Enriching leukapheresis improves T cell activation and transduction efficiency during CAR T processing

The majority of CD19-directed CAR T cell products are manufactured using an autologous process. Although using a patient's leukapheresis reduces the risks of rejection, it introduces variability in starting material composition and the presence of cell populations that might negatively affect production of chimeric antigen receptor (CAR) T cells, such as myeloid cells. In this work, the effect of monocytes (CD14) on the level of activation, growth, and transduction efficiency was monitored across well plate and culture bag platforms using healthy donor leukapheresis. Removal of monocytes from leukapheresis improved the level of activation 2-fold, achieving the same level of activation as when initiating the process with a purified T cell starting material. Two activation reagents were tested in well plate cultures, revealing differing sensitivities to starting material composition. Monocyte depletion in culture bag systems had a significant effect on transduction efficiency, improving consistency and increasing the level of CAR expression by up to 64% compared to unsorted leukapheresis. Cytotoxicity assays revealed that CAR T cell products produced from donor material depleted of monocytes and isolated T cells consistently outperformed those made from unsorted leukapheresis. Analysis of memory phenotypes and gene expression indicated that CAR T cells produced using depleted starting material displayed a more rested and naive state. The success of CAR T cell manufacturing and final product function is influenced by the composition of the donor starting material. In this work, we show that upstream depletion of specific cell populations can enhance processing outcomes such as activation, transduction, and phenotype of the therapeutic product

Towards modular bone tissue engineering using Ti-Co-doped phosphate glass microspheres: cytocompatibility and dynamic culture studies

The production of large quantities of functional vascularized bone tissue ex vivo still represent an unmet clinical challenge. Microcarriers offer a potential solution to scalable manufacture of bone tissue due to their high surface area-to-volume ratio and the capacity to be assembled using a modular approach. Microcarriers made of phosphate bioactive glass doped with titanium dioxide have been previously shown to enhance proliferation of osteoblast progenitors and maturation towards functional osteoblasts. Furthemore, doping with cobalt appears to mimic hypoxic conditions that have a key role in promoting angiogenesis. This characteristic could be exploited to meet the clinical requirement of producing vascularized units of bone tissue. In the current study, the human osteosarcoma cell line MG-63 was cultured on phosphate glass microspheres doped with 5% mol titanium dioxide and different concentrations of cobalt oxide (0%, 2% and 5% mol), under static and dynamic conditions (150 and 300 rpm on an orbital shaker). Cell proliferation and the formation of aggregates of cells and microspheres were observed over a period of two weeks in all glass compositions, thus confirming the biocompatibility of the substrate and the suitability of this system for the formation of compact micro-units of tissue. At the concentrations tested, cobalt was not found to be cytotoxic and did not alter cell metabolism. On the other hand, the dynamic environment played a key role, with moderate agitation having a positive effect on cell proliferation while higher agitation resulting in impaired cell growth. Finally, in static culture assays, the capacity of cobalt doping to induce vascular endothelial growth factor (VEGF) upregulation by osteoblastic cells was observed, but was not found to increase linearly with cobalt oxide content. In conclusion, Ti–Co phosphate glasses were found to support osteoblastic cell growth and aggregate formation that is a necessary precursor to tissue formation and the upregaulation of VEGF production can potentially support vascularization

A parameterised mathematical model to elucidate osteoblast cell growth in a phosphate-glass microcarrier culture

Tissue engineering has the potential to augment bone grafting. Employing microcarriers as cell-expansion vehicles is a promising bottom-up bone tissue engineering strategy. Here we propose a collaborative approach between experimental work and mathematical modelling to develop protocols for growing microcarrier-based engineered constructs of clinically relevant size. Experiments in 96-well plates characterise cell growth with the model human cell line MG-63 using four phosphate glass microcarrier materials. Three of the materials are doped with 5 mol% TiO2 and contain 0%, 2% or 5% CoO, and the fourth material is doped only with 7% TiO2 (0% CoO). A mathematical model of cell growth is parameterised by finding material-specific growth coefficients through data-fitting against these experiments. The parameterised mathematical model offers more insight into the material performance by comparing culture outcome against clinically relevant criteria: maximising final cell number starting with the lowest cell number in the shortest time frame. Based on this analysis, material 7% TiO2 is identified as the most promising

Characterisation of osteogenic and vascular responses of hMSCs to Ti-Co doped phosphate glass microspheres using a microfluidic perfusion platform

Using microspherical scaffolds as building blocks to repair bone defects of specific size and shape has been proposed as a tissue engineering strategy. Here, phosphate glass (PG) microcarriers doped with 5 mol % TiO2 and either 0 mol % CoO (CoO 0%) or 2 mol % CoO (CoO 2%) were investigated for their ability to support osteogenic and vascular responses of human mesenchymal stem cells (hMSCs). Together with standard culture techniques, cell-material interactions were studied using a novel perfusion microfluidic bioreactor that enabled cell culture on microspheres, along with automated processing and screening of culture variables. While titanium doping was found to support hMSCs expansion and differentiation, as well as endothelial cell-derived vessel formation, additional doping with cobalt did not improve the functionality of the microspheres. Furthermore, the microfluidic bioreactor enabled screening of culture parameters for cell culture on microspheres that could be potentially translated to a scaled-up system for tissue-engineered bone manufacturing

Assessing behaviour of osteoblastic cells in dynamic culture conditions using titanium-doped phosphate glass microcarriers

Tissue engineering is a promising approach for bone regeneration; yet challenges remain that limit successful translation to patients. It is necessary to understand how real-world manufacturing processes will affect the constituent cells and biomaterials that are needed to create engineered bone. Bioactive phosphate glasses processed into microspheres are an attractive platform for expanding bone-forming cells and also for driving their osteogenic differentiation and maturation. The aim of this study was to assess whether Ti-doped phosphate glass microspheres could support osteoblastic cell responses in dynamic cell culture environments. Dynamic culture conditions were achieved using microwell studies under orbital agitation. Dimensionless parameters such as the Froude number were used to inform the choice of agitation speeds, and the impact on cell proliferation and microunit formation was quantified. We found that phosphate glass microspheres doped with titanium dioxide at both 5 and 7 mol% provided a suitable biomaterial platform for effective culture of MG63 osteoblastic cells and was not cytotoxic. Dynamic culture conditions supported expansion of MG63 cells and both 150 and 300 rpm orbital shake resulted in higher cell yield than static cultures at the end of the culture (day 13). The Froude number analysis provided insight into how the microunit size could be manipulated to enable an appropriate agitation speed to be used, while ensuring buoyancy of the microunits. These small-scale experiments and analyses provide understanding of the impact of fluid flow on cell expansion that will have increasing importance when scaling up to process technologies that can deliver clinical quantities of cell-microsphere units. Such knowledge will enable future engineering of living bone-like material using processing systems such as bioreactors that use mixing and agitation for nutrient transfer, therefore introducing cells to dynamic culture conditions

Characterisation of osteogenic and vascular responses of hMSCs to Ti-Co doped phosphate glass microspheres using a microfluidic perfusion platform

Using microspherical scaffolds as building blocks to repair bone defects of specific size and shape has been proposed as a tissue engineering strategy. Here, phosphate glass (PG) microcarriers doped with 5 mol % TiO2 and either 0 mol % CoO (CoO 0%) or 2 mol % CoO (CoO 2%) were investigated for their ability to support osteogenic and vascular responses of human mesenchymal stem cells (hMSCs). Together with standard culture techniques, cell-material interactions were studied using a novel perfusion microfluidic bioreactor that enabled cell culture on microspheres, along with automated processing and screening of culture variables. While titanium doping was found to support hMSCs expansion and differentiation, as well as endothelial cell-derived vessel formation, additional doping with cobalt did not improve the functionality of the microspheres. Furthermore, the microfluidic bioreactor enabled screening of culture parameters for cell culture on microspheres that could be potentially translated to a scaled-up system for tissue-engineered bone manufacturing

CAR T cells with dual targeting of CD19 and CD22 in pediatric and young adult patients with relapsed or refractory B cell acute lymphoblastic leukemia: a phase 1 trial

Chimeric antigen receptor (CAR) T cells targeting CD19 or CD22 have shown remarkable activity in B cell acute lymphoblastic leukemia (B-ALL). The major cause of treatment failure is antigen downregulation or loss. Dual antigen targeting could potentially prevent this, but the clinical safety and efficacy of CAR T cells targeting both CD19 and CD22 remain unclear. We conducted a phase 1 trial in pediatric and young adult patients with relapsed or refractory B-ALL (n = 15) to test AUTO3, autologous transduced T cells expressing both anti-CD19 and anti-CD22 CARs (AMELIA trial, EUDRA CT 2016-004680-39). The primary endpoints were the incidence of grade 3-5 toxicity in the dose-limiting toxicity period and the frequency of dose-limiting toxicities. Secondary endpoints included the rate of morphological remission (complete response or complete response with incomplete bone marrow recovery) with minimal residual disease-negative response, as well as the frequency and severity of adverse events, expansion and persistence of AUTO3, duration of B cell aplasia, and overall and event-free survival. The study endpoints were met. AUTO3 showed a favorable safety profile, with no dose-limiting toxicities or cases of AUTO3-related severe cytokine release syndrome or neurotoxicity reported. At 1 month after treatment the remission rate (that is, complete response or complete response with incomplete bone marrow recovery) was 86% (13 of 15 patients). The 1 year overall and event-free survival rates were 60% and 32%, respectively. Relapses were probably due to limited long-term AUTO3 persistence. Strategies to improve CAR T cell persistence are needed to fully realize the potential of dual targeting CAR T cell therapy in B-ALL

Microscale tissue engineering: a modular approach for vascularized bone regeneration

Four million surgeries involving bone grafting or bone substitutes for the treatment of bone defects are performed yearly worldwide. However, limited donor tissue availability, pain and the risk of infection and immune rejection, have led to the development of alternative strategies for bone repair. Tissue engineering represents an alternative to current treatments as it consists of using a biomaterial scaffold alone or in combination with proteins, genes or cells, as a bioactive implant to stimulate bone repair. Microspherical scaffolds have been proposed as a potential modular unit for bone tissue engineering applications as their shape could facilitate filling of irregular shaped defects. Furthermore, microspheres could be used as a support for ex vivo expansion of adherent cells as well as a carrier to directly deliver cells to the defect site. In this study, the use of phosphate glass microcarriers for bone tissue engineering applications was investigated. As this material is completely soluble and non-toxic, it can be implanted in the patient together with cells. Furthermore, the tuneable glass composition can be easily engineered to induce specific structural and biological properties. Here, the effect of culturing MG-63 and hBM-MSCs on titanium-doped phosphate glass microspheres containing increasing concentration of cobalt (0, 2 and 5%) was investigated, as these ions have been shown to induce osteogenesis and angiogenesis, respectively. Furthermore, as part of this study a novel perfusion microfluidic bioreactor was fabricated to culture cells on microspheres under perfusion and to enable parallel screening of multiple culture variables. Cells proliferation on the microspheres as well as secretion of ECM proteins in response to the substrate was observed over time, thus confirming the biocompatibility of all compositions tested. Upregulation of osteogenic markers by MSCs also occurred in response to the microspheres in the absence of exogenous supplements. However, this effect was suppressed when cobalt was added to the glass composition. On the other hand, while cobalt doping was found to induce key angiogenic responses (i.e. VEGF secretion), this did not translate into improved functional vascularization in comparison to the cobalt-free microspheres. Successful MSCs culture on the microspheres within the microfluidic reactor was achieved and it was possible to efficiently quantify functional outputs, such as the expression of ECM proteins as a function of microspheres substrates and nutrient feeds under perfusion. In conclusion, titanium-doped phosphate glass microspheres were identified as a potential substrate for bone tissue engineering applications in terms of MSCs expansion and differentiation, as well as to support endothelial cells migration towards the scaffold and vessel formation, while additional doping with cobalt was not found to improve the functionality of the microspheres. Furthermore, the microfluidic bioreactor enabled to identify optimal parameters for perfused cell culture on microspheres that could be potentially translated to a scaled-up system for tissue-engineered bone manufacturing