Gohil, Satyen Harish; (2018) Pre-clinical development of novel ROR1 chimeric antigen receptor T cells and bispecific T cell engagers. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Receptor tyrosine kinase like orphan receptor 1 (ROR1) is an onco-embryonic antigen present on a range of solid and haematological malignancies, including chronic lymphocytic leukaemia. Additionally, limited, low level expression on normal tissues makes it an attractive therapeutic target. Chimeric antigen receptor T cells and Bispecific T Cell Engagers have emerged as exciting immunotherapeutic approaches, utilising the inherent cytotoxic potential of autologous T cells to yield demonstrable benefit for patients. We therefore aimed to generate novel ROR1 CAR T cells and BiTEs. Following a rat ROR1 immunisation programme, we screened over 150 single cell hybridoma clones to isolate 13 novel antibodies of which 10 bound in a single chain variable fragment format. Iterative optimisation led to two lead candidates that imparted superior cytotoxicity and cytokine secretion. To minimise immunogenicity we screened 50 humanised ROR1 scFv variants and selected a final humanised candidate, hF(1x1), which maintained effector function, specificity and demonstrated broad applicability against a panel of cell lines representing various tumour subtypes. Focusing specifically on haematological cell lines and CLL, our humanised CAR demonstrates superior cytotoxicity compared to previously reported ROR1 constructs. However, low ROR1 antigen density limits efficacy in B cell malignancies, compared to CD19 CAR T cells and strategies to overcome this are in development. Our ROR1 BiTE mediates cytotoxicity at low concentrations (ng/ml) and effector to target ratios against cell lines, but in untreated and relapsed CLL patients, inherent T cell dysfunction limits efficacy. This can be overcome with the BTK inhibitor Ibrutinib, with T cells isolated from patients on treatment, showing markedly improved cytotoxicity against autologous CLL cells. This work has laid the preclinical foundations for translation of these novel agents into clinical trials for a range of tumours including CLL and many of which have high unmet therapeutic need.

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