Rampotas, Alexandros; (2024) Towards a CAR-T cell therapy approach for Calreticulin mutated Myelofibrosis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mutant calreticulin (mutCALR), a key oncogene in approximately one-third of essential thrombocythemia and myelofibrosis cases, presents a viable target for immunotherapy, interacting aberrantly with the thrombopoietin receptor (TPO-R) to activate it, it can be targeted as a cancer neoantigen on TPO-R expressing cells. Recent advances have seen therapeutic antibodies and bispecific T-cell engagers against mutCALR enter clinical trials. In this thesis, we report on the pre-clinical validation of a novel second-generation, 4-1BB chimeric antigen receptor T-cell (CAR-T) therapy targeting mutCALR-driven myelofibrosis. We identified a binding domain targeting the common mutant C-terminus of mutCALR, confirmed through immunohistochemical staining of myelofibrosis bone marrow biopsies and cell lines bearing mutCALR. Low and high expressing mutCALR cell lines, alongside patient peripheral blood samples, were utilized to test the CAR-T’s efficacy in 2D liquid cultures and 3D bone marrow organoids, simulating the myelofibrotic environment. For in vivo assessments, NSG mice were inoculated with mutCALR+ luciferase+ MARIMO cells, and therapeutic success was monitored via bioluminescent imaging. The CAR-T effectively eradicated both low and high mutCALR expressing cell lines, including a MARIMO line from acute myeloid leukaemia and a megakaryocytic UT7-TPO line expressing the del52 mutCALR variant. Of note, it selectively depleted 50-75% of CD34+ stem/progenitor cells from chronic-phase myelofibrosis patients, displaying high specificity with no observed off-target effects. Variability in cytotoxicity was noted, particularly in samples from accelerated/blast phase disease, likely due to differences in mutCALR-TPO-R expression. In vivo, CAR-T significantly reduced leukemic burden and improved survival in NSG mice xenografted with mutCALR+ TpoR+ MARIMO cells, with extensive CAR-T infiltration and tumour ablation confirmed in bone marrow. In summary, we have developed a pioneering CAR-T therapy targeting mutCALR-driven myelofibrosis, demonstrating potent efficacy in challenging myelofibrotic environments. Ongoing studies will further explore the therapeutic potential of this CAR-T in ameliorating disease features, aiming towards future clinical trials.

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