Car Drivers and Fuel Sources: How Distinct Signaling Domains in Chimeric Antigen Receptors Reprogram T Cells

With breakthroughs in synthetic biology, improved cell culture techniques and advanced genetic engineering, it has now become possible to generate bi-specific primary human T cells with desired specificities. One mode of redirecting specificity is the modification of T cells to express chimeric antigen receptors (CARs). Recent studies indicate that natural T cells have distinct biochemical and metabolic features that endow them with short lived effector or long lived memory fates. The central objective of this thesis was to investigate whether the signaling endodomain of CARs could reprogram T cells with pre-specified effector and memory fates. This thesis describes a novel technique that allows for detailed investigation of the impact of CAR design on the fate of T cells. Specifically, it compares the short-term and long-term signaling effects of CD28 and 4-1BB costimulatory domains in the CAR architecture. These two signaling domains have been most extensively employed in CAR therapy trials against a wide variety of malignancies. Incorporation of 4-1BB signaling domain imparts superior proliferative and survival benefits as compared to the CD28-containing CAR T cells. This increased persistence correlates with clinical observations. 4-1BB CARs T cells show an enrichment of central memory phenotype along with relative increase in fatty acid based metabolism. This is accompanied by a relative increase in mitochondrial mass, upregulation of key metabolic enzymes and increased spare respiratory capacity. Furthermore, stimulation of CD28-containing CARs promotes rapid induction of biochemical signaling events that are associated with T cell activation. Specifically, the phosphorylation of key proximal and distal signaling proteins between the two CAR models have been compared. Inclusion of CD28 domain in the CAR structure dramatically reduces activation threshold and leads to increased and sustained calcium flux. Taken together, this thesis work uncovers some key differences triggered by the different costimulatory domains. This thesis establishes that the choice of CAR signaling domain can be used to dictate the fate of engineered T cells. Moving forward, the ability of CARs to reprogram T cell metabolism and induce differential activation patterns will need to be considered when designing future CAR trials

The Genetics of Anti-Neutrophil Cytoplasmic Antibody Associated Vasculitis (AAV)

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a multi-systemic autoimmune disorder with evidence of circulating pathogenic ANCA. There are two main antigenic targets: proteinase 3 (PR3) and myeloperoxidase (MPO). Previous genome-wide association studies (GWAS) have provided evidence that PR3-AAV and MPO-AAV are genetically distinct autoimmune syndromes, though only three loci specific to PR3-AAV and one to MPO-AAV have been identified to date. With the European Vasculitis Genetics Consortium, we conducted a larger GWAS, powered to discover additional risk loci in both PR3-AAV and MPO-AAV independently. A meta-analysis of two European cohorts was conducted, comprising 1,610 PR3-AAV cases, 870 MPO-AAV cases and 11,947 controls. For PTPN22 (rs6679677), a further replication cohort, previously genotyped using the Sequenom MassARRAY platform, and comprising 1,122 PR3-AAV and 347 MPO-AAV cases and 1,531 controls was included in the combined analysis. This is the largest genome-wide association study of AAV to date and we have identified a total of 12 AAV susceptibility loci. Previously genome-wide significant loci were confirmed, including HLA class II, SERPINA1, PRTN3 and PTPN22. Seven new genome-wide significant loci were identified: three associated with PR3-AAV (BCL2L11-MIR4435-2HG, EBF3-MGMT, IGHV1-69), two with MPO-AAV (BACH2, ANKRD11-SPG7) and two shared by both (CTLA-4 and DGUOK-TET3). Further analyses based on common variants suggested that a substantial component of the genetic architecture was shared between PR3-AAV and MPO-AAV, similar to that observed between ulcerative colitis and Crohn’s disease. In addition, Mendelian randomisation analysis confirmed that a higher eosinophil count increased the risk of PR3-AAV but not MPO-AAV, and this effect might, in part, be modulated by MIR4435-2HG through prolongation of eosinophil survival. MIR4435-2HG encodes a long non-coding RNA that plays a critical role in the regulation of BCL2L11 transcription (a Bcl2 family member essential for controlling apoptosis) in myeloid cells and hence their lifespan. We have also identified a missense variant in IGHV1-69 (rs11845244) that leads to a loss in neutralising function of antibodies generated against the NEAT2 domain of Staphylococcus aureus. This therefore provides a plausible host genetic factor in determining the susceptibility to infectious disease and as a potential driver of PR3-AAV. Overall, this study provides key novel insights into disease biology for AAV and potential therapeutic targets.Wellcome Trust Clinical PhD Fellowshi

From harmful to useful: exploiting a leukemia-associated transcription factor for large-scale manufacture of functional human macrophages

Expansion of hematopoietic stem and progenitor cells outside the body is hardly possible due to spontaneous differentiation. Therefore, ex vivo production of immune cells in sufficient quantities for cell therapeutic approaches is limited. Exploitation of oncogenic fusion proteins represents a new, promising possibility for ex vivo propagation of human blood progenitor cells. These chromosomal translocation-derived chimeric transcription factors are capable of driving hematopoietic progenitor cell expansion by blocking differentiation. In this context, it was investigated to what extent a controllable designed leukemia-associated fusion protein can be exploited for ex vivo proliferation of human hematopoietic progenitor cells to generate functional macrophages on a large scale after switching off the protein. As such a fusion gene, MLL-ENL was stably integrated into the genome of human hematopoietic progenitor cells via retroviral gene transfer. A destabilization domain fused to MLL-ENL was used to target protein stability using the specific small molecule ligand Shield-1. In the presence of Shield-1, late monocytic progenitor cells were expanded in a large scale in ex vivo cultures in a controlled manner. The cells exhibited an immature monocyte immunophenotype, a normal karyotype, and permanent Shield-1 dependence. Genome-wide sequencing and viral integration site analysis also confirmed the absence of copy number alterations, mutations, or integration-activated proto-oncogenes that could promote cell proliferation. Shield-1 withdrawal allowed the expanded progenitor cells to be specifically differentiated into functional macrophages with appropriate cytokines. These phagocytes expressed macrophage-associated cell surface proteins and upregulated a variety of genes that play critical roles in the innate immune response. Functionally, adhesion under shear stress, migration along a chemokine gradient, and clearance of inactivated bacteria and apoptotic cells were demonstrated. Furthermore, macrophages were able to efficiently phagocytose antibody-loaded lymphoma or leukemia cells derived from patient blood. In summary, functional human phagocytes were produced for the first time from MLL-ENL expanded monocytic progenitor cells and characterized in detail by molecular and cell biology. This successful proof of concept suggests that such acute leukemia-derived and modified transcription factors have the potential to be used as molecular tools to generate functional immune cells for cell therapy approaches

From molecular targets to antitumor immunity

This thesis aimed at the development of strategies that could contribute to the evaluation of the clinical potential of new anticancer therapies. The work was divided into two main sections comprising the development of a therapeutic approach to target cancer genetic vulnerabilities and the development of 3D tumor models incorporating cues from the stromal and immune microenvironments. (...

Development and Preclinical Evaluation of Enzyme Prodrug Therapies Targeted to the Tumor Vasculature

Breast cancer is a global health concern of high prevalence that lacks safe and effective therapies for advanced cases. A targeted enzyme prodrug therapy aims to address this issue using an enzyme localized to the tumor to convert a systemically administered nontoxic prodrug into a toxic anticancer agent exclusively in the tumor. The target of the presented enzyme prodrug systems, phosphatidylserine, exists on cancer cells and the cells of the tumor vasculature. Annexin V binds to phosphatidylserine with high affinity and was successfully fused to three enzymes for the targeted delivery of the enzyme prodrug systems to the tumor. Development of the purine nucleoside phosphorylase fusion with annexin V is described, and results showing strong in vitro binding and promising cytotoxicity are presented. This system is compared in vivo with targeted cytosine deaminase and targeted methioninase enzyme prodrug systems. The methioninase system produced the strongest antitumor results showing tumor regression for the duration of treatment. Further engineering of the system resulted in the generation of a mammalian cystathionine-γ-lyase protein with methioninase activity to prevent the immune response anticipated against foreign methioninase. Successful transition to immune competent models without incurring an immune response led to studies with combination therapies to achieve an enhanced therapeutic effect. Antitumor synergism was observed when the enzyme prodrug therapy was combined with rapamycin to address the hypoxic response. Combination with immunostimulatory levels of cyclophosphamide produced an anti-metastatic response and enhanced survival. Combination of the enzyme prodrug therapy with both rapamycin and cyclophosphamide effectively reduced tumor volumes, inhibited metastatic progression, and enhanced survival

Radiation Response Biomarkers for Individualised Cancer Treatments

Personalised medicine is the next step in healthcare, especially when applied to genetically diverse diseases such as cancers. Naturally, a host of methods need to evolve alongside this, in order to allow the practice and implementation of individual treatment regimens. One of the major tasks for the development of personalised treatment of cancer is the identification and validation of a comprehensive, robust, and reliable panel of biomarkers that guide the clinicians to provide the best treatment to patients. This is indeed important with regards to radiotherapy; not only do biomarkers allow for the assessment of treatability, tumour response, and the radiosensitivity of healthy tissue of the treated patient. Furthermore, biomarkers should allow for the evaluation of the risks of developing adverse late effects as a result of radiotherapy such as second cancers and non-cancer effects, for example cardiovascular injury and cataract formation. Knowledge of all of these factors would allow for the development of a tailored radiation therapy regime. This Special Issue of the Journal of Personalised Medicine covers the topic of Radiation Response Biomarkers in the context of individualised cancer treatments, and offers an insight into some of the further evolution of radiation response biomarkers, their usefulness in guiding clinicians, and their application in radiation therapy

Genotypic and phenotypic determinants of immunity across gynecological malignancies

Immunotherapy is emerging as oncological treatment strategy. To enhance immunotherapy efficacy in gynecological cancers, the studies in this dissertation aimed to improve understanding of immunological aspects in gynecological cancers. To this end, initiation of the anti-tumor response and determinants of this response were analyzed at both a genetic and protein level. Moreover, a novel manner to study cancer immunotherapy in the preclinical setting is proposed. Expression of STING, a protein with an important role in the initiation of immunity against cancer cells with DNA damage or infection, appeared to be decreased in patients with cervical cancer. This decrease coincided with worse prognosis. Therapeutic intervention to activate STING may be beneficial in the treatment of cervical cancer. Furthermore, patients with specific genetic variations in the STING-encoding gene also had worse prognoses. Genetic screening to determine prognosis could be considered. The CD8+ T cell is an important type of immune cell in the elimination of cancer cells. Various markers such as CD103, PD-1 and CD39 enable identification of anti-cancer reactive CD8+ T cells. In this dissertation, transcriptional profiles were obtained for these cells. Notably, CD103 marked cells that secreted the chemokine CXCL13, thereby linking T cells, B cells and formation of tertiary lymphoid structures.Induced pluripotent stem cells, established from the urine of cancer patients, showed potential to create novel preclinical models for immunotherapy in (gynecological) cancer