Qing, Chen; (2023) Investigating secondary resistance to immunotherapy in a murine melanoma model: characterisation and potential therapeutic strategies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Immunotherapy has transformed the clinical landscape in oncology. However, the majority of responding patients experience a period of stable disease before progressing. The mechanisms underlying this process of secondary resistance are not well known. We set out to explore mechanisms of secondary resistance, using a murine model of melanoma treated with a novel non-IL-2 blocking regulatory T cell (Treg)-depleting antibody (anti-CD25NIB) combined with a cancer cell vaccine (GVAX). We profiled the activation and differentiation landscape of infiltrating T cells in this model via single-cell RNA sequencing (scRNAseq) and flow cytometry. Combined anti-CD25/GVAX therapy resulted in three different clinical response patterns – no response, partial response, and secondary resistance, with characteristic immune phenotypes. 90% of partially responsive tumours relapsed between day 35-45. Reasoning that loss of immune control precedes clinical progression, we characterised the evolution of the immune landscape in pre-relapse tumours and found a decrease in the abundance of 4-1BB+TIM-3+TCF1- CD8+ effector T cells (Teffs) and Ki67+ CD4 effector cells. Prior to relapse, there was a simultaneous increase in resting TCF1+ and Treg cells. ScRNAseq and scTCRseq analyses of pre-relapse and relapse tumours revealed that resting CD4 Teffs accumulating at relapse were derived from previously activated effector cells. In contrast, little overlap in CDR3 usage was found between CD8 activated and resting populations at relapse, indicating that the accumulating resting CD8 T population represented newly entered, likely non-reactive clones. Additionally, we observed that in pre-relapse tumours, the percentage of Fas+ cells in activated Teffs was higher than that in resting Teffs. Blocking Fas-Fasl interactions with an anti-Fasl antibody synergised with anti-CD25NIB to prevent relapse. In conclusion, combined Treg depletion and tumour vaccine therapy is effective in a poorly infiltrated B16 model. Most mice that achieve partial response eventually relapse, mimicking what is often seen in human disease. By characterising the evolution of the immune landscape within partially controlled tumours, we revealed that progression is associated with a loss of immune fitness characterised by deactivation and death of activated infiltrating Teffs that can be partly intercepted by Fas-Fasl blockade.

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