Validation of an ICH Q2 Compliant Flow Cytometry-Based Assay for the Assessment of the Inhibitory Potential of Mesenchymal Stromal Cells on T Cell Proliferation.

Mesenchymal stromal cells (MSCs) have the potential to suppress pathological activation of immune cells and have therefore been considered for the treatment of Graft-versus-Host-Disease. The clinical application of MSCs requires a process validation to ensure consistent quality. A flow cytometry-based mixed lymphocyte reaction (MLR) was developed to analyse the inhibitory effect of MSCs on T cell proliferation. Monoclonal antibodies were used to stimulate T cell expansion and determine the effect of MSCs after four days of co-culture based on proliferation tracking with the violet proliferation dye VPD450. Following the guidelines of the International Council for Harmonisation (ICH) Q2 (R1), the performance of n = 30 peripheral blood mononuclear cell (PBMC) donor pairs was assessed. The specific inhibition of T cells by viable MSCs was determined and precision values of <10% variation for repeatability and <15% for intermediate precision were found. Compared to a non-compendial reference method, a linear correlation of r = 0.9021 was shown. Serial dilution experiments demonstrated a linear range for PBMC:MSC ratios from 1:1 to 1:0.01. The assay was unaffected by PBMC inter-donor variability. In conclusion, the presented MLR can be used as part of quality control tests for the validation of MSCs as a clinical product.

A simple, rapid and low-cost qPCR assay for evaluating the severity of exosomal PD-L1-mediated T cell exhaustion in blood samples.

A novel dual-aptamer activated proximity-induced qPCR assay was developed for the quantitative analysis of exosomal PD-L1 on T cell-exosome complexes in blood samples.

Using DNA sequencing data to quantify T cell fraction and therapy response.

The immune microenvironment influences tumour evolution and can be both prognostic and predict response to immunotherapy1,2. However, measurements of tumour infiltrating lymphocytes (TILs) are limited by a shortage of appropriate data. Whole-exome sequencing (WES) of DNA is frequently performed to calculate tumour mutational burden and identify actionable mutations. Here we develop T cell exome TREC tool (T cell ExTRECT), a method for estimation of T cell fraction from WES samples using a signal from T cell receptor excision circle (TREC) loss during V(D)J recombination of the T cell receptor-α gene (TCRA (also known as TRA)). TCRA T cell fraction correlates with orthogonal TIL estimates and is agnostic to sample type. Blood TCRA T cell fraction is higher in females than in males and correlates with both tumour immune infiltrate and presence of bacterial sequencing reads. Tumour TCRA T cell fraction is prognostic in lung adenocarcinoma. Using a meta-analysis of tumours treated with immunotherapy, we show that tumour TCRA T cell fraction predicts immunotherapy response, providing value beyond measuring tumour mutational burden. Applying T cell ExTRECT to a multi-sample pan-cancer cohort reveals a high diversity of the degree of immune infiltration within tumours. Subclonal loss of 12q24.31-32, encompassing SPPL3, is associated with reduced TCRA T cell fraction. T cell ExTRECT provides a cost-effective technique to characterize immune infiltrate alongside somatic changes.

Application of a simple unstructured kinetic and cost of goods models to support T-cell therapy manufacture.

Manufacturing of cell therapy products requires sufficient understanding of the cell culture variables and associated mechanisms for adequate control and risk analysis. The aim of this study was to apply an unstructured ordinary differential equation-based model for prediction of T-cell bioprocess outcomes as a function of process input parameters. A series of models were developed to represent the growth of T-cells as a function of time, culture volumes, cell densities, and glucose concentration using data from the Ambr®15 stirred bioreactor system. The models were sufficiently representative of the process to predict the glucose and volume provision required to maintain cell growth rate and quantitatively defined the relationship between glucose concentration, cell growth rate, and glucose utilization rate. The models demonstrated that although glucose is a limiting factor in batch supplied medium, a delivery rate of glucose at significantly less than the maximal specific consumption rate (0.05 mg 1 × 106  cell h-1 ) will adequately sustain cell growth due to a lower glucose Monod constant determining glucose consumption rate relative to the glucose Monod constant determining cell growth rate. The resultant volume and exchange requirements were used as inputs to an operational BioSolve cost model to suggest a cost-effective T-cell manufacturing process with minimum cost of goods per million cells produced and optimal volumetric productivity in a manufacturing settings. These findings highlight the potential of a simple unstructured model of T-cell growth in a stirred tank system to provide a framework for control and optimization of bioprocesses for manufacture.

Immune Checkpoint Inhibitors-Related Thyroid Dysfunction: Epidemiology, Clinical Presentation, Possible Pathogenesis, and Management.

Immune checkpoint inhibitors (ICIs) are a group of drugs employed in the treatment of various types of malignant tumors and improve the therapeutic effect. ICIs blocks negative co-stimulatory molecules, such as programmed cell death gene-1 (PD-1) and its ligand (PD-L1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), reactivating the recognition and killing effect of the immune system on tumors. However, the reactivation of the immune system can also lead to the death of normal organs, tissues, and cells, eventually leading to immune-related adverse events (IRAEs). IRAEs involve various organs and tissues and also cause thyroid dysfunction. This article reviews the epidemiology, clinical manifestations, possible pathogenesis, and management of ICIs-related thyroid dysfunction.

Cost of decentralized CAR T-cell production in an academic nonprofit setting.

Chimeric antigen receptor (CAR) T-cell therapy is a promising immunotherapy with high acquisition costs, and it has raised concerns about affordability and sustainability in many countries. Furthermore, the current centralized production paradigm for the T cells is less than satisfactory. Therefore, several countries are exploring alternative T-cell production modes. Our study is based on the T-cell production experience in a nonprofit setting in Germany. We first identified the work steps and main activities in the production process. Then we determined the fixed costs and variable costs. Main cost components included personnel and technician salaries, expenditure on equipment, a clean room, as well as production materials. All costs were calculated in 2018 euros and converted into U.S. dollars. For a clean room with one machine for closed and automated manufacturing installed, annual fixed costs summed up to approximately €438 098 ($584 131). The variable cost per production was roughly €34 798 ($46 397). At the maximum capacity of one machine, total cost per product would be close to €60 000 ($78 849). As shown in the scenario analysis, if three machines were to be installed in the clean room, per production cost could be as low as €45 000 (roughly $59905). If a cheaper alternative to lentivirus was used, per production total cost could be further reduced to approximately €33 000 (roughly $44309). Decentralized T-cell production might be a less costly and more efficient alternative to the current centralized production mode that requires a high acquisition cost.

Chimeric Antigen Receptor T Cell Therapy During the COVID-19 Pandemic.

The SARS-CoV-2 coronavirus (COVID-19) pandemic has significantly impacted the delivery of cellular therapeutics, including chimeric antigen receptor (CAR) T cells. This impact has extended beyond patient care to include logistics, administration, and distribution of increasingly limited health care resources. Based on the collective experience of the CAR T-cell Consortium investigators, we review and address several questions and concerns regarding cellular therapy administration in the setting of COVID-19 and make general recommendations to address these issues. Specifically, we address (1) necessary resources for safe administration of cell therapies; (2) determinants of cell therapy utilization; (3) selection among patients with B cell non-Hodgkin lymphomas and B cell acute lymphoblastic leukemia; (4) supportive measures during cell therapy administration; (5) use and prioritization of tocilizumab; and (6) collaborative care with referring physicians. These recommendations were carefully formulated with the understanding that resource allocation is of the utmost importance, and that the decision to proceed with CAR T cell therapy will require extensive discussion of potential risks and benefits. Although these recommendations are fluid, at this time it is our opinion that the COVID-19 pandemic should not serve as reason to defer CAR T cell therapy for patients truly in need of a potentially curative therapy.

Histopathologic assessment of cultured human thymus.

The maintenance and propagation of complex mixtures of cells in vitro in the form of native organs or engineered organoids has contributed to understanding mechanisms of cell and organ development and function which can be translated into therapeutic benefits. For example, allogeneic cultured postnatal human thymus tissue has been shown to support production of naïve recipient T cells when transplanted into patients with complete DiGeorge anomaly and other genetic defects that result in congenital lack of a thymus. Patients receiving such transplants typically exhibit reversal of their immunodeficiency and normalization of their peripheral blood T cell receptor V-beta repertoire, with long-term survival. This study was designed to assess the histopathologic changes that occur in postnatal human thymus slices when cultured according to protocols used for transplanted tissues. Results showed that as thymic organ cultures progressed from days 0 through 21, slices developed increasing amounts of necrosis, increasing condensation of thymic epithelium, and decreasing numbers of residual T cells. The architecture of the thymic epithelial network remained generally well-preserved throughout the 21 days of culture, with focal expression of cytokeratin 14, a putative biomarker of thymic epithelial cells with long-term organ-repopulating potential. All organ slices derived from the same donor thymus closely resembled one another, with minor differences in size, shape, and relative content of cortex versus medulla. Similarly, slices derived from different donors showed similar histopathologic characteristics when examined at the same culture time point. Taken together, these results demonstrate that diagnostic criteria based on structural features of the tissue identifiable via hematoxylin and eosin staining and cytokeratin immunohistochemistry can be used to evaluate the quality of slices transplanted into patients with congenital athymia.

CART manufacturing process and reasons for academy-pharma collaboration.

The success of genetically engineered T-cells modified with a chimeric antigen receptor as an adoptive cell immunotherapy and the subsequent last regulatory approvals of products based on this therapy are leading to a crescent number of both academic and pharmaceutical industry clinical trials testing new approaches of this "living drugs". The aim of this review is to outline the latest developments and regulatory considerations in this field, with a particular emphasis to differences and similarities between academic and industry approaches and the role they should play to coexist and move forward together. To do that, the main considerations for the manufacturing process are firstly discussed, from the chimeric antigen receptor design to final production steps, passing through ex vivo T-cell handling, gene delivery methods, patient´s final product infusion observations or possible associated side effects of this treatment.

Costimulation Blockade in Transplantation.

T cells play a pivotal role in orchestrating immune responses directed against a foreign (allogeneic) graft. For T cells to become fully activated, the T-cell receptor (TCR) must interact with the major histocompatibility complex (MHC) plus peptide complex on antigen-presenting cells (APCs), followed by a second "positive" costimulatory signal. In the absence of this second signal, T cells become anergic or undergo deletion. By blocking positive costimulatory signaling, T-cell allo-responses can be aborted, thus preventing graft rejection and promoting long-term allograft survival and possibly tolerance (Alegre ML, Najafian N, Curr Mol Med 6:843-857, 2006; Li XC, Rothstein DM, Sayegh MH, Immunol Rev 229:271-293, 2009). In addition, costimulatory molecules can provide negative "coinhibitory" signals that inhibit T-cell activation and terminate immune responses; strategies to promote these pathways can also lead to graft tolerance (Boenisch O, Sayegh MH, Najafian N, Curr Opin Organ Transplant 13:373-378, 2008). However, T-cell costimulation involves an incredibly complex array of interactions that may act simultaneously or at different times in the immune response and whose relative importance varies depending on the different T-cell subsets and activation status. In transplantation, the presence of foreign alloantigen incites not only destructive T effector cells but also protective regulatory T cells, the balance of which ultimately determines the fate of the allograft (Lechler RI, Garden OA, Turka LA, Nat Rev Immunol 3:147-158, 2003). Since the processes of alloantigen-specific rejection and regulation both require activation of T cells, costimulatory interactions may have opposing or synergistic roles depending on the cell being targeted. Such complexities present both challenges and opportunities in targeting T-cell costimulatory pathways for therapeutic purposes. In this chapter, we summarize our current knowledge of the various costimulatory pathways in transplantation and review the current state and challenges of harnessing these pathways to promote graft tolerance (summarized in Table 10.1).

Regular monitoring of cytomegalovirus-specific cell-mediated immunity in intermediate-risk kidney transplant recipients: predictive value of the immediate post-transplant assessment.

OBJECTIVE: Previous studies on monitoring of post-transplant cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) are limited by single-centre designs and disparate risk categories. We aimed to assess the clinical value of a regular monitoring strategy in a large multicentre cohort of intermediate-risk kidney transplant (KT) recipients. METHODS: We recruited 124 CMV-seropositive KT recipients with no T-cell-depleting induction pre-emptively managed at four Spanish institutions. CMV-specific interferon-γ-producing CD4+ and CD8+ T cells were counted through the first post-transplant year by intracellular cytokine staining after stimulation with pp65 and immediate early-1 peptides (mean of six measurements per patient). The primary outcome was the occurrence of any CMV event (asymptomatic infection and/or disease). Optimal cut-off values for CMV-specific T cells were calculated at baseline and day 15. RESULTS: Twelve-month cumulative incidence of CMV infection and/or disease was 47.6%. Patients with pre-transplant CMV-specific CD8+ T-cell count <1.0 cells/µL had greater risk of CMV events (adjusted hazard ratio (aHR) 2.84; p 0.054). When the CMI assessment was performed in the immediate post-transplant period (day 15), the presence of <2.0 CD8+ T cells/µL (aHR 2.18; p 0.034) or <1.0 CD4+ T cells/µL (aHR 2.43; p 0.016) also predicted the subsequent development of a CMV event. In addition, lower counts of CMV-specific CD4+ (but not CD8+) T cells at days 60 and 180 were associated with a higher incidence of late-onset events. CONCLUSIONS: Monitoring for CMV-specific CMI in intermediate-risk KT recipients must be regular to reflect dynamic changes in overall immunosuppression and individual susceptibility. The early assessment at post-transplant day 15 remains particularly informative.

Autoreactive, Low-Affinity T Cells Preferentially Drive Differentiation of Short-Lived Memory B Cells at the Expense of Germinal Center Maintenance.

B cell fate decisions within a germinal center (GC) are critical to determining the outcome of the immune response to a given antigen. Here, we characterize GC kinetics and B cell fate choices in a response to the autoantigen myelin oligodendrocyte glycoprotein (MOG) and compare the response with a standard model foreign antigen. Both antigens generate productive primary responses, as evidenced by GC development, circulating antigen-specific antibodies, and differentiation of memory B cells. However, in the MOG response, the status of the cognate T cell partner drives preferential B cell differentiation to a memory phenotype at the expense of GC maintenance, resulting in a truncated GC. Reduced plasma cell differentiation is largely independent of T cell influence. Interestingly, memory-phenotype B cells formed in the MOG GC are not long lived, resulting in a failure of the B cell response to secondary challenge.

Flow cytometric assessment of leukocyte kinetics for the monitoring of tissue damage.

Leukocyte populations quickly respond to tissue damage, but most leukocyte kinetic studies are not based on multiparameter flow cytometry. We systematically investigated several blood leukocyte populations after controlled tissue damage. 48 patients were assigned to either an anterior or posterolateral total hip arthroplasty. Peripheral blood was collected pre-operatively and at 2 h, 24 h, 48 h, 2 and 6 weeks postoperatively and assessed by flow cytometry for absolute counts of multiple leukocyte populations using standardized EuroFlow protocols. Absolute counts of leukocyte subsets differed significantly between consecutive time points. Neutrophils increased instantly after surgery, while most leukocyte subsets initially decreased, followed by increasing cell counts until 48 h. At two weeks all leukocyte counts were restored to pre-operative counts. Immune cell kinetics upon acute tissue damage exhibit reproducible patterns, which differ between the leukocyte subsets and with "opposite kinetics" among monocyte subsets. Flow cytometric leukocyte monitoring can be used to minimally invasively monitor tissue damage.

InterCells: A Generic Monte-Carlo Simulation of Intercellular Interfaces Captures Nanoscale Patterning at the Immune Synapse.

Molecular interactions across intercellular interfaces serve to convey information between cells and to trigger appropriate cell functions. Examples include cell development and growth in tissues, neuronal and immune synapses (ISs). Here, we introduce an agent-based Monte-Carlo simulation of user-defined cellular interfaces. The simulation allows for membrane molecules, embedded at intercellular contacts, to diffuse and interact, while capturing the topography and energetics of the plasma membranes of the interface. We provide a detailed example related to pattern formation in the early IS. Using simulation predictions and three-color single molecule localization microscopy (SMLM), we detected the intricate mutual patterning of T cell antigen receptors (TCRs), integrins and glycoproteins in early T cell contacts with stimulating coverslips. The simulation further captures the dynamics of the patterning under the experimental conditions and at the IS with antigen presenting cells (APCs). Thus, we provide a generic tool for simulating realistic cell-cell interfaces, which can be used for critical hypothesis testing and experimental design in an iterative manner.

The Functional Assessment of T cells.

It is important to know what kind of T cell populations is involved in various disease states and to know the state of T cell functions involved in the disease. When T cell antigen receptors (TCR) recognize a specific antigen, the cell transmits a signal by a transduction mechanism within the T cell's cytoplasm. This signal initiates gene transcription essential for differentiation and activation of T cells. In this chapter, we will describe the methods of analyzing the transcribed mRNA and detecting the translated product in order to know the activation state of T cells.

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue.

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

Nonclinical Safety Assessment of the γ-Secretase Inhibitor Avagacestat.

The toxicity of avagacestat, a sulfonamide-based gamma (γ)-secretase inhibitor that was in development as a treatment for Alzheimer's disease, was evaluated in a comprehensive nonclinical toxicology program that included 6-month and 1-year repeat-dose toxicity studies in rats and dogs, respectively. There was a spectrum of mechanism-based changes attributed to inhibition of Notch signaling that regulates the differentiation and proliferation of cells throughout development and in adult tissues. In both rats and dogs, ovarian follicular degeneration and atrophy and a low incidence of granulosa cell hyperplasia and benign granulosa-thecal cell tumors were observed. Gastrointestinal (GI) findings, including goblet cell metaplasia, dilatation of intestinal crypts/glands, mucosal epithelial necrosis and regeneration, and villous atrophy, were limited to dogs that had clinical evidence of GI toxicity. Other avagacestat-related findings attributed to interference with Notch signaling included decreases in peripheral lymphocytes (T and/or B cells) and lymphoid depletion in lymph nodes and the spleen in both species, as well as epiphyseal cartilage and trabecular bone changes in rats. Pharmacologically mediated decreases in brain and cerebrospinal fluid levels of ß-amyloid (Aß) peptides Aß40 and Aß42 and decreased expression of white blood cell mRNA levels of the Notch-regulated gene hairy and enhancer of split-1 confirmed target engagement at all doses. Reductions in brain Aß peptide levels (22 to 34%) in dogs after 1 year at exposures up to the no-observed-effect level for GI toxicity of 1.1× the human plasma exposure, and reversible GI changes at a 3.2× multiple, indicated that a sustained pharmacodynamic effect was attained at exposures without dose-limiting toxicity.

Assessment of interactions of efavirenz solid drug nanoparticles with human immunological and haematological systems.

BACKGROUND: Recent work has developed solid drug nanoparticles (SDNs) of efavirenz that have been demonstrated, preclinically, improved oral bioavailability and the potential to enable up to a 50% dose reduction, and is currently being studied in a healthy volunteer clinical trial. Other SDN formulations are being studied for parenteral administration, either as intramuscular long-acting formulations, or for direct administration intravenously. The interaction of nanoparticles with the immunological and haematological systems can be a major barrier to successful translation but has been understudied for SDN formulations. Here we have conducted a preclinical evaluation of efavirenz SDN to assess their potential interaction with these systems. Platelet aggregation and activation, plasma coagulation, haemolysis, complement activation, T cell functionality and phenotype, monocyte derived macrophage functionality, and NK cell function were assessed in primary healthy volunteer samples treated with either aqueous efavirenz or efavirenz SDN. RESULTS: Efavirenz SDNs were shown not to interfere with any of the systems studied in terms of immunostimulation nor immunosuppression. Although efavirenz aqueous solution was shown to cause significant haemolysis ex vivo, efavirenz SDNs did not. No other interaction with haematological systems was observed. Efavirenz SDNs have been demonstrated to be immunologically and haematologically inert in the utilised assays. CONCLUSIONS: Taken collectively, along with the recent observation that lopinavir SDN formulations did not impact immunological responses, these data indicate that this type of nanoformulation does not elicit immunological consequences seen with other types of nanomaterial. The methodologies presented here provide a framework for pre-emptive preclinical characterisation of nanoparticle safety.

Regulatory perspective on in vitro potency assays for human T cells used in anti-tumor immunotherapy.

The adaptive immune system is known to play an important role in anti-neoplastic responses via induction of several effector pathways, resulting in tumor cell death. Because of their ability to specifically recognize and kill tumor cells, the potential use of autologous tumor-derived and genetically engineered T cells as adoptive immunotherapy for cancer is currently being explored. Because of the variety of potential T cell-based medicinal products at the level of starting material and manufacturing process, product-specific functionality assays are needed to ensure quality for individual products. In this review, we provide an overview of in vitro potency assays suggested for characterization and release of different T cell-based anti-tumor products. We discuss functional assays, as presented in scientific advices and literature, highlighting specific advantages and limitations of the various assays. Because the anticipated in vivo mechanism of action for anti-tumor T cells involves tumor recognition and cell death, in vitro potency assays based on the cytotoxic potential of antigen-specific T cells are most evident. However, assays based on other T cell properties may be appropriate as surrogates for cytotoxicity. For all proposed assays, biological relevance of the tests and correlation of the read-outs with in vivo functionality need to be substantiated with sufficient product-specific (non-)clinical data. Moreover, further unraveling the complex interaction of immune cells with and within the tumor environment is expected to lead to further improvement of the T cell-based products. Consequently, increased knowledge will allow further optimized guidance for potency assay development.

Challenges and opportunities in the manufacture and expansion of cells for therapy.

INTRODUCTION: Laboratory-based ex vivo cell culture methods are largely manual in their manufacturing processes. This makes it extremely difficult to meet regulatory requirements for process validation, quality control and reproducibility. Cell culture concepts with a translational focus need to embrace a more automated approach where cell yields are able to meet the quantitative production demands, the correct cell lineage and phenotype is readily confirmed and reagent usage has been optimized. Areas covered: This article discusses the obstacles inherent in classical laboratory-based methods, their concomitant impact on cost-of-goods and that a technology step change is required to facilitate translation from bed-to-bedside. Expert opinion: While traditional bioreactors have demonstrated limited success where adherent cells are used in combination with microcarriers, further process optimization will be required to find solutions for commercial-scale therapies. New cell culture technologies based on 3D-printed cell culture lattices with favourable surface to volume ratios have the potential to change the paradigm in industry. An integrated Quality-by-Design /System engineering approach will be essential to facilitate the scaled-up translation from proof-of-principle to clinical validation.