Digital path to Industry 4.0: The role of data sciences in the cell & gene therapy space

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Novel Assays For Immunotherapy Product Characterisation And Potency Measurement

The use of adoptive T-cell therapy for the treatment of haematological cancers and solid tumours is one of the fastest growing areas in the cell and gene therapy field, with oncology targets accounting for approximately 40% of all cell therapy clinical trials currently being performed. A significant number of these immunotherapies use genetic modifications of T-cells using viral vectors to engineer their specificity or enhance their function. Examples of these products include gene modified T-cells expressing Chimeric Antigen Receptors (CAR) which direct specificity against cancer cell surface markers, or engineered T-cell receptors (TCR) which can target intracellular proteins through the presentation of their fragments on the cell surface by HLA molecules. Various strategies are applied for the manufacture of gene modified immunotherapies but the use of patient cells as a starting material and the use of viruses to deliver the CAR or TCR construct can lead to variability in terms of transduction efficiency, CAR/TCR expression and product potency. Characterisation is therefore critical both during manufacture to maintain consistency, and post manufacture to ensure sufficient function. Strategies for the characterisation of gene modified immunotherapies are continually evolving. However, a number of the methods commonly used for measurement of viral transduction and potency are complex, semi-quantitative, require complex pre-labelling of cells or are based on the detection of surrogate markers. In this paper we demonstrate two novel approaches for the characterisation of a gene modified TCR immunotherapy product targeting the Wilms-tumour 1 (WT1) protein. WT1 expression has been demonstrated to be elevated in haematological malignancies such as acute myeloid leukaemia (AML) chronic myeloid leukaemia (CML) and myelodysplastic syndrome (MDS). The first approach uses single cell analysis to directly measure viral copy number integration into the genome and the expression of the WT1-TCR mRNA following transduction. This assay offers advantages over currently used techniques. From a safety perspective it provides high level characterisation of viral integration which can be used to optimise the manufacture process to control the number of integration events within the genome. It also offers a method to optimise the amount of virus used during manufacture which could have a significant positive impact on the cost of goods for product manufacture. Single cell mRNA analysis offers a direct functional measurement of TCR expression following viral transduction which overcomes the limitations of available antibodies specific for the antigen recognised by the TCR. The second approach demonstrated in this paper is for a novel potency assay which uses impedance spectroscopy to give a label free, real time measurement of cell killing by the WT1-TCR gene modified T-cells. This has many advantages over commonly used alternative methods such as the chromium-51 killing assay or surrogate assays looking at the stimulation of cytokine release. Firstly it is label free and does not require pre-loading of the target cells with a radioactive isotopes or other detection labels which can interfere with the assay readout. Secondly it can be performed with established cell lines which can act as antigen presenting cells reducing the assay variability associated with the use of primary cells. Thirdly, the impedance assay provides real time data showing the kinetics of the killing response rather than just a single end-point measurement. These new assays are a valuable addition to the repertoire of techniques which can be applied to characterise immunotherapy products and while this paper demonstrates their use with a gene modified TCR products they are equally as applicable for CAR T-cell therapies and for measuring lentiviral based immunotherapy products

Glycaemic benefit of iGlarLixi in insulin-naive type 2 diabetes patients with high HbA1c or those with inadequate glycaemic control on two oral antihyperglycaemic drugs in the LixiLan-O randomized trial

In this post hoc analysis of the randomized controlled LixiLan‐O trial in insulin‐naive type 2 diabetes mellitus (T2DM) patients not controlled on metformin with or without a second oral antihyperglycaemic drug (OAD), the efficacy and safety of the fixed‐ratio combination, iGlarLixi (insulin glargine 100 U [iGlar] and lixisenatide [Lixi]), compared to its individual components was assessed in two patient subgroups: (1) a baseline HbA1c ≥9% (n = 134); (2) inadequate control (HbA1c ≥7.0% and ≤9.0%) despite administration of two OADs at screening (n = 725). Treatment with iGlarLixi resulted in a significantly greater reduction in least squares mean HbA1c compared with iGlar or Lixi alone in both subgroups (HbA1c ≥9% group: 2.9%, 2.5%, 1.7%; two OADs group: 1.5%, 1.2%, 0.7%, respectively). Target HbA1c 70% of patients on iGlarLixi in both subgroups, while mitigating the weight gain observed with iGlar alone. Rates of hypoglycaemic events were low overall. These results suggest that iGlarLixi achieves superior glycaemic control compared with iGlar or Lixi alone in T2DM patients with HbA1c ≥9% or those inadequately controlled on two OADs

Development of a cost efficient platform for the industrial manufacturing of pluripotent stem cell derived products for cell therapy: Cell expansion is the starting point

The development of stem cell-derived allogeneic therapeutics requires manufacturing processes able to generate high-density cultures of pluripotent stem cells (PSCs) to be further differentiated to target somatic cells. The Cell Plasticity platform of The Cell and Gene Therapy Catapult (CGT) is a core program that focuses on the cost efficient development of bioprocesses for the industrial manufacture of PSC-derived products in 2D and 3D culture systems. We started this program by establishing banks of PSCs adapted to defined culture systems and used conventional analytical techniques to characterise the cells to industry standards. Defined media were evaluated for the expansion of induced pluripotent stem cells (iPSC) in adherent culture. Scale-down high-throughput tools along with Design of Experiment methodology have been employed to establish a baseline process for the expansion of PSC as cellular aggregates in stirred-suspension culture and targeting cell yield \u3e 5x106 viable cells/mL. We are currently investigating bioengineering parameters for scale-up and evaluating cell retention devices for the dissociation of PSC aggregates in a closed and automated fashion. In parallel, a framework of analytical assays comprising imaging, flow-cytometry and gene expression is under development for process monitor and control using a proprietary multi-parametric analysis approach

Albuminuria is associated with too few glomeruli and too much testosterone

Normally, the glomerular filtration barrier almost completely excludes circulating albumin from entering the urine. Genetic variation and both pre- and postnatal environmental factors may affect albuminuria in humans. Here we determine whether glomerular gene expression in mouse strains with naturally occurring variations in albuminuria would allow identification of proteins deregulated in relatively 'leaky' glomeruli. Albuminuria increased in female B6 to male B6 to female FVB/N to male FVB/N mice, whereas the number of glomeruli/kidney was the exact opposite. Testosterone administration led to increased albuminuria in female B6 but not female FVB/N mice. A common set of 39 genes, many expressed in podocytes, were significantly differentially expressed in each of the four comparisons: male versus female B6 mice, male versus female FVB/N mice, male FVB/N versus male B6 mice, and female FVB/N versus female B6 mice. The transcripts encoded proteins involved in oxidation/reduction reactions, ion transport, and enzymes involved in detoxification. These proteins may represent novel biomarkers and even therapeutic targets for early kidney and cardiovascular disease

Complementary actions of dopamine D2 receptor 1 agonist and anti-Vegf therapy on tumoral vessel normalization in a transgenic mouse model

International audienceAngiogenesis contributes in multiple ways to disease progression in tumors and reduces treatment efficiency. Molecular therapies targeting Vegf signaling combined with chemotherapy or other drugs exhibit promising results to improve efficacy of treatment. Dopamine has been recently proposed to be a novel safe anti-angiogenic drug that stabilizes abnormal blood vessels and increases therapeutic efficacy. Here, we aimed to identify a treatment to normalize tumoral vessels and restore normal blood perfusion in tumor tissue with a Vegf receptor inhibitor and/or a ligand of dopamine G protein-coupled receptor D2 (D2R). Dopamine, via its action on D2R, is an endogenous effector of the pituitary gland, and we took advantage of this system to address this question. We have used a previously described Hmga2/T mouse model developing haemorrhagic prolactin-secreting adenomas. In mutant mice, blood vessels are profoundly altered in tumors, and an aberrant arterial vascularization develops leading to the loss of dopamine supply. D2R agonist treatment blocks tumor growth, induces regression of the aberrant blood supply and normalizes blood vessels. A chronic treatment is able to restore the altered balance between pro- and anti-angiogenic factors. Remarkably, an acute treatment induces an upregulation of the stabilizing factor Angiopoietin 1. An anti-Vegf therapy is also effective to restrain tumor growth and improves vascular remodeling. Importantly, only the combination treatment suppresses intratumoral hemorrhage and restores blood vessel perfusion, suggesting that it might represent an attractive therapy targeting tumor vasculature. Similar strategies targeting other ligands of GPCRs involved in angiogenesis may identify novel therapeutic opportunities for cancer

Loss of endogenous thymosin β4 accelerates glomerular disease

Glomerular disease is characterized by morphologic changes in podocyte cells accompanied by inflammation and fibrosis. Thymosin $\beta_4$ regulates cell morphology, inflammation, and fibrosis in several organs and administration of exogenous thymosin $\beta_4$ improves animal models of unilateral ureteral obstruction and diabetic nephropathy. However, the role of endogenous thymosin $\beta_4$ in the kidney is unknown. We demonstrate that thymosin β4 is expressed prominently in podocytes of developing and adult mouse glomeruli. Global loss of thymosin $\beta_4$ did not affect healthy glomeruli, but accelerated the severity of immune-mediated nephrotoxic nephritis with worse renal function, periglomerular inflammation, and fibrosis. Lack of thymosin $\beta_4$ in nephrotoxic nephritis led to the redistribution of podocytes from the glomerular tuft toward the Bowman capsule suggesting a role for thymosin $\beta_4$ in the migration of these cells. Thymosin $\beta_4$ knockdown in cultured podocytes also increased migration in a wound-healing assay, accompanied by F-actin rearrangement and increased RhoA activity. We propose that endogenous thymosin $\beta_4$ is a modifier of glomerular injury, likely having a protective role acting as a brake to slow disease progression

Au service de l'archéologie gallo-romaine. Les photographies aériennes de l'aviation interalliée déposées à l'Ecole française de Rome

Baradez Jean. Au service de l'archéologie gallo-romaine. Les photographies aériennes de l'aviation interalliée déposées à l'Ecole française de Rome. In: Mélanges d'archéologie et d'histoire, tome 78, n°1, 1966. pp. 267-272

Le port marchand de Carthage

Baradez Jean. Le port marchand de Carthage. In: Comptes rendus des séances de l'Académie des Inscriptions et Belles-Lettres, 99ᵉ année, N. 3, 1955. pp. 299-300

Deux missions de recherche sur le limes de Tingitane

Baradez Jean. Deux missions de recherche sur le limes de Tingitane. In: Comptes rendus des séances de l'Académie des Inscriptions et Belles-Lettres, 99ᵉ année, N. 2, 1955. pp. 288-298