Exploring T Cell Immunotherapeutic Strategies to Target Breast Cancer

Abstract

Introduction: Breast cancer is the most common malignancy affecting women in the UK. Despite a multi-modality treatment approach, it remains the most common cause of death in women aged 25-59 worldwide, and as such, there remains a significant clinical unmet need. Recent developments within the field of cancer immunotherapy have radically improved outcomes in solid cancers such as melanoma. However, this improvement in clinical outcome has yet to be translated into many other solid cancers such as breast cancer. Tumour infiltrating lymphocytes (TIL) have been found to be an important prognostic biomarker in breast cancer. Adoptive cell therapy (ACT) using ex vivo expanded TIL has shown clinical efficacy in some solid cancers. However despite the role of TIL in breast cancer prognostication, their use as a therapeutic strategy remains novel. The primary aim of this thesis was to investigate the feasibility of an ACT approach for breast cancer treatment, by optimising the TIL expansion protocol and defining the phenotype of isolated and expanded TIL. This thesis also explored the peripheral T cell compartment of breast cancer patients undergoing neoadjuvant chemotherapy (NACT), in an attempt to identify a readily accessible predictor of response to therapy. Methods: TIL were isolated from breast tumour samples using a combined enzymatic and mechanical isolation protocol. They were subsequently expanded using anti-CD3/CD28 paramagnetic beads and high dose IL2. A comprehensive flow cytometry assay was designed and used to examine the expression of immune checkpoint proteins on isolated TIL and additionally cytokine potential of expanded TIL. The anti-cancer functionality of breast cancer TIL was assessed by co-culture of TIL and autologous tumour and subsequent ELISA analysis of the supernatant. To investigate the peripheral T cell compartment of patients undergoing NACT, peripheral blood samples were taken throughout the course of treatment and analysed by flow cytometry. Results: Following optimisation of the protocol, TIL were successfully expanded from 91.1% of breast cancer tumour samples. Phenotypic analysis of isolated (day 0) TIL revealed low expression frequency of the inhibitory immune checkpoint markers CTLA4, LAG 3 and TIM 3. Expression of PD1 correlated with markers of T cell activation. Expanded TIL subjected to phenotypic analysis demonstrated retained potential cytokine production. In CD8 positive TIL, PD1 positivity selected for cells capable of an increased mean number of cytokines/cell when compared to PD1 negative cells. Co-culture of TIL and autologous tumour demonstrated anti-cancer functionality in 2/7 samples. Concerns over tumour cell viability led to the development of a successful, novel in vitro organoid co-culture model. Finally, investigation of the peripheral T cell population of treatment naïve breast cancer patients demonstrated a pattern of expression of immune checkpoint molecules consistent with systemic immunosuppression. Additionally the cytokine IFN was found to be expressed in a significantly smaller percentage of CD8 positive peripheral T cells in patients who progressed through NACT, suggesting it will be a useful biomarker of response to systemic therapy. Conclusion: This thesis has developed a successful ex vivo expansion protocol for breast cancer TIL and demonstrates feasibility of an ACT therapy approach. This thesis also investigated the expression of immune checkpoint proteins on TIL and correlated this with functionality. Notably, PD1 is not a marker of exhausted T cells, and expanded TIL retain cytotoxic potential. It is anticipated that this, alongside the optimised TIL expansion protocol will guide future research, and translate into a successful clinical ACT strategy. Additionally, IFN expression of peripheral T cells is a potential biomarker of response to NACT, providing the rationale for a larger study to validate this novel finding.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Anne Armstrong (Supervisor), Robert Clarke (Supervisor) & Gray Kueberuwa (Supervisor)