Exploiting Adoptive T Cell Therapy as a Therapeutic Strategy in Epithelial Ovarian Cancer

UoM administered thesis: Phd

Abstract

Introduction: Epithelial ovarian cancer is the fifth most common cause of cancer-related mortality in women in the UK. Despite advances in surgical techniques and improvements in the efficacy of cytotoxic treatments, there has been no appreciable increase in overall survival in the last three decades. Thus, novel therapeutic strategies are urgently needed to improve survival and prevent recurrence. Epithelial ovarian cancers are frequently infiltrated with T lymphocytes. Adoptive cell therapy (ACT) using tumour-infiltrating lymphocytes (TIL) has shown remarkable efficacy in other immunogenic tumours, particularly malignant melanoma. Early clinical trials of TIL therapy in ovarian cancer demonstrated mixed results; however, none of these trials used current T cell expansion strategies. The primary aims of this thesis were to optimise the generation of TIL from ovarian tumour samples, define the phenotype and anti-tumour function of TIL, and evaluate expanded TIL responses to putative mutated neoantigens. Genetic modification of T cells to direct their specificity through the use of a chimeric antigen receptor (CAR), is an attractive strategy in patients who lack TIL. This thesis also explored the feasibility of CAR T cell therapy targeting the oncofetal protein, 5T4. Methods: TIL were expanded from resected ovarian tumours and ascites using anti-CD3/CD28 coated magnetic beads and high-dose IL-2. In later experiments, TIL were also expanded in the presence of immune checkpoint inhibitors. TIL phenotype was characterised at baseline (day 0) and following expansion by flow cytometry. Functional activity was determined by co-culturing TIL against autologous tumour and measuring IFNγ production and cytotoxicity. To determine whether expanded ovarian TIL are reactive to autologous somatic mutated neoantigens, two tumours were subjected to whole exome and transcriptome sequencing. Candidate neoantigens were filtered using HLA prediction algorithms and synthesised. Peptide-pulsed antigen presenting cells were then co-cultured with TIL and T cell responses identified by IFNγ secretion. Peripheral blood T cells were transduced with two anti-5T4 CAR constructs and co-cultured against 5T4+ cell lines and autologous tumour cells, to determine cytotoxicity. Results: Ovarian TIL were successfully expanded from 75 of 84 (89.2%) clinical specimens. Expanded TIL demonstrated features associated with central- and effector-memory phenotypes, and showed strong functional activity when co-cultured with autologous tumour cells. Small-scale REP showed that TIL could be expanded to clinically relevant numbers in 87% of donors. Next generation sequencing of two patient samples identified a total of 101 non-synonymous mutations, which were predicted to give rise to 93 unique neoepitopes. Subsequent peptide screening assays identified a strong T cell response to a HLA-C*07:01 restricted neoepitope, FSFPTSDTY. Increased expression of PD-1, TIM-3 and LAG-3 were noted on freshly isolated ovarian TIL. Further analysis revealed that TIL expressing PD-1 and TIM-3 had an increased capacity to secrete effector molecules compared to their negative counterparts. Immune checkpoint blockade during ex vivo expansion significantly increased TIL yields at day 19 but failed to demonstrate any difference in the phenotype or cytotoxicity of the final TIL product compared to untreated controls. Finally, T cells isolated from the peripheral blood of patients with epithelial ovarian cancer were effectively transduced with two different anti-5T4 CAR constructs which differed in their affinity for the target antigen. Co-culture of CAR T cells with 5T4+ cell lines and autologous tumour cells resulted in antigen-specific secretion of IFNγ and IL-2. Conclusion: This thesis has shown that ACT utilising ex vivo expanded TIL or autologous T cells transduced with an anti-5T4 CAR are feasible immunotherapeutic strategies in epithelial ovarian cancer. Based on the results outlined, funding has been secured for a phase I/II clinical trial to establish the safety and efficacy of TIL in patients with relapsed ovarian cancer. Additionally, the results show that expanded ovarian TIL contain T cells capable of recognising neoantigens arising from somatic mutations within the tumour.

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Original languageEnglish
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Award date1 Aug 2019