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Introduction: In the UK in 2016, approximately 16,000 new cases of oesophagogastric cancer were diagnosed. Chemotherapy and surgery are the mainstay of treatment but are associated with considerable morbidity and overall survival rates are dismally low. Worryingly the incidence of oesophageal adenocarcinoma is predicted to rise in the UK. Promising results using immunotherapies, including adoptive cell therapy and immune checkpoint inhibition, have been observed in other solid malignancies such as melanoma, and hold potential promise for oesophagogastric cancers. The tumour microenvironment and detectable changes in the peripheral blood of patients with oesophagogastric adenocarcinoma have not been widely researched in a Western population, potentially due to prevalence being higher in Eastern countries such as Japan and China. A better understanding of the tumour immune relationship in oesophagogastric adenocarcinoma may lead to the identification of potential biomarkers and development of novel treatment strategies. Aims Based on previous research (including the host laboratory), the primary aim was to quantify levels of regulatory T-cells, T helper 17 and 22 cells; relate expression to prognosis; and explore as potential predictive biomarkers. The secondary aim was to assess the feasibility of expanding tumour infiltrating lymphocytes (TIL) and perform in vitro experiments to evidence the potential for ACT in the treatment of oesophagogastric adenocarcinoma. Methods: T-cell phenotype markers levels were determined using multi-colour flow cytometry in the peripheral blood of patients with oesophagogastric cancer before and after initiation of chemotherapy, which was compared to healthy volunteers. Experiments to isolate and expand TIL from tumour specimens were performed using the GentleMACs system of dissociation. Results: Chapter 3 (Results 1) characterised 100 patients with oesophagogastric adenocarcinoma who were enrolled for the peripheral blood phenotyping arm of this thesis and reported poor survival patterns confirmatory with the literature. Chapter 4 (Results 2) reported that higher levels of Th17, Th22 and TCRgd cells were demonstrated in the peripheral T-cells of patients with cancer compared to healthy controls. Th17 levels appear to be predictive of survival outcome at 21-months for patients with oesophageal adenocarcinoma. Chapter 5 (Results 3) reported higher levels of Treg, CD4+OX40+, TregICOS+ and CD8+ICOS+ cells in analysis of the same patient cohort; however, levels were unrelated to site or stage. A decrease in Treg and Th22 levels were also observed in patients after initiation of chemotherapy. Chapter 6 (Results 4) showed that cytotoxic TILs were successfully extracted and expanded from three of thirteen tumour specimens. Th17 levels were significantly increased in these expanded samples. Conclusion: This thesis has performed complex peripheral T-cell phenotyping in potentially the largest group of patients with oesophageal adenocarcinoma from the UK, this is unique. This research demonstrated interesting differences in the immune phenotype of circulating PBMCs in patients with oesophagogastric adenocarcinoma using simultaneous expression of multiple markers included in Panel A and B. Th17 levels have been identified as a marker which correlates with clinical outcome and this finding should be expanded in further studies. Although this study identified problems in extracting and expanding TIL, it demonstrated that the technique is possible and therefore future work should optimise this protocol further to evidence the potential for ACT in oesophagogastric adenocarcinoma. By continuing to monitor the changes in the immune system and research into the potential role of adoptive cell therapy in this disease, potentially novel immunotherapies will change the prognosis of what is an extremely deadly disease.

T cells are a subset of the adaptive immune system that have the capacity to eliminate transforming cells by recognition of minute differences from healthy tissue. Recently, T cell therapies have become an incredible example of scientific progress as human ingenuity is applied to our growing understanding of immunology. In particular, T cells engineered with chimeric antigen receptors (CAR-T cells) have been successful in the treatment of hematological malignancies including B cell leukemias, B cell lymphomas, and multiple myeloma. CARs allow T cells to utilize their anti-tumor functions while bypassing typical T cell constraints such as activation by the innate immune system and human leukocyte antigen dependence. While CAR-T cells are highly efficacious in the treatment of several malignancies, the field is rapidly expanding in their use and application with innumerable strategies remaining to be tested. Here we describe two strategic methods to improve the efficacy and accessibility of CAR-T cell therapy. First, an automated manufacturing method is described that allows in-house generation of clinical grade CAR-T cells from non-Hodgkin’s lymphoma (NHL) patients outside a cleanroom environment with a shortened manufacturing time from 12 to 8 days. Second, single-cell RNA sequencing and high dimensional flow cytometry profiling of CAR-T cells derived from the autologous products or blood of patients treated for NHL were used to identify the T cell immunoreceptor with Ig and ITIM domains (TIGIT) as a marker of CAR-T cell dysfunction, particularly in patients with stable or progressive disease. TIGIT antibody blockade was then demonstrated to improve CAR-T cell function with in vitro and in vivo models of NHL. While these strategies were studied in the context of NHL, they may be translated to a variety of disease contexts including solid tumors, as is discussed in this work.

Multiple sclerosis (MS) is considered a prototypic autoimmune disease of the central nervous system. It is the leading cause of chronic neurological disability in young adults, and not only T and B cells, but also regulatory T cells (Tregs) have been suggested to play important roles in its pathogenesis. Several studies have shown Treg dysfunction in patients with MS, but underlying mechanisms remain poorly understood. By combining immunophenotyping, functional assays, high-throughput T cell receptor (TCR) Vβ-chain-, and RNA sequencing we examined the Treg compartments in 41 patients with relapsing-remitting MS (RRMS) and 17 healthy donors (HDs). Patients with MS showed a reduced frequency of CD3+CD4+ cells and Foxp3+ Tregs and an increased frequency of CD39+ Tregs. The expression of CD127 was augmented, whereas the expression of CD49d was decreased in Tregs derived from patients. By using a novel staining strategy based on CD45RA and GPA33, we observed low t-Tregs in younger patients. When we determined Treg function, we found that this was reduced only in natalizumab-treated patients. Accordingly, only in this group we detected an altered gene expression in t-Tregs, but not in p-Tregs. Here we provide insights into the possible mechanisms underlying the functional Treg impairment in some patients with MS, such as an impaired proliferation and homeostasis of t-Tregs, leading to their depletion, and an increased Teff autoproliferation. In addition, we detected potential compensatory mechanisms, that might be beneficial or detrimental, such as CD39, CD127 and CD45RA upregulation. Our findings prompted us to investigate strategies to reestablish immune tolerance by adoptive transfer of Tregs. We provide the first description of suppressive, Foxp3 positive T cell clones specific for myelin basic protein (MBP) that could be used as source of TCRs for adoptive Treg therapy in selected patients.

Chimeric antigen receptor (CAR) T cell therapy, among other adoptive cell transfer approaches, has emerged as a promising strategy to introduce tumor-specific T cells into patients. CAR T cell therapy, in particular targeting CD19, has had great success in the treatment of hematological malignancies. However, CAR T cell treatment of solid tumors has been limited by several challenges—(1) target antigen selection, complicated by tumor heterogeneity and on- target, off-tumor toxicity; (2) CAR T cell exhaustion, limiting their persistence and function; and (3) interaction with and engagement of the endogenous immune system. This thesis addresses these topics with a biomaterials-centered approach, with the hope of tackling barriers to therapy from several perspectives. Chapter 2 tackles the problem of tumor heterogeneity by developing a strategy to deliver CAR T cell ligands to tumors using the membrane-inserting properties of amphiphile-ligands. We demonstrate that intratumorally- administered amph-ligand labels tumor cell membranes, recruits ligand-specific CAR T cells, and triggers potent antitumor efficacy in both syngeneic and xenograft tumor models. Furthermore, this therapy primes endogenous tumor-specific T cells. In Chapter 3, we apply the amph-ligand approach to vaccine boosting CAR T cells as a potential solution to CAR T cell exhaustion and limitations of CAR T cell manufacturing, and turn to characterization of the vaccine strategy and differences as a result of CAR design. Finally, Chapter 4 describes an approach to recruit endogenous virus-specific T cells to tumors via the delivery of viral peptides to tumors. Taken together, these strategies demonstrate the potential of biomaterials to transform CAR T cell therapy, promoting not only greater response rates and lasting tumor control, but also broadening the number of patients and cancer types that can be treated.

Adoptive immunotherapy with ex vivo-expanded tumor-infiltrating lymphocytes (TILs) has achieved objective clinical responses in a significant number of patients with cancer. The failure of many patients to develop long-term tumor control may be, in part, due to exhaustion of transferred T cells in the presence of a hostile tumor microenvironment. In several tumor types, growth and survival of carcinoma cells appear to be sustained by a network of receptors/ligands of the ErbB family. We speculated that if transferred T cells could benefit from EGFR ligands produced by the tumor, they might proliferate better and exert their anti-tumor activities more efficiently. We found that CD8+ T cells transduced with a retrovirus to express EGFR responded to EGFR ligands activating the EGFR signaling pathway. These EGFR-expressing effector T cells proliferated better and produced more IFN-γ and TNF-α in the presence of EGFR ligands produced by tumor cells in vitro. EGFR-expressing CD8 T cells from OT-1 mice were more efficient killing B16-OVA cells than control OT-1 CD8 T cells. Importantly, EGFR-expressing OT-1 T cells injected into B16-OVA tumor bearing mice were recruited into the tumor, expressed lower levels of the exhaustion markers PD1, TIGIT, and LAG3, and were more efficient in delaying tumor growth. Our results suggest that genetic modification of CD8+ T cells to express EGFR might be considered in immunotherapeutic strategies based on adoptive transfer of anti-tumor T cells against cancers expressing EGFR ligands.

With the establishment of clinically effective adoptive T-cell therapy for metastatic cancer, efforts are growing to advance T-cell therapy for new indications, especially solid tumors. A major challenge, however, is that the antitumor activity of infused T cells can be suppressed by the cytokine transforming growth factor beta (TGFβ). To counteract TGFβ-mediated immunosuppression, a TGFβ-specific chimeric antigen receptor (TGFβ-CAR) was engineered and characterized in vitro. The TGFβ-CAR converted TGFβ from a suppressive signal to a stimulatory signal, enabling CD4+ and CD8+ T cells to overcome TGFβ-mediated dysfunction by blocking endogenous TGFβ signaling and triggering TGFβ-specific T-cell activation, Th1 cytokine production, and robust proliferation. These properties suggest the TGFβ-CAR could be incorporated into tumor-reactive T cells to boost antitumor activity. Alternatively, the TGFβ-CAR could be a tool for robust T-cell expansion ex vivo. To our knowledge, this is the first CAR specifically designed to respond to a soluble antigen.

Magnetic and plasmonic properties combined in a single nanostructure provide a synergy that is advantageous in a number of biomedical applications, such as contrast enhancement in multimodal imaging, simultaneous capture and detection of circulating tumor cells, and photothermal therapy of cancer. These applications have stimulated significant interest in development of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared region and a strong magnetic moment. In this dissertation, we addressed this need to create a novel immunotargeted magneto-plasmonic nanoparticle platform. The nanostructures were synthetized by self-assembly of primary 6 nm iron oxide core-gold shell particles, resulting in densely packed spherical nanoclusters. The close proximity of the primary particles in the nanoclusters generates a greatly improved response to an external magnetic field and strong near-infrared plasmon resonances. A procedure for antibody conjugation and PEGylation to the hybrid nanoparticles was developed for biomedical applications which require molecular and biocompatible targeting. Furthermore, we presented two biomedical applications based on the immunotargeted hybrid nanoparticle platform, including circulating tumor cell (CTC) detection and cell-based immunotherapy of cancer. In the CTC detection assays, rare cancer cells were specifically targeted by antibody-conjugated nanoparticles and efficiently separated from normal blood cells by a magnetic force in a microfluidic chamber. The experiments in whole blood showed capture efficiency greater than 90% for a variety of cancers. We also explored photoacoustic imaging to detect nanoparticle-labeled CTCs in whole blood. The results showed excellent sensitivity to delineate the distribution of hybrid nanoparticles on the cancer cells. Thus, these works paves the way for a novel CTC detection approach which utilizes immunotargeted magneto-plasmonic nanoclusters for a simultaneous magnetic capture and photoacoustic detection of CTCs. In another application, we introduced a novel approach to label cytotoxic T cells using the magnetic nanoparticles with an expectation to enhance T cell recruitment in tumor under external magnetic stimulus. A series of in vitro experiments demonstrated highly controllable manipulation of labeled T cells. Thus, these results highlight the promise of using our nanoparticle platform as a multifunctional probe to manipulate and track immune cells in vivo and further improve the efficacy of cell-based cancer immunotherapy.