A quality-by-design approach to improve process understanding and optimise the production and quality of CAR-T cells in automated stirred-tank bioreactors.

Ex vivo genetically-modified cellular immunotherapies, such as chimeric antigen receptor T cell (CAR-T) therapies, have generated significant clinical and commercial outcomes due to their unparalleled response rates against relapsed and refractory blood cancers. However, the development and scalable manufacture of these novel therapies remains challenging and further process understanding and optimisation is required to improve product quality and yield. In this study, we employ a quality-by-design (QbD) approach to systematically investigate the impact of critical process parameters (CPPs) during the expansion step on the critical quality attributes (CQAs) of CAR-T cells. Utilising the design of experiments (DOE) methodology, we investigated the impact of multiple CPPs, such as number of activations, culture seeding density, seed train time, and IL-2 concentration, on CAR-T CQAs including, cell yield, viability, metabolism, immunophenotype, T cell differentiation, exhaustion and CAR expression. Initial studies undertaken in G-Rex® 24 multi-well plates demonstrated that the combination of a single activation step and a shorter, 3-day, seed train resulted in significant CAR-T yield and quality improvements, specifically a 3-fold increase in cell yield, a 30% reduction in exhaustion marker expression and more efficient metabolism when compared to a process involving 2 activation steps and a 7-day seed train. Similar findings were observed when the CPPs identified in the G-Rex® multi-well plates studies were translated to a larger-scale automated, controlled stirred-tank bioreactor (Ambr® 250 High Throughput) process. The single activation step and reduced seed train time resulted in a similar, significant improvement in CAR-T CQAs including cell yield, quality and metabolism in the Ambr® 250 High Throughput bioreactor, thereby validating the findings of the small-scale studies and resulting in significant process understanding and improvements. This study provides a methodology for the systematic investigation of CAR-T CPPs and the findings demonstrate the scope and impact of enhanced process understanding for improved CAR-T production.

LIVECell-A large-scale dataset for label-free live cell segmentation.

Light microscopy combined with well-established protocols of two-dimensional cell culture facilitates high-throughput quantitative imaging to study biological phenomena. Accurate segmentation of individual cells in images enables exploration of complex biological questions, but can require sophisticated imaging processing pipelines in cases of low contrast and high object density. Deep learning-based methods are considered state-of-the-art for image segmentation but typically require vast amounts of annotated data, for which there is no suitable resource available in the field of label-free cellular imaging. Here, we present LIVECell, a large, high-quality, manually annotated and expert-validated dataset of phase-contrast images, consisting of over 1.6 million cells from a diverse set of cell morphologies and culture densities. To further demonstrate its use, we train convolutional neural network-based models using LIVECell and evaluate model segmentation accuracy with a proposed a suite of benchmarks.

The BlueScreen-384 assay as an indicator of genotoxic hazard potential in early-stage drug discovery.

High-throughput cell-based techniques that permit early detection of compound-induced genotoxic damage have recently become available. Methods based on induction of the GADD45a promoter are attractive because multiple intracellular mechanisms that detect genetic damage intersect at this checkpoint gene. Consequently, assays such as GreenScreen HC, which uses p53-competant human TK6 lymphoblastoid cells and a GADD45a-GFP reporter, have been developed. GreenScreen HC allows weekly testing of dozens of compounds using 96-well microplates, with high interassay consistency. BlueScreen HC is a recent advancement, coupling GADD45a to Gaussia luciferase, with several advantages over GADD45a-GFP including the potential for miniaturization. Here we describe implementation of a 384-well BlueScreen assay. For drug discovery programs carrying out iterative analogue synthesis around a chemical lead series, these assays permit assessment of compound genotoxic potential in parallel to, rather than subsequent to, determination of activity at a therapeutic target. We demonstrate comparability of BlueScreen-384 to GreenScreen HC and illustrate the use of BlueScreen-384 to explore the structure-activity relationship around a genotoxic lead molecule to identify nongenotoxic analogues. BlueScreen-384 can reduce the need for costly and time-consuming analogue testing in more traditional genotoxicity tests, such as the Ames test.

In vivo activity of an azole series of CCR2 antagonists.

Optimisation of a series of biaryl sulphonamides resulted in the identification of compound 14 [corrected] which demonstrated dose-dependent and strain-specific inhibition of monocyte recruitment in a thioglycollate-induced peritonitis model of inflammation. [Formula: see text]. [corrected].

The discovery of long-acting saligenin ß2 adrenergic receptor agonists incorporating hydantoin or uracil rings.

A series of novel, potent and selective human ß(2) adrenoceptor agonists incorporating a hydantoin or a uracil ring on the right-hand side phenyl ring of (R)-salmeterol is presented. Hydantoin 12a had long duration of action in vitro on guinea pig trachea, and 12h in guinea pigs in vivo at its EC(90) 25 µM. It had lower oral absorption than salmeterol in rats, and lower bioavailability than salmeterol in vivo in both rats and dogs (2% and 5%, respectively). An improved method for measuring the absorbed fraction of analogues dosed to rats, which considers the glucuronidated fraction is presented. Compound 12a was metabolised in human liver microsomes and hepatocytes to the active hydantoic acid 12m.

Synthesis and structure-activity relationships of long-acting beta2 adrenergic receptor agonists incorporating metabolic inactivation: an antedrug approach.

A series of saligenin beta(2) adrenoceptor agonist antedrugs having high clearance were prepared by reacting a protected saligenin oxazolidinone with protected hydroxyethoxyalkoxyalkyl bromides, followed by removal of the hydroxy-protecting group, alkylation, and final deprotection. The compounds were screened for beta(2), beta(1), and beta(3) agonist activity in CHO cells. The onset and duration of action in vitro of selected compounds were assessed on isolated superfused guinea pig trachea. Compound 13f had high potency, selectivity, fast onset, and long duration of action in vitro and was found to have long duration in vivo, low oral bioavailability in the rat, and to be rapidly metabolized. Crystalline salts of 13f (vilanterol) were identified that had suitable properties for inhaled administration. A proposed binding mode for 13f to the beta(2)-receptor is presented.

Synthesis and structure-activity relationships of long-acting beta2 adrenergic receptor agonists incorporating arylsulfonamide groups.

A series of saligenin alkoxyalkylphenylsulfonamide beta(2) adrenoceptor agonists were prepared by reacting a protected saligenin oxazolidinone with alkynyloxyalkyl bromides, followed by Sonogashira reaction, hydrogenation, and deprotection. The meta-substituted primary sulfonamide was more potent than the para- and the ortho-analogues. Primary sulfonamides were more potent than the secondary and tertiary analogues. The onset and duration of action in vitro of selected compounds was assessed on isolated superfused guinea pig trachea. Sulfonamide 29b had the best profile of potency, selectivity, onset, and duration of action on both guinea pig trachea and human bronchus. Furthermore, 29b was found to have low oral bioavailability in rat and dog and also to have long duration of action in an in vivo model of bronchodilation. Crystalline salts of 29b were identified that had suitable properties for inhaled administration. A proposed binding mode for 29b to the beta(2)-receptor is presented.

Pharmacological characterisation and inhibitory effects of (2R,3R,4S,5R)-2-(6-amino-2-{[(1S)-2-hydroxy-1-(phenylmethyl)ethyl]amino}-9H-purin-9-yl)-5-(2-ethyl-2H-tetrazol-5-yl)tetrahydro-3,4-furandiol, a novel ligand that demonstrates both adenosine A(2A) receptor agonist and adenosine A(3) receptor antagonist activity.

The pharmacological properties of the novel ligand, (2R,3R,4S,5R)-2-(6-amino-2-{[(1S)-2-hydroxy-1-(phenylmethyl)ethyl]amino}-9H-purin-9-yl)-5-(2-ethyl-2H-tetrazol-5-yl)tetrahydro-3,4-furandiol (I), at the human adenosine receptors were investigated using Chinese hamster ovary cell lines recombinantly expressing these receptors. Functional studies were performed using a cyclic AMP-coupled reporter gene system. Binding studies were performed using membranes from these cells. The effects of ligand (I) were also determined on functional responses of human neutrophils and eosinophils. Ligand (I) had a high affinity for the adenosine A(2A) receptor (pKi 7.8+/-0.2) and was a potent agonist at this receptor (pEC(50) 9.0+/-0.2). Ligand (I) had a similar affinity for the adenosine A(3) receptor (pKi 7.8+/-0.1) but displayed no agonist activity, acting instead as a competitive antagonist (pA(2) 8.3+/-0.04). Ligand (I) had lower affinity for adenosine A(1) and A(2B) receptors (pKi

Subcellular distribution of GABA(B) receptor homo- and hetero-dimers.

GBRs (GABA(B) receptors; where GABA stands for gamma-aminobutyric acid) are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. In vitro assays have previously demonstrated that these receptors are heterodimers assembled from two homologous subunits, GBR1 and GBR2, neither of which is capable of producing functional GBR on their own. We have used co-immunoprecipitation in combination with bioluminescence and fluorescence resonance energy transfer approaches in living cells to assess directly the interaction between GBR subunits and determine their subcellular localization. The results show that, in addition to forming heterodimers, GBR1 and GBR2 can associate as stable homodimers. Confocal microscopy indicates that, while GBR1/GBR1 homodimers are retained in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartment, both GBR2/GBR2 homodimers and GBR1/GBR2 heterodimers are present at the plasma membrane. Although these observations shed new light on the assembly of GBR complexes, they raise questions about the potential functional roles of GBR1 and GBR2 homodimers.