Characterisation of breast and endometrial tumour infiltrating lymphocytes: investigating their responsiveness to tumour antigens

Abstract

Introduction: The most common cancers affecting women worldwide are breast cancer, followed by endometrial cancer. Despite advances in surgical techniques and the effectiveness of treatments, the mortality rate remains high and novel therapeutic approaches are therefore needed to increase survival rate and prevent recurrence. The field of cancer immunotherapy has significantly improved outcomes in other types of cancer such as melanoma. Adoptive cellular therapy (ACT) using tumour-infiltrating lymphocytes (TILs) has shown extraordinary results mainly in melanoma. However, despite the prognostic role of TILs in breast and endometrial cancer, the use of TILs as a therapeutic approach remains novel. The aim of this thesis was to determine the feasibility of expanding TILs ex vivo for the treatment of breast and endometrial cancer, to evaluate the TIL phenotype and functionality, and investigate the expanded TILs response to neoantigens encoded by somatically mutated genes in cancer. Methods: TILs were isolated and expanded from breast and endometrial tumours using anti-CD3/CD28 magnetic beads and a high dose of IL-2. TIL phenotype was characterised before and after expansion using flow cytometry. Anti-tumour functional activity of the expanded breast and endometrial TILs was determined by co-culture with autologous tumour and measuring IFNγ secretion. Additionally, to study whether expanded breast TILs can recognise autologous mutated neoantigens, two tumours were analysed by whole exome and transcriptome sequencing. Candidate neoantigens were then filtered by applying HLA prediction algorithms and synthesised. Peptide pulsed antigen-presenting cells were co-cultured with expanded TILs and their response was detected by IFNγ production. Results: TILs isolated from breast and endometrial tumours were successfully expanded from 72.6% and from 90% of samples, respectively. A rapid expansion protocol (REP) showed that both breast and endometrial TILs could be expanded to clinically relevant numbers. Expanded breast and endometrial TILs were demonstrated to be more active against autologous tumour cells than PBMCs, associated with greater central and effector memory phenotypes and a higher expression frequency of co-stimulatory markers such as CD69 and OX40 with more ability to produce cytokines. Finally, analysis of two breast cancer patient samples identified non-synonymous mutations that were predicted to give rise to unique neoantigens. Consequent peptide screening assays detected a strong T cell response in the second sample (B75) to all of HLA A29, B40, B4, C03, C16 restricted pools. In the HLA A11 restricted pool, only two of five pools showed a response. Conclusion: The results presented in this thesis demonstrate that ACT using expanded TILs from breast or endometrial cancer is a feasible therapeutic strategy. Additionally, this thesis evaluated the presence of co-inhibitory receptors on TILs in both breast and endometrial cancer and demonstrated their functionality. The data from tumour sequencing show that expanded breast TILs are able to recognise neoantigens arising from somatic mutations within the tumour.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Anne Armstrong (Supervisor), Robert Clarke (Supervisor) & Milena Kalaitsidou (Supervisor)