Project description:Natural killer group 2D (NKG2D) is a natural killer (NK) cell-activating receptor that recognizes different stress-induced ligands that are overexpressed in a variety of childhood and adult tumors. NKG2D chimeric antigen receptor (CAR) T cells have shown potent anticancer effects against different cancer types. A second-generation NKG2D CAR was generated by fusing full-length human NKG2D to 4-1BB costimulatory molecule and CD3? signaling domain. Patient-derived CAR T cells show limitations including inability to manufacture CAR T cells from the patients' own T cells, disease progression, and death prior to return of engineered cells. The use of allogeneic T cells for CAR therapy could be an attractive alternative, although undesirable graft vs. host reactions may occur. To avoid such adverse effects, we used CD45RA- memory T cells, a T-cell subset with less alloreactivity, as effector cells to express NKG2D CAR. In this study, we developed a protocol to obtain large-scale NKG2D CAR memory T cells for clinical use by using CliniMACS Prodigy, an automated closed system compliant with Good Manufacturing Practice (GMP) guidelines. CD45RA+ fraction was depleted from healthy donors' non-mobilized apheresis using CliniMACS CD45RA Reagent and CliniMACS Plus device. A total of 108 CD45RA- cells were cultured in TexMACS media supplemented with 100 IU/mL IL-2 and activated at day 0 with T Cell TransAct. Then, we used NKG2D-CD8TM-4-1BB-CD3? lentiviral vector for cell transduction (MOI = 2). NKG2D CAR T cells expanded between 10 and 13 days. Final cell products were analyzed to comply with the specifications derived from the quality and complementary controls carried out in accordance with the instructions of the Spanish Regulatory Agency of Medicines and Medical Devices (AEMPS) for the manufacture of investigational advanced therapy medicinal products (ATMPs). We performed four validations. The manufacturing protocol here described achieved large numbers of viable NKG2D CAR memory T cells with elevated levels of NKG2D CAR expression and highly cytotoxic against Jurkat and 531MII tumor target cells. CAR T cell final products met release criteria, except for one showing myc overexpression and another with viral copy number higher than five. Manufacturing of clinical-grade NKG2D CAR memory T cells using CliniMACS Prodigy is feasible and reproducible, widening clinical application of CAR T cell therapies.
Project description:Powder metallurgy (P/M) technique is usually used for manufacturing porous metal materials. However, some P/M materials are limitedly used in engineering for their performance deficiency. A novel 304 stainless steel P/M material was produced by a solid-state sintering of 304 stainless steel powders and 304 short stainless steel fibers, which were alternately laid in layers according to mass ratio. In this paper, the compressive properties of the P/M materials were characterized by a series of uniaxial compression tests. The effects of fiber content, compaction pressure and high temperature nitriding on compressive properties were investigated. The results indicated that, without nitriding, the samples changed from cuboid to cydariform without damage in the process of compression. The compressive stress was enhanced with increasing fiber content ranging from 0 to 8 wt.%. For compaction pressure from 55 to 75 MPa, greater compaction pressure improved compressive stress. Moreover, high temperature nitriding was able to significantly improve the yield stress, but collapse failure eventually occurred.
Project description:Immunotherapy using chimeric antigen receptor-modified T cells has demonstrated high response rates in patients with B cell malignancies, and chimeric antigen receptor T cell therapy is now being investigated in several hematologic and solid tumor types. Chimeric antigen receptor T cells are generated by removing T cells from a patient's blood and engineering the cells to express the chimeric antigen receptor, which reprograms the T cells to target tumor cells. As chimeric antigen receptor T cell therapy moves into later-phase clinical trials and becomes an option for more patients, compliance of the chimeric antigen receptor T cell manufacturing process with global regulatory requirements becomes a topic for extensive discussion. Additionally, the challenges of taking a chimeric antigen receptor T cell manufacturing process from a single institution to a large-scale multi-site manufacturing center must be addressed. We have anticipated such concerns in our experience with the CD19 chimeric antigen receptor T cell therapy CTL019. In this review, we discuss steps involved in the cell processing of the technology, including the use of an optimal vector for consistent cell processing, along with addressing the challenges of expanding chimeric antigen receptor T cell therapy to a global patient population.
Project description:The volume of slags generated from the steel industry is a source of possible resources which is constantly increasing. Specifically, in the production of stainless steel, specific and singular slags with unique characteristics are obtained, which allows considering an approach aimed at their use in new recycling ways. This work shows the feasibility of using stainless steel slag as a substitute for limestone filler in the manufacture of self-compacting concrete. The influence of different treatments applied to slags on physical and chemical properties was studied. On the other hand, the mechanical behaviour, as well as the durability acquired in self-compacting concrete, has been analysed. Very encouraging results were obtained, since this research demonstrates the possible application of this stainless steel slag as a construction material, improving sustainability and promoting circular economy processes, which are achieved through the minimisation of the waste disposal and accumulation.
Project description:The next generation of therapeutic products to be approved for the clinic is anticipated to be cell therapies, termed "living drugs" for their capacity to dynamically and temporally respond to changes during their production ex vivo and after their administration in vivo. Genetically engineered chimeric antigen receptor (CAR) T cells have rapidly developed into powerful tools to harness the power of immune system manipulation against cancer. Regulatory agencies are beginning to approve CAR T cell therapies due to their striking efficacy in treating some hematological malignancies. However, the engineering and manufacturing of such cells remains a challenge for widespread adoption of this technology. Bioengineering approaches including biomaterials, synthetic biology, metabolic engineering, process control and automation, and in vitro disease modeling could offer promising methods to overcome some of these challenges. Here, we describe the manufacturing process of CAR T cells, highlighting potential roles for bioengineers to partner with biologists and clinicians to advance the manufacture of these complex cellular products under rigorous regulatory and quality control.
Project description:As of April 2021, there are five commercially available chimeric antigen receptor (CAR) T cell therapies for hematological malignancies. With the current transition of CAR T cell manufacturing from academia to industry, there is a shift toward Good Manufacturing Practice (GMP)-compliant closed and automated systems to ensure reproducibility and to meet the increased demand for cancer patients. In this review we describe current CAR T cells clinical manufacturing models and discuss emerging technological advances that embrace scaling and production optimization. We summarize measures being used to shorten CAR T-cell manufacturing times and highlight regulatory challenges to scaling production for clinical use.Statement of significance ∣As the demand for CAR T cell cancer therapy increases, several closed and automated production platforms are being deployed, and others are in development.This review provides a critical appraisal of these technologies that can be leveraged to scale and optimize the production of next generation CAR T cells.
Project description:Small objects of an alloy tool steel were built by selective laser melting at different scan speeds, and their microstructures were analyzed using electron backscatter diffraction (EBSD). To present an explicit correlation with the local thermal cycles in the objects, prior austenite grains were reconstructed using the EBSD mapping data. Extensive growth of austenitic grains after solidification could be detected by the disagreement between the networks of carbides and austenite grain boundaries. A rapid laser scan at 2000 mm/s led to less growth, but retained a larger amount of austenite than a slow one at 50 mm/s. The rapid scan also exhibited definite evolution of Goss-type textures in austenite, which could be attributed to the growth of austenitic grains under a steep temperature gradient. The local variations in the microstructures and the textures enabled us to speculate the locally different thermal cycles determined by the different process conditions, that is, scan speeds.
Project description:ObjectivesThe aim of this study was to couple fused deposition modelling 3D printing with melt extrusion technology to produce core-shell-structured controlled-release tablets with dual-mechanism drug-release performance in a simulated intestinal fluid medium. Coupling abovementioned technologies for personalized drug delivery can improve access to complex dosage formulations at a reasonable cost. Compared with traditional pharmaceutical manufacturing, this should facilitate the following: (1) the ability to manipulate drug release by adjusting structures, (2) enhanced solubility and bioavailability of poorly water-soluble drugs and (3) on-demand production of more complex structured dosages for personalized treatment.MethodsAcetaminophen was the model drug and the extrusion process was evaluated by a series of physicochemical characterizations. The geometries, morphologies, and in vitro drug-release performances were compared between directly compressed and 3D-printed tablets.Key findingsInitially, 3D-printed tablets released acetaminophen more rapidly than directly compressed tablets. Drug release became constant and steady after a pre-determined time. Thus, rapid effectiveness was ensured by an initially fast acetaminophen release and an extended therapeutic effect was achieved by stabilizing drug release.ConclusionsThe favourable drug-release profiles of 3D-printed tablets demonstrated the advantage of coupling HME with 3D printing technology to produce personalized dosage formulations.
Project description:Tisagenlecleucel is a CD19-specific chimeric antigen receptor (CAR)-T cell therapy approved for patients aged ≤25 years with relapsed or refractory B cell precursor acute lymphoblastic leukemia (B-ALL) and adults with relapsed or refractory diffuse large B cell lymphoma (DLBCL). The initial tisagenlecleucel manufacturing process technology was developed at an academic center and was subsequently transferred, optimized, validated, and scaled out to supply large global trials before commercialization. Tisagenlecleucel manufactured in two centralized facilities has been successfully used in global multicenter trials for B-ALL and DLBCL (>50 clinical centers in 12 countries). In this paper, we describe some of the continuous process improvements made to tisagenlecleucel manufacturing over time to meet global demand while maintaining and improving product quality. During early tisagenlecleucel clinical trials, process enhancements were made to address logistical challenges related to manufacturing for multicenter trials and to accommodate the variability observed in patient starting cellular material. These enhancements resulted in improvements in manufacturing capacity, process robustness, manufacturing success rates, and product quality, and reductions in throughput time. In summary, through continuous evaluation and improvements based on experience during global trials, a consistent and robust commercial manufacturing process for tisagenlecleucel has been developed, leading to improvements in manufacturing success when compared to the initial processes.
Project description:Since the FDA approval of two Chimeric Antigen Receptor (CAR) T cell therapies against CD19+ malignancies, there has been significant interest in adapting CAR technology to other diseases. As such, the ability to simultaneously monitor manufacturing criteria and functional characteristics of multiple CAR T cell products by a single instrument would likely accelerate the development of candidate therapies. Here, we demonstrate that image-based cytometry yields high-throughput measurements of CAR T cell proliferation and size, and captures the kinetics of in vitro antigen-specific CAR T cell-mediated killing. The data acquired and analyzed by the image cytometer are congruent with results derived from conventional technologies when tested contemporaneously. Moreover, the use of bright-field and fluorescence microscopy by the image cytometer provides kinetic measurements and rapid data acquisition, which are direct advantages over industry standard instruments. Together, image cytometry enables fast, reproducible measurements of CAR T cell manufacturing criteria and effector function, which can greatly facilitate the evaluation of novel CARs with therapeutic potential.