Commercial Scale Manufacturing of Allogeneic Cell Therapy

Allogeneic cell therapy products are generating encouraging clinical and pre-clinical results. Pluripotent stem cell (PSC) derived therapies, in particular, have substantial momentum and the potential to serve as treatments for a wide range of indications. Many of these therapies are also expected to have large market sizes and require cell doses of ≥109 cells. As therapeutic technologies mature, it is essential for the cell manufacturing industry to correspondingly develop to adequately support commercial scale production. To that end, there is much that can be learned and adapted from traditional manufacturing fields. In this review, we highlight key areas of allogeneic cell therapy manufacturing, identify current gaps, and discuss strategies for integrating new solutions. It is anticipated that cell therapy scale-up manufacturing solutions will need to generate batches of up to 2,000 L in single-use disposable formats, which constrains selection of currently available upstream hardware. Suitable downstream hardware is even more limited as processing solutions from the biopharmaceutical field are often not compatible with the unique requirements of cell therapy products. The advancement of therapeutic cell manufacturing processes to date has largely been developed with a cell biology driven approach, which is essential in early development. However, for truly robust and standardized production in a maturing field, a highly controlled manufacturing engineering strategy must be employed, with the implementation of automation, process monitoring and control to increase batch consistency and efficiency

Development of a closed CAR-T manufacturing process

The field of immunotherapy has emerged as a promising new type of treatment for cancer with the approval of the first two CAR-T therapies. The clinical success of T-cell based immunotherapies necessitates a robust manufacturing process for these products to be consistently produced at commercial scale. Our CAR-T workflow combines unit operation specific solutions for thaw of an apheresis unit, wash, CD3 selection, T-cell activation, lentiviral transduction, incubator- and reactor-based expansion culture, harvest, formulation, cryopreservation and thaw of CAR-T product. We have evaluated the impact of both serum-containing and xeno-free culture media, commercially available T-cell selection and activation reagents, closed small-scale culture vessel options, alternative solutions to enhance transduction, and the specific timing of process steps to develop a modular platform process that is robust and flexible for the varied needs of CAR-T developers. Frozen apheresis units are processed using the SmartWash protocol on the SepaxTM 2 and T-cells are isolated with EasySepTM Release CD3 Positive Selection Kit. The cells are then activated with ImmunoCult CD3/CD28/CD2 T-cell activator before being transduced 24 hours later using the SepaxTM 2. Expansion of Tcells are carried out in two stages: incubator-based culture before going into the XuriTM Cell Expansion System W25 with a perfusion feeding regime. Cultured cells are then harvested and washed in Plasmalyte-A with human serum albumin and formulated with CryoStor® CS10 using the FlexCell protocol on the Sefia™ Cell Processing System. The final cell products are cryopreserved using the VIA Freeze controlled-rate freezer. We have also accessed a point-of-care thawing strategy using the VIA Thaw. Our CAR-T process achieves greater than 1.0E10 expanded T-cells with \u3e80% eGFP transduction efficiency across an 8-day manufacturing process

Dynamic interaction networks in a hierarchically organized tissue

We have integrated gene expression profiling with database and literature mining, mechanistic modeling, and cell culture experiments to identify intercellular and intracellular networks regulating blood stem cell self-renewal.Blood stem cell fate in vitro is regulated non-autonomously by a coupled positive–negative intercellular feedback circuit, composed of megakaryocyte-derived stimulatory growth factors (VEGF, PDGF, EGF, and serotonin) versus monocyte-derived inhibitory factors (CCL3, CCL4, CXCL10, TGFB2, and TNFSF9).The antagonistic signals converge in a core intracellular network focused around PI3K, Raf, PLC, and Akt.Model simulations enable functional classification of the novel endogenous ligands and signaling molecules

Ex vivo Manufactured Neutrophils for Treatment of Neutropenia—A Process Economic Evaluation

Neutropenia is a common side-effect of acute myeloid leukemia (AML) chemotherapy characterized by a critical drop in neutrophil blood concentration. Neutropenic patients are prone to infections, experience poorer clinical outcomes, and require expensive medical care. Although transfusions of donor neutrophils are a logical solution to neutropenia, this approach has not gained clinical traction, primarily due to challenges associated with obtaining sufficiently large numbers of neutrophils from donors whilst logistically managing their extremely short shelf-life. A protocol has been developed that produces clinical-scale quantities of neutrophils from hematopoietic stem and progenitor cells (HSPC) in 10 L single-use bioreactors (1). This strategy could be used to mass produce neutrophils and generate sufficient cell numbers to allow decisive clinical trials of neutrophil transfusion. We present a bioprocess model for neutrophil production at relevant clinical-scale. We evaluated two production scenarios, and the impact on cost of goods (COG) of multiple model parameters including cell yield, materials costs, and process duration. The most significant contributors to cost were consumables and raw materials, including the cost of procuring HSPC-containing umbilical cord blood. The model indicates that the most cost-efficient culture volume (batch size) is ~100 L in a single bioreactor. This study serves as a framework for decision-making and optimization strategies when contemplating the production of clinical quantities of cells for allogeneic therapy

PERT: A Method for Expression Deconvolution of Human Blood Samples from Varied Microenvironmental and Developmental Conditions

The cellular composition of heterogeneous samples can be predicted using an expression deconvolution algorithm to decompose their gene expression profiles based on pre-defined, reference gene expression profiles of the constituent populations in these samples. However, the expression profiles of the actual constituent populations are often perturbed from those of the reference profiles due to gene expression changes in cells associated with microenvironmental or developmental effects. Existing deconvolution algorithms do not account for these changes and give incorrect results when benchmarked against those measured by well-established flow cytometry, even after batch correction was applied. We introduce PERT, a new probabilistic expression deconvolution method that detects and accounts for a shared, multiplicative perturbation in the reference profiles when performing expression deconvolution. We applied PERT and three other state-of-the-art expression deconvolution methods to predict cell frequencies within heterogeneous human blood samples that were collected under several conditions (uncultured mono-nucleated and lineage-depleted cells, and culture-derived lineage-depleted cells). Only PERT's predicted proportions of the constituent populations matched those assigned by flow cytometry. Genes associated with cell cycle processes were highly enriched among those with the largest predicted expression changes between the cultured and uncultured conditions. We anticipate that PERT will be widely applicable to expression deconvolution strategies that use profiles from reference populations that vary from the corresponding constituent populations in cellular state but not cellular phenotypic identity

Development of Gene Expression Markers of Acute Heat-Light Stress in Reef-Building Corals of the Genus Porites

Coral reefs are declining worldwide due to increased incidence of climate-induced coral bleaching, which will have widespread biodiversity and economic impacts. A simple method to measure the sub-bleaching level of heat-light stress experienced by corals would greatly inform reef management practices by making it possible to assess the distribution of bleaching risks among individual reef sites. Gene expression analysis based on quantitative PCR (qPCR) can be used as a diagnostic tool to determine coral condition in situ. We evaluated the expression of 13 candidate genes during heat-light stress in a common Caribbean coral Porites astreoides, and observed strong and consistent changes in gene expression in two independent experiments. Furthermore, we found that the apparent return to baseline expression levels during a recovery phase was rapid, despite visible signs of colony bleaching. We show that the response to acute heat-light stress in P. astreoides can be monitored by measuring the difference in expression of only two genes: Hsp16 and actin. We demonstrate that this assay discriminates between corals sampled from two field sites experiencing different temperatures. We also show that the assay is applicable to an Indo-Pacific congener, P. lobata, and therefore could potentially be used to diagnose acute heat-light stress on coral reefs worldwide

3 years of liraglutide versus placebo for type 2 diabetes risk reduction and weight management in individuals with prediabetes: a randomised, double-blind trial

Background: Liraglutide 3·0 mg was shown to reduce bodyweight and improve glucose metabolism after the 56-week period of this trial, one of four trials in the SCALE programme. In the 3-year assessment of the SCALE Obesity and Prediabetes trial we aimed to evaluate the proportion of individuals with prediabetes who were diagnosed with type 2 diabetes. Methods: In this randomised, double-blind, placebo-controlled trial, adults with prediabetes and a body-mass index of at least 30 kg/m2, or at least 27 kg/m2 with comorbidities, were randomised 2:1, using a telephone or web-based system, to once-daily subcutaneous liraglutide 3·0 mg or matched placebo, as an adjunct to a reduced-calorie diet and increased physical activity. Time to diabetes onset by 160 weeks was the primary outcome, evaluated in all randomised treated individuals with at least one post-baseline assessment. The trial was conducted at 191 clinical research sites in 27 countries and is registered with ClinicalTrials.gov, number NCT01272219. Findings: The study ran between June 1, 2011, and March 2, 2015. We randomly assigned 2254 patients to receive liraglutide (n=1505) or placebo (n=749). 1128 (50%) participants completed the study up to week 160, after withdrawal of 714 (47%) participants in the liraglutide group and 412 (55%) participants in the placebo group. By week 160, 26 (2%) of 1472 individuals in the liraglutide group versus 46 (6%) of 738 in the placebo group were diagnosed with diabetes while on treatment. The mean time from randomisation to diagnosis was 99 (SD 47) weeks for the 26 individuals in the liraglutide group versus 87 (47) weeks for the 46 individuals in the placebo group. Taking the different diagnosis frequencies between the treatment groups into account, the time to onset of diabetes over 160 weeks among all randomised individuals was 2·7 times longer with liraglutide than with placebo (95% CI 1·9 to 3·9, p<0·0001), corresponding with a hazard ratio of 0·21 (95% CI 0·13–0·34). Liraglutide induced greater weight loss than placebo at week 160 (–6·1 [SD 7·3] vs −1·9% [6·3]; estimated treatment difference −4·3%, 95% CI −4·9 to −3·7, p<0·0001). Serious adverse events were reported by 227 (15%) of 1501 randomised treated individuals in the liraglutide group versus 96 (13%) of 747 individuals in the placebo group. Interpretation: In this trial, we provide results for 3 years of treatment, with the limitation that withdrawn individuals were not followed up after discontinuation. Liraglutide 3·0 mg might provide health benefits in terms of reduced risk of diabetes in individuals with obesity and prediabetes. Funding: Novo Nordisk, Denmark

Feedback-mediated Regulation of Human Hematopoietic Stem Cell Fate

Umbilical cord blood (UCB) cells fill a clinical need for hematopoietic stem and progenitor cell (HSPC) transplantation, and the ex vivo expansion of these cells provides an approach to increase their clinical relevance. Ex vivo HSPC expansion has been limited by a poor understanding of the interplay between stem cell autonomous and feedback mediated regulators. We hypothesized that developing strategies to modulate the HSPC microenvironment would enable enhanced expansion of these cells and a greater insight into the underlying biologic mechanisms. We developed a system for the optimized delivery of the transcription factor HOXB4 to human hematopoietic culture and revealed new insights into the context-dependent potentials and limitations of HOXB4. We investigated approaches for the global reduction of inhibitory feedback signaling and developed a fed-batch system that minimizes endogenously produced factors to create a more supportive environment for HSPC self-renewal. This strategy led to an 11-fold expansion of long-term repopulating HSCs in a clinically relevant bioreactor, producing a novel system for ex vivo expansion and generating a platform to assess HSPC enhancing factors. By combining the fed-batch system with the Notch Delta-1 ligand (DL1), we identified a mechanism whereby DL1 initiated a conversion from IL-6 cis-signaling to trans-signaling, resulting in the modulation of mature cell population production. This demonstrated the impact of cell lineage skewing on microenvironment regulation and the expansion of HSPCs. This work demonstrates how cell-cell interactions and feedback mediated signaling are critical regulators of HSPCs and the manipulation of these regulators can be used both to engineer HSPC expansion processes and to identify novel mechanisms within the hematopoietic system. This provides an important step towards the development of robust methods for the ex vivo expansion of UCB-derived cells and the ultimate goal of achieving cures in hematologic disease.Ph

PERT recovers compositions of uncultured human cord blood mono-nucleated and lineage-depleted (Lin-) cells.

<p>(A) Schematic compositions of mono-nucleated cell samples and Lin- cell samples. (B) Model predicted proportions of 11 homogeneous blood cell lineages, namely granulocytes (GRAN), erythrocytes (ERY), monocytes (MONO), precursor B cells (PREB), megakaryocyte-erythrocyte progenitors (MEP), megakaryocytes (MEGA), primitive progenitor cells (PPC), eosinophils (EOS), granulocyte-monocyte progenitors (GMP), common myeloid progenitors (CMP), and basophils (BASO) in uncultured human mono-nucleated cord blood cell samples. (C) Flow cytometry measured proportions of the 11 blood cell lineages in the uncultured human mono-nucleated cord blood cell samples shown in (B). (D) Model predicted proportions in uncultured human Lin- cord blood cell samples. (E) Flow cytometry measured proportions in the uncultured human Lin- cord blood cell samples shown in (D). (F) R² calculated from the Pearson's correlation coefficients between the model predicted cell proportions and the ones assigned by flow cytometry. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002838#pcbi-1002838-t002" target="_blank">Table 2</a> for the associated t-statistics and P-values. (G) Averaged absolute differences of model predicted cell proportions. Error bars show standard deviations of the absolute differences between model predicted and flow cytometry assigned proportions of the 11 blood cell lineages. (H) The Bayesian information criterion (BIC) calculated from the parameters in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002838#pcbi-1002838-t001" target="_blank">Table 1</a>.</p