CAR-T Cell Therapy: Mechanism, Management, and Mitigation of Inflammatory Toxicities

Engineered T cell therapies such as chimeric antigen receptor (CAR) expressing T cells (CAR-T cells) have great potential to treat many human diseases; however, inflammatory toxicities associated with these therapies present safety risks and can greatly limit its widespread use. This article briefly reviews our current understanding of mechanisms for inflammatory toxicities during CAR T-cell therapy, current strategies for management and mitigation of these risks and highlights key areas of knowledge gap for future research

Automated Microraft Array Platform for Immune Cell Assays and Cell Sorting

Immunology research and cell therapies have provided great advancements in recent years and have highlighted the need to understand the effects of single cell heterogeneity within immune cell populations. Single cell platforms currently used in immune cell analysis often include tedious manual work, are not able to measure immune cell function in a time-dependent manner, or are not selective. The following work describes the development of an automated microraft array platform for analyzing the function of individual immune cells, including helper T cells and chimeric antigen receptor T cells (CAR-T cells), and for the sorting of cells of interest. In this dissertation, an automated microraft array platform was developed and adapted to address the challenges seen in immune cell research. In Chapter 2, an automated microraft array platform was developed to assay thousands of single cells in parallel and sort individual cells of interest. The platform was used to assay single CD4+ T cells, isolate cells displaying proliferation in response to allogeneic cell stimulation, and sequence their T cell receptor genes. In Chapter 3, a next-generation magnetic microbead-based microraft array was developed as an alternative to nanoparticle-based microraft arrays. The microbead-based arrays were shown to substantially reduce fabrication time compared to nanoparticle-based microraft arrays and improve performance in imaging of fluorescently labeled cells. Chapter 4 focused on the development and application of the automated microraft array platform to assay CD19 CAR-T cells for cell-mediated cytotoxicity and isolate T cells of interest for gene expression analysis. CAR-T cells were shown to participate in serial-killing of target cells and T cells demonstrating high cytotoxicity were isolated for future gene expression analysis using single-cell multiplex qPCR. The findings presented in this dissertation demonstrate the capabilities of an automated microraft array platform and its uses in immunology research. The studies described in each chapter provided valuable insight into the behavior and phenotype of immune cells at the single cell level.Doctor of Philosoph

Chimeric Antigen Receptor engineered gamma delta T cells for neuroblastoma immunotherapy

Gamma delta (γδ) T cells are a unique subset of lymphocytes that combine both innate and adaptive immune properties. They are primed for rapid function including tumour cell cytotoxicity, and following activation, have professional antigen presenting function. Chimeric antigen receptors (CARs) are synthetically engineered receptors that combine the specific antigen-binding region of a monoclonal antibody with a signalling domain responsible for T cell activation and cytotoxicity. Given the natural tissue tropism and innate cellular responses of γδ T cells, it was hypothesised that transduction with a CAR would enhance antigen-specific cytotoxicity, whilst maintaining direct antigen presenting function and the ability to migrate towards tumours. Using the tumour antigen GD2 as a model system, we demonstrated that Vδ1 and Vδ2 cells could be activated, propagated and transduced to sufficient number for use in clinical studies in paediatric patients. The addition of GD2-CAR, enhanced γδ T cell innate cytotoxicity through specific killing of GD2-expressing neuroblastoma cell lines. Migration towards tumour cells was not impaired by the presence of the CAR. Following activation, GD2-CAR transduced Vδ2 cells, retained the ability to take up exogenous tumour antigen, and cross-presented processed peptide to responder αβ T cells. This study provides evidence to support the emerging role of CAR γδ T cells as a safe and efficacious immunotherapy for neuroblastoma

A single-cell multi-omic approach to the analysis of T cell differentiation

This thesis aims to investigate T cell differentiation through the bioinformatic analysis of single-cell multi-omic data. T cells are an important part of the adaptive immune system, involved in the immune response to infections and cancer. Single-cell technologies have advanced to the point where multiple modes of data, such as gene and protein expression, can be assayed on the same cells. Greater understanding of T cell differentiation pathways at the single-cell level can help in the design of immunotherapies to treat cancer and autoimmune disease. The thesis begins by presenting a multi-omic workflow that combines scRNA-seq and T cell receptor (TCR) sequence extraction. The principles developed for this workflow were applied to investigate T cell differentiation in two scenarios. The first scenario was an application of single-cell multi-omics to the study of CD8+ T cell peripheral tolerance mechanisms in a mouse model. This work demonstrated that tolerance is a distinct differentiation program to functional effector responses, and T cells progressively commit to the tolerised state over the first 60hrs post exposure to triggering antigen. A gene signature for the tolerised state was identified, containing genes uniquely upregulated in tolerised cells. Quiescent and Proliferating clusters were found in tolerised cells, indicating that a proportion of cells exit cell cycle within each division. The second scenario was an investigation of the differentiation of CD4+ CAR T cells in vivo, and the evolution of a lymphoma derived from these cells. Three cell types, proliferating, cytotoxic and resting, were observed within the malignant CAR T-cells, and these types were also observed within non-malignant CAR T and endogenous CD4+ T cells. The lymphoma was characterised by expression of the NF-κB transcription factor in all three cell types, while each cell type had differing expression levels for several other known oncogenes. This thesis has contributed to the understanding of T cell differentiation in tolerance and CAR T therapy, and has helped meet the challenge of increasingly large and complex single- cell datasets through the development of bioinformatic workflows to integrate samples from multiple patients and sequencing technologies, and integrate gene, protein, TCR sequence, cell division count and somatic mutation data at the single-cell level

Deploying single-cell transcriptomics for assessing CAR T cell generation: alleviating antiviral restriction factors enhances gene transfer

Lentiviral vectors (LV) have become the dominant tool for stable gene transfer into lymphocytes including chimeric antigen receptor (CAR) gene delivery to T cells, a major breakthrough in cancer therapy. Yet, room for improvement remains, especially for the latest LV generations delivering genes selectively into T cell subtypes, a key requirement for in vivo CAR T cell generation. Towards improving gene transfer rates with these vectors, transcriptome analyses on human T lymphocytes after exposure to CAR-encoding conventional vector VSV-LV, and vectors targeted to CD8+ (CD8-LV) or CD4+ T cells (CD4-LV) was conducted. Genes related to quiescence and antiviral restriction were found to be upregulated in CAR-negative cells exposed to all types of LVs. Down-modulation of various antiviral restriction factors including the interferon-induced transmembrane proteins (IFITMs) was achieved with rapamycin as verified by mass spectrometry (LC-MS). Strikingly, rapamycin enhanced transduction by up to 7-fold for CD8-LV and CD4-LV without compromising CAR T cell activities, but did not improve VSV-LV. When administered to humanized mice, CD8-LV resulted in higher rates of GFP gene delivery as well as faster in vivo CAR T cell generation and tumor control. The data favor multi-omics approaches for improvements in gene delivery

Development of Novel Transient Delivery Systems for Gene Therapy

Gene and cell therapies hold enormous promise for many presently incurable diseases. Although the efficiency and safety of site-specific genome editing systems have improved tremendously over the past decade, the delivery of these novel technologies to specific target cells remains a key technical bottleneck for the clinical translation. Since long-term expression of DNA-modifying enzymes can be associated with cytotoxicity, transient delivery is desirable to reduce the risk of mutagenesis from off-target activity. A main target for gene and cell therapies are T cells. In order to achieve gene transfer into human T cells, current gene delivery techniques involve significant levels of T cell activation and ex vivo expansion prior to lentiviral or retroviral transduction. However, ex vivo stimulation and expansion have been shown to significantly alter the natural physiology of T cells. To address these challenges, we have developed a non-integrative lentiviral gene therapy platform to focus on genetic modifications of resting CD4+ T cells. To overcome barriers, present in this notoriously hard to transduce cell population, we have re-engineered lentiviral particles from the inside-out. Firstly, we have identified key viral glycoproteins that can mediate cytosolic delivery of transgenes across resting CD4+ T cell plasma membranes. Secondly, by replacing HIV nucleocapsid with the coat protein of the MS2 bacteriophage and substituting lentiviral RNA packaging motifs (LTR and packaging signal Ψ) with a series of MS2 stem loops that facilitate RNA binding to the MS2 coat protein, we have enabled the packaging of CRISPR/Cas9 components into these particles. In this setting, we observed expression of biologically active mRNA in up to 65% of resting CD4+ T cells. Furthermore, with our CRISPR/Cas9 delivery platform we have achieved genome editing efficiencies of >85% in human cell lines, and >20% in primary CD4+ T cells. With the emergence of SARS-CoV-2, we have shifted our focus on the development of a novel viral vector for gene delivery. Using a SARS-CoV-2 VLP scaffold, we have enabled the packaging and transient delivery of mRNA into target cells by tethering a reporter gene to the recently identified SARS-CoV-2 packaging signal. Equipping these VLPs with Spike glycoproteins from emerging SARS-CoV-2 variants, we observed an increase in transient mRNA delivery to >50% of target cells. This platform may give us a novel set of viral vector solutions to use for many respiratory diseases, including coronavirus disease (COVID-19) itself. Taken together, this thesis describes the development of robust delivery platforms for the transient delivery of biologically active transgenes to multiple cell types