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

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

Image processing analysis of stem cell antigens

This thesis aims to investigate the automation of an image processing driven analysis of antigen distributions in the membrane of early human Haematopoietic StemIProgenitor Cells (HSPCs ) imaged by Laser Scanning Confocal Microscopy (LSCM). LSCM experiments generated a vast amount of images of both single and dual labelled HSPCs. Special focus was given to the analysis of colocalised antigen distributions, as colocalisation may involve functional relationships. However, quantitative methods are also investigated to characterise both single and dual labelled antigen distributions. Firstly, novel segmentation algorithms are developed and assessed for their performances in automatically achieving fast fluorescence signal identification. Special attention is given to global histogram-based thresholding methods due to their potential use in real time applications. A new approach to fluorescence quantification is proposed and tested. Secondly, visualisation techniques are developed in order to further assist the analysis of the antigen distributions in cell membranes. They include 3D reconstruction of the fluorescence, newly proposed 2D Antigen Density Maps (ADMs) and new 3D graphs of the spatial distributions (sphere models). Thirdly, original methods to quantitatively characterise the fluorescence distributions are developed. They are applied to both single and dual/colocalised distributions. For the latest, specific approaches are investigated and applied to colocalised CD34/CD164 distributions and to colocalised CD34[sup]class I CD34[sup]class II and CD34[sup]c1ass I CD34[sup]class III epitopes distributions (two combinations of the three known different isoforms of the CD34 molecule, a major clinical marker for HSPCs). The visualisation tools revealed that HSPC membrane antigens are often clustered within membrane domains. Three main types of clusters were identified: small clusters, large patch-like clusters and newly identified meridian-shaped crest-like (MSCL) clusters. Quantitative analysis of antigen distributions showed heterogeneous distributions of the various measured features (such as polarity or colocalisation patterns) within the HSPC populations analysed. Finally, the proposed methodology to characterise membrane antigen distributions is discussed, and its potential application to other biomedical studies is commented. The potential extensions of the innovative linear diffusion-based MultiScale Analysis (MSA) algorithm to other applications are outlined. Visual and quantitative analyses of antigen membrane distributions are eventually used to generate hypotheses on the potential, yet unknown roles of these early antigens and are discussed in the context of haematopoietic theories

Mapping the distribution of HDF cells on the surface of microcarrier beads.

<p><i>(A)</i> Maximum intensity projection from confocal image Z-stack with microcarriers identified by Hough transform (circles). <i>(B)</i> Extraction of sub-volume from Z-stack containing 3D fluorescence associated with a single microcarrier. <i>(C)</i> Top and side projection images calculated from sub-volume used to locate X-Y-Z coordinates of microcarrier (dashed circles and arrows). <i>(D)</i> Iterative fluorescence intensity measurements in the vicinity of the microcarrier surface (sphere) using 30 sampling spherical grids (horizontal planes in magnified sampling volume, extended to the whole microcarrier surface). <i>(E)</i> Cell distribution map (bottom) computed from unwrapped stack of sampling grids (top, M<i>_x,y,z</i>).</p

Measurement techniques used to monitor the quality of adherent cells in bioreactor culture.

<p>Measurement techniques used to monitor the quality of adherent cells in bioreactor culture.</p

Validation of distribution mapping process with <i>in silico</i> modelling of cells adhered to microcarrier beads.

<p><i>(A)</i> Flow diagram for <i>in silico</i> modelling (top compartment) and validation of confluence measurement (bottom compartment). <i>(B) 1</i>- Maximum intensity top projection confocal images of real HDF cells adhered to microcarrier beads. <i>2</i> – Synthetic maximum intensity top projections of cells distributions around microcarrier beads generated by <i>in silico</i> modelling. <i>3</i> – 3D rendering of the synthetic cell distribution in B2 to show cell localisation and comparability to real image data.</p

Comparison of cell number measurments.

<p>Graph to the show the linear relationship between cell number measurements obtained using the cell distribution mapping process and the commercial Cyquant assay.</p