSC line that is currently in expansion (banking) in the GMP facility of our institute. hPSCs-derived β-like cells are a promising future alternative to donor islets for the treatment of type 1 diabetes.

Method for Isolating Extracellular Vesicles from Human Neural Stem Cells Expanded Under Neurosphere Culture.

Neural stem cells (NSCs) transplantation enhances plasticity and restores functions in neurological diseases. Therapeutic benefits of NSCs are due to their ability to replace the lost neurons and glial cells and also secreting a wide array of free and membrane-bound bioactive molecules that can reduce the hostility of diseased microenvironment, resolve inflammation, and rescue damaged neural cells. Membrane-encircled spherical nanostructures that are collectively known as extracellular vesicles (EVs) contain mRNA, miRNA, lipids, and specific proteins that affect different biological processes in cells located nearby or at far distances. Using EVs as an alternative non-cell-based therapy has gained huge attention, and developing methods for large-scale production of EVs is of great clinical importance. Here, we describe an efficient method to yield significant quantity of EVs from human NSCs that are expanded under free floating neurosphere assay culture system. Using the neurosphere assay in bioreactors under GMP-compliant conditions can result in scalable NSC-EVs required for human trials.

Application of a novel bioreactor system for automated expansion of adipose- derived mesenchymal stem cells under gmp-compliant conditions

Application of a novel bioreactor system for automated expansion of adipose- derived mesenchymal stem cells under gmp-compliant conditions

GMP-grade neural progenitor derivation and differentiation from clinical-grade human embryonic stem cells

BackgroundA major challenge for the clinical use of human pluripotent stem cells is the development of safe, robust and controlled differentiation protocols. Adaptation of research protocols using reagents designated as research-only to those which are suitable for clinical use, often referred to as good manufacturing practice (GMP) reagents, is a crucial and laborious step in the translational pipeline. However, published protocols to assist this process remain very limited.MethodsWe adapted research-grade protocols for the derivation and differentiation of long-term neuroepithelial stem cell progenitors (lt-NES) to GMP-grade reagents and factors suitable for clinical applications. We screened the robustness of the protocol with six clinical-grade hESC lines deposited in the UK Stem Cell Bank.ResultsHere, we present a new GMP-compliant protocol to derive lt-NES, which are multipotent, bankable and karyotypically stable. This protocol resulted in robust and reproducible differentiation of several clinical-grade embryonic stem cells from which we derived lt-NES. Furthermore, GMP-derived lt-NES demonstrated a high neurogenic potential while retaining the ability to be redirected to several neuronal sub-types.ConclusionsOverall, we report the feasibility of derivation and differentiation of clinical-grade embryonic stem cell lines into lt-NES under GMP-compliant conditions. Our protocols could be used as a flexible tool to speed up translation-to-clinic of pluripotent stem cells for a variety of neurological therapies or regenerative medicine studies.

A phase I/II clinical trial of autologous CMV-specific T cells in glioblastoma (GBM) patients to reveal a lack of immune effector function.

2515 Background: Cytomegalovirus (CMV) antigens are present in > 90% of GBMs but not in normal brain making it an attractive immunological target. Methods: Highly functional autologous polyclonal CMV pp65 specific T cells were expanded under GMP-compliant conditions from GBM patients and administered after 3 weeks of lymphodepleting dose-dense temozolomide (ddTMZ, 100 mg/m2). The phase I component used a 3+3 design ascending through four dose levels (5 x 106 cells to 1 x 108 cells). Treatment was repeated every 6 weeks for a total of 4 cycles. Dose expansion was conducted in recurrent GBM patients undergoing resection and in newly diagnosed GBM patients following concurrent chemoradiation. In vivo persistence and effector function of the adoptively transferred CMV-specific T cells was determined by dextramer staining and multiparameter flow cytometry in serially-sampled peripheral blood and in the tumor microenvironment. Results: 65 patients were screened, 25 underwent leukapheresis, and 20 completed at least 1 cycle. Median age 48 (27-69), 35% were MGMT methylated, and 10% were IDH mutated. No dose limiting toxicities (DLTs) observed. Complete radiographic response was observed in 1 patient, partial responses in 2, stable disease in 9, and progressive disease in 8. The median PFS time was 1.3 months (95% CI: 0-8.3 months) and the median OS time was 12 months (95% CI: 6 months to not reached). Repeated infusions of CMV-TC were associated with significant increase in circulating CMV+ CD8+ T cells, but cytokine production reflective of effector activity (CD107a, TNFα, IFNγ, IL2) was suppressed in these cells including directly from the GBM microenvironment. Conclusions: Adoptive infusion of CMV-specific T cells after lymphodepleting ddTMZ was well-tolerated. However, effector function of the adoptively transferred T cells was attenuated indicating further modulation of the T cell is required to prevent its dysfunction prior to proceeding to large scale clinical studies. Clinical trial information: NCT02661282 .

Cell manufacturing innovation facility: use of GMP-compliant space to accelerate advances in therapeutic cell production

Background & Aim Production space for cell therapy products is in high demand, with the recent FDA approvals of the first cell therapy products in the U.S. Hospitals are expanding their qualified production spaces to support the growing number of cell and gene therapy clinical trials. Simultaneously, a pronounced need to improve engineering and controlled manufacturing principles has been made clear. Initiatives have emerged to accelerate cell therapy manufacturing innovations at academic institutions, including the NSF Engineering Research Center for Cell Manufacturing Technologies (CMaT) and the Marcus Center for Therapeutic Cell Characterization and Manufacturing (MC3M). In order for researchers, particularly in engineering and manufacturing disciplines, to have an understanding of restrictions placed on therapeutic cell production, exposure to compliant environments without disrupting production is necessary. The MC3M facility at Georgia Tech provides the community access to facilities operating under GMP-compliant conditions and the ability to develop next generation technologies. Methods, Results & Conclusion The MC3M facility contains two isolated ISO 7/GMP suites for viral and viral-free work, and a large ISO 8 research lab. GMP rooms are adjacent to analytical suites, with pass-thrus to enable real-time analytics and feedback control. MC3M houses various bioreactors, including hollow fiber, vertical wheel, and stirred batch systems, as well as analytics equipment to enable at-line and in-line monitoring within minutes from sampling directly from culture vessels. Additionally, automation technologies that measure and control process parameters such as glucose and lactate are being developed and employed. Faculty, students, and industry can pilot new process technologies for cell manufacturing needs. Users learn proper gowning procedures and operational requirements like material quarantine and environmental monitoring. These experiences serve to better inform researchers in the practical applications of their early technology or culture process improvements, and advance the translation of discoveries to products. The facility is utilized for training purposes beyond the university's community to inform and equip the future workforce of the growing cell manufacturing industry. Facilities like MC3M are imperative to meet the demands of the cell manufacturing industry, and ensure clinically and industry relevant, impactful research is conducted, even at the earliest stages of discovery.

Initial experience in synthesis of (2S,4R)-4-[18 F]fluoroglutamine for clinical application.

We report initial experience in synthesis of (2S,4R)-4-[18 F]fluoroglutamine, [18 F]FGln, which has been used as a tool for monitoring glutamine metabolism in cancer patients. [18 F]FGln was prepared by a fully automated PET-MF-2V-IT-I synthesizer under GMP-compliant conditions for routine clinical studies. The total radiosynthesis time was about 65minutes, the decay-corrected radiochemical yield was 18.0±4.2% (n=59; failure n=15), and the radiochemical purity was greater than 90%. In some situations, the yields were low (less than 5%), and the most likely cause of this problem is the initial fluorination step; the fluoride ion might not have been fully activated. In other occasions, low final radiochemical purity was often associated with the failure of the second step-removal of protection groups by anhydrous trifluoroacetic acid. A trace amount of water led to production of undesired 4-[18 F]fluoroglutamic acid. Knowledge learned from the successes and failures of synthesis may be helpful to identify critical steps and pitfalls for preparation of this clinically useful metabolic probe, [18 F]FGln, for imaging glutamine utilization in tumor of cancer patients.

A bioreactor system for the manufacture of a genetically modified Plasmodium falciparum blood stage malaria cell bank for use in a clinical trial

BackgroundAlthough the use of induced blood stage malaria infection has proven to be a valuable tool for testing the efficacy of vaccines and drugs against Plasmodium falciparum, a limiting factor has been the availability of Good Manufacturing Practice (GMP)—compliant defined P. falciparum strains for in vivo use. The aim of this study was to develop a cost-effective method for the large-scale production of P. falciparum cell banks suitable for use in clinical trials.MethodsGenetically-attenuated parasites (GAP) were produced by targeted deletion of the gene encoding the knob associated histidine rich protein (kahrp) from P. falciparum strain 3D7. A GAP master cell bank (MCB) was manufactured by culturing parasites in an FDA approved single use, closed system sterile plastic bioreactor. All components used to manufacture the MCB were screened to comply with standards appropriate for in vivo use. The cryopreserved MCB was subjected to extensive testing to ensure GMP compliance for a phase 1 investigational product.ResultsTwo hundred vials of the GAP MCB were successfully manufactured. At harvest, the GAP MCB had a parasitaemia of 6.3%, with 96% of parasites at ring stage. Testing confirmed that all release criteria were met (sterility, absence of viral contaminants and endotoxins, parasite viability following cryopreservation, identity and anti-malarial drug sensitivity of parasites).ConclusionLarge-scale in vitro culture of P. falciparum parasites using a wave bioreactor can be achieved under GMP-compliant conditions. This provides a cost-effective methodology for the production of malaria parasites suitable for administration in clinical trials.

Development of Large Scale Silkworm-rearing Technologies for the GMP Production of Biologics

There exists an increasing need to produce useful proteins in recombinant technologies. In particular, most biologics for medical purposes are produced as recombinant proteins. Various host cell/vector systems have been developed, but it remains difficult to efficiently produce large molecular weight proteins with complex structures. As a result of breeding for several thousand years, the silkworm has acquired the ability to synthesize bulk amounts of silk proteins. To utilize this capacity for the mass production of useful proteins, transgenic silkworms have been generated that synthesize recombinant proteins in the silk gland and secrete them into the silk cocoon. Using this transgenic silkworm system, various proteins, including antibodies, collagen, and fibrinogen, have been successfully produced and are being developed as materials for diagnostic or research-use reagents, as well as for cosmetics. Moreover, several silkworm-produced proteins are being developed as biologics for therapeutic use. Transgenic silkworms need to be reared under good manufacturing practices (GMP)-compliant conditions to produce biologics. Therefore, we have constructed a GMP-compliant pilot plant for producing biologics using transgenic silkworm, and are now developing silkworm-rearing technology under GMP-compliant conditions.

19F-perfluorocarbon-labeled human peripheral blood mononuclear cells can be detected in vivo using clinical MRI parameters in a therapeutic cell setting

A 19Fluorine (19F) perfluorocarbon cell labeling agent, when employed with an appropriate cellular MRI protocol, allows for in vivo cell tracking. 19F cellular MRI can be used to non-invasively assess the location and persistence of cell-based cancer vaccines and other cell-based therapies. This study was designed to determine the feasibility of labeling and tracking peripheral blood mononuclear cells (PBMC), a heterogeneous cell population. Under GMP-compliant conditions human PBMC were labeled with a 19F-based MRI cell-labeling agent in a manner safe for autologous re-injection. Greater than 99% of PBMC labeled with the 19F cell-labeling agent without affecting functionality or affecting viability. The 19F-labeled PBMC were detected in vivo in a mouse model at the injection site and in a draining lymph node. A clinical cellular MR protocol was optimized for the detection of PBMC injected both at the surface of a porcine shank and at a depth of 1.2 cm, equivalent to depth of a human lymph node, using a dual 1H/19F dual switchable surface radio frequency coil. This study demonstrates it is feasible to label and track 19F-labeled PBMC using clinical MRI protocols. Thus, 19F cellular MRI represents a non-invasive imaging technique suitable to assess the effectiveness of cell-based cancer vaccines.

Isolation and prolonged expansion of oral mesenchymal stem cells under clinical-grade, GMP-compliant conditions differentially affects \u201cstemness\u201d properties

BackgroundDevelopment of clinical-grade cell preparations is central to meeting the regulatory requirements for cellular therapies under good manufacturing practice-compliant (cGMP) conditions. Since addition of animal serum in culture media may compromise safe and efficient expansion of mesenchymal stem cells (MSCs) for clinical use, this study aimed to investigate the potential of two serum/xeno-free, cGMP culture systems to maintain long-term “stemness” of oral MSCs (dental pulp stem cells (DPSCs) and alveolar bone marrow MSCs (aBMMSCs)), compared to conventional serum-based expansion.MethodsDPSC and aBMMSC cultures (n = 6/cell type) were established from pulp and alveolar osseous biopsies respectively. Three culture systems were used: StemPro_MSC/SFM_XenoFree (Life Technologies); StemMacs_MSC/XF (Miltenyi Biotek); and α-MEM (Life Technologies) with 15% fetal bovine serum. Growth (population doublings (PDs)), immunophenotypic (flow cytometric analysis of MSC markers) and senescence (β-galactosidase (SA-β-gal) activity; telomere length) characteristics were determined during prolonged expansion. Gene expression patterns of osteogenic (ALP, BMP-2), adipogenic (LPL, PPAR-γ) and chondrogenic (ACAN, SOX-9) markers and maintenance of multilineage differentiation potential were determined by real-time PCR.ResultsSimilar isolation efficiency and stable growth dynamics up to passage 10 were observed for DPSCs under all expansion conditions. aBMMSCs showed lower cumulative PDs compared to DPSCs, and when StemMacs was used substantial delays in cell proliferation were noted after passages 6–7. Serum/xeno-free expansion produced cultures with homogeneous spindle-shaped phenotypes, while serum-based expansion preserved differential heterogeneous characteristics of each MSC population. Prolonged expansion of both MSC types but in particular the serum/xeno-free-expanded aBMMSCs was associated with downregulation of CD146, CD105, Stro-1, SSEA-1 and SSEA-4, but not CD90, CD73 and CD49f, in parallel with an increase of SA-gal-positive cells, cell size and granularity and a decrease in telomere length. Expansion under both serum-free systems resulted in “osteogenic pre-disposition”, evidenced by upregulation of osteogenic markers and elimination of chondrogenic and adipogenic markers, while serum-based expansion produced only minor changes. DPSCs retained a diminishing (CCM, StemPro) or increasing (StemMacs) mineralization potential with passaging, while aBMMSCs lost this potential after passages 6–7 under all expansion conditions.ConclusionsThese findings indicate there is still a vacant role for development of qualified protocols for clinical-grade expansion of oral MSCs; a key milestone achievement for translation of research from the bench to clinics.

ID: 1016 Production of endothelial progenitor cells from skin fibroblasts by direct reprogramming for clinical usages

Endothelial progenitor cells (EPCs) play an important role in angiogenesis. However, they exist in limited numbers in the human body. This study was aimed to produce EPCs, for autologous transplantation, using direct reprogramming of skin fibroblasts under GMP-compliant conditions. Fibroblasts were collected and cultured from the skin in DMEM/F12 medium supplemented with 5% activated platelet-rich plasma and 1% antibiotic-antimycotic solution. They were then transfected with mRNA ETV2 and incubated in culture medium under hypoxia (5% oxygen) for 14 d. Phenotype analysis of transfected cells confirmed that single-factor ETV2 transfection successfully reprogrammed dermal fibroblasts into functional EPCs. Our results showed that ETV2 mRNA combined with hypoxia can give rise to functional EPCs. The cells exhibited functional phenotypes similar to endothelial cells derived from umbilical cord vein; they expressed CD31 and VEGFR2, and formed capillary-like structures in vitro. Moreover, these EPCs could significantly improve hindlimb ischemia in mouse models. Although the direct conversion efficacy was low (3.12 ± 0.98%), altogether our study demonstrates that functional EPCs can be produced from fibroblasts and can be used in clinical applications.

The development of sexual stage malaria gametocytes in a Wave Bioreactor

BackgroundBlocking malaria gametocyte development in RBCs or their fertilization in the mosquito gut can prevent infection of the mosquito vector and passage of disease to the human host. A ‘transmission blocking’ strategy is a component of future malaria control. However, the lack of robust culture systems for producing large amounts of Plasmodium falciparum gametocytes has limited our understanding of sexual-stage malaria biology and made vaccine or chemotherapeutic discoveries more difficult.MethodsThe Wave BioreactorTM 20/50 EHT culture system was used to develop a convenient and low-maintenance protocol for inducing commitment of P. falciparum parasites to gametocytogenesis. Culture conditions were optimised to obtain mature stage V gametocytes within 2 weeks in a large-scale culture of up to a 1 l.ResultsWe report a simple method for the induction of gametocytogenesis with N-acetylglucosamine (10 mM) within a Wave Bioreactor. By maintaining the culture for 14–16 days as many as 100 million gametocytes (stage V) were produced in a 1 l culture. Gametocytes isolated using magnetic activated cell sorting (MACS) columns were frozen in aliquots for storage. These were revitalised by thawing and shown to retain their ability to exflagellate and infect mosquitoes (Anopheles stephansi).ConclusionsThe production of gametocytes in the Wave Bioreactor under GMP-compliant conditions will not only facilitate cellular, developmental and molecular studies of gametocytes, but also the high-throughput screening for new anti-malarial drugs and, possibly, the development of whole-cell gametocyte or sporozoite-based vaccines.

Full preclinical validation of the 123I-labeled anti-PSMA antibody fragment ScFvD2B for prostate cancer imaging.

PurposeIn the context of prostate cancer (PCa) imaging, the aim of this study was to optimize (in vitro) the specificity and assess preclinically (in vivo) the tumor targeting properties of the 123I-scFvD2B antibody specific for prostate-specific membrane antigen (PSMA).Experimental DesignThe 123I-labeling conditions of the antibody fragment scFvD2B, produced in an eukaryotic system under GMP-compliant conditions, were optimized and assessed for purity and immunoreactivity. The specificity and potency of tumor uptake were tested in three preclinical in vivo models of subcutaneously xenografted human tumors expressing different levels of PSMA (LNCaP, naturally expressing PSMA; PC3-PIP and LS174T-PSMA, transfected with PSMA) or PC3 and LS174T, as negative controls, to assess the clearance, biodistribution and imaging potential of 123I-scFvD2B.ResultsThe set conditions of production and radiolabeling yielded a reagent suitable for human delivery thanks to the purity of the formulation and the high immunoreactivity. In all preclinical models 123I-scFvD2B showed specific targeting only to PSMA-positive tumors with the final specific activity ranging up to 1500 MBq/mg. Despite different levels of PSMA expression, biodistribution analyses and SPECT/CT imaging demonstrated similar results and maximal signal-to-background ratios 24 hours after injection.ConclusionsDue to its in vitro and in vivo properties, 123I-scFvD2B could be a promising tool for the early diagnosis of PCa, and may represent a molecular imaging option to monitor disease progression and assist in the clinical management of PCa patients.

Production of endothelial progenitor cells from skin fibroblasts by direct reprogramming for clinical usages.

Endothelial progenitor cells (EPCs) play an important role in angiogenesis. However, they exist in limited numbers in the human body. This study was aimed to produce EPCs, for autologous transplantation, using direct reprogramming of skin fibroblasts under GMP-compliant conditions. Fibroblasts were collected and cultured from the skin in DMEM/F12 medium supplemented with 5% activated platelet-rich plasma and 1% antibiotic-antimycotic solution. They were then transfected with mRNA ETV2 and incubated in culture medium under hypoxia (5% oxygen) for 14d. Phenotype analysis of transfected cells confirmed that single-factor ETV2 transfection successfully reprogrammed dermal fibroblasts into functional EPCs. Our results showed that ETV2 mRNA combined with hypoxia can give rise to functional EPCs. The cells exhibited functional phenotypes similar to endothelial cells derived from umbilical cord vein; they expressed CD31 and VEGFR2, and formed capillary-like structures in vitro. Moreover, these EPCs could significantly improve hindlimb ischemia in mouse models. Although the direct conversion efficacy was low (3.12±0.98%), altogether our study demonstrates that functional EPCs can be produced from fibroblasts and can be used in clinical applications.

444. Large Scale Culture and Differentiation of Induced Pluripotent Stem Cells for Neutrophil Replacement Therapies

Neutrophils are a key component in the innate immune system and are crucial in the protection against bacterial and fungal infections. Patients with neutropenias are at high risk to develop serious life threatening bacterial and fungal infections. Transfusion of donor neutrophils into neutropenic patients may help alleviate disease burden, but difficulty in collecting and transfusing sufficient quantities of viable donor neutrophils has limited the clinical adaptation of neutrophil replacement therapies. Induced pluripotent stem cells (iPSC) are an attractive alternative source to donor derived neutrophils due to their ability to self-renew and differentiate into cells of the three embryonic germ layers, including neutrophils. Successful implementation of iPSC for neutrophil replacement therapies will require a) derivation of iPSC, b) large scale culture of iPSC, and c) efficient differentiation of these iPSC into functional neutrophils in xeno-fee, GMP-compliant conditions. First, normal human fibroblasts were reprogrammed in xeno-free, GMP compliant conditions though nucleofection of a non-integrating 3 plasmid system carrying OCT4, SOX2, KLF-4, l-MYC, Lin28 and shp53. The reprogrammed fibroblasts were cultured in defined conditions on Vitronectin XF in TeSR-E7 media until colony formation when they were transitioned to E8 medium. We generated 3 lines under these conditions and all iPSC derived and cultured under these conditions retain a normal karyotype, express pluripotency markers SSEA-4, Tra-1-60, Tra-1-81, and CD9 at >85% as determined by FACs analysis, and differentiate into all three germ layers in vitro. Once iPSC lines were established, cultures were adapted from adherent cultures to non-adherent, aggregate suspension cultures in spinner flask bioreactor systems in mTeSR or E8 medium. Briefly, adherent iPSC are dissociated into single cells and seeded into a spinner flask at a concentration of 2×105 cells/ml and spun at 60 RPM. We evaluated 2 lines in 3 independent experiments and cells readily formed clusters and were able to achieve 4-log expansion (range: 2-4 log) within approximately 40 days (10 passages). All lines retained a normal karyotype, expressed pluripotency markers SSEA-4, Tra-1-60, Tra-1-81, and CD9 (>85%), and displayed a normal morphology when re-plated in adherent culture. We next adapted a neutrophil differentiation system for differentiation from iPSC from the defined media/spinnerflask system. Clusters of cells from spinner flask cultures were induced to form embryoid bodies (EBs) by transitioning the cultures to EB specification medium for 5 days, followed by a neutrophil specification medium containing IL-3 and G-CSF in adherent conditions. After 7-14 days in neutrophil specification conditions, EBs attached to the plate and CD45+CD11b+CD16+ neutrophils were shed into the medium and were harvested twice weekly for 30-60 days. In 10 experiments, we collected an average of 6.5×10^5 CD45+ cells per well of differentiation representing a 6.5 fold expansion (range: 3-10 fold). In conclusion, iPSC have been derived in GMP compliant conditions and can be cultured at a large scale using spinner flask bioreactors, and the iPSC from spinner flask bioreactors can then successfully be differentiated into CD45+ cells. Thus, iPSC represent an attractive, self-renewing resource of neutrophils for neutrophil replacement therapies.

Cord Blood Derived Natural Killer Cells Engineered with a Chimeric Antigen Receptor Targeting CD19 and Expressing IL-15 Have Long Term Persistence and Exert Potent Anti-Leukemia Activity

Natural killer (NK) cells are a major component of the innate immune system, possessing the ability to lyse their targets without the need for prior sensitization or specificity for antigen. Besides their classical role in providing potent antitumor and antiviral immunity, NK cells can reduce the risk of graft-versus-host disease (GVHD) by targeting host antigen-presenting cells, as well as activated alloreactive donor T cells, indicating that NK-mediated graft-versus-leukemia (GVL) responses may occur in the absence of GVHD. Although most groups have relied on autologous or adult peripheral blood donor-derived NK cells, we have studied umbilical CB as a potential source of NK cells because of their availability as an "off-the-shelf" frozen product and their potent preclinical activity against leukemia cells. To overcome the obstacle of limited numbers of NK cells in a single CB unit, we have established GMP-compliant conditions for the ex vivo expansion of clinically relevant doses of such cells. By using GMP grade K562-based artificial antigen-presenting cells (aAPCs), which express membrane-bound IL-21 (clone 9.mbIL21), to numerically expand highly functional and mature CB-derived NK cells.To further enhance the GVL effect independent of KIR-ligand mismatch, we have genetically modified human CB-derived NK cells with a retroviral vector, CAR19-CD28-zeta-2A-IL15 (CAR19/IL15), which incorporates the genes for CAR-CD19, IL-15 to enhance proliferation and survival, and the inducible caspase-9 molecule. CB-NK cells could be stably transduced with CAR19/IL15, proliferated efficiently in vitro and maintained superior effector function against CD19-expressing leukemia cell lines and primary CLL cells. Moreover, the effector functions of both NK-CAR and nontransduced NK cells against K562 were comparable, indicating that the genetic modification of CB-NK cells does not alter their intrinsic cytotoxicity against NK-sensitive targets. Because of concerns over autonomous, uncontrolled NK cell growth due to autocrine production of IL15, we also incorporated into our construct a suicide gene based on the inducible caspase-9 (IC9) gene. The addition of as little as 10 nM of the small molecule dimerizer CID AP20187 to cultures of iC9/CAR19/IL15+ NK cells induced apoptosis/necrosis of >60% of transgenic cells within 4 hours as assessed by annexin-V-7AAD staining. The infusion of CAR.CD19.IL15-transduced CB-NK cells into a NOD-SCID-gamma null model of lymphoblastic lymphoma (Raji model) resulted in impressive anti-tumor responses (Fig. 1). Moreover, adoptively infused CAR-transduced CB NK persisted for up to 70 days post-infusion (Fig. 2), supporting our hypothesis that IL-15 enhances the proliferation and survival of the engineered CB-NK cells. Based on these promising data, we now propose to manufacture a GMP-grade CAR19-CD28-zeta-2A-IL15 vector for a phase 1 dose escalation trial in patients with high risk B-cell leukemia. [Display omitted] [Display omitted] DisclosuresWierda:Celgene Corp.: Consultancy; Glaxo-Smith-Kline Inc.: Research Funding. Rezvani:Pharmacyclics: Research Funding.