Burke, Christina Lynne; (2023) Engineering cellular therapy for rhabdomyosarcoma: Design of CAR T-cells and characterisation of the tumour immune microenvironment. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and young adults. Despite this, the prognosis remains dismal for patients with high-risk disease, with fewer than 30% achieving cure. The aim of this work was therefore to develop a chimeric antigen receptor (CAR) T-cell immunotherapy for RMS, to serve as a more effective and tolerable treatment option. Six candidate surface target antigens were investigated for targeting with a CAR. Notably, B7-H3 was homogeneously expressed on five of five RMS cell lines and four of four primary RMS samples tested. Five anti-B7-H3 scFv binders generated by collaborators and their derivatives were tested for binding to RMS and reference cell lines, from which candidate CAR T-cells were generated. The CAR “A10” showed significant activity in vitro, achieving between 43.0 and 66.5% specific cell lysis of RMS cell lines after four hours of co-culture in a chromium release cytotoxicity assay. In addition, appreciable inflammatory cytokine secretion was observed against RMS targets in vitro. A second antigen, the foetal acetylcholine receptor (fAChR) was also taken forward for targeting with a CAR. Though highly selective for RMS, a significantly lower inflammatory and cytotoxic response was observed from anti-fAChR CAR T-cells when compared with the anti-B7-H3 CAR. Mechanisms of immunosuppression within the solid tumour microenvironment (TME) may dampen CAR T-cell efficacy. Presented here also is an interrogation of the RMS tumour immune microenvironment using complementary scRNA-seq and Chip Cytometry approaches, the latter being the first multiplex immunofluorescence study performed on RMS. Findings in a preliminary cohort of seven RMS tumours indicate a myeloid cell-dominated TME and absence of T-cell infiltrate, with the exception of paratesticular tumours. The results published herein are ultimately combined to give recommendations for the design of a next-generation CAR for treating RMS, which will be the focus of future studies.

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