Towards a CAR T-cell therapy approach for Calreticulin-mutated Myelofibrosis

Myelofibrosis (MF) is a malignant bone marrow disease, driven by a population of abnormal, clonal megakaryocyte progenitor cells. It has an incidence of 1 in 100,000 in the UK and is characterised by extensive bone marrow fibrosis which leads to ineffective blood production, anaemia, uncontrolled infection, bleeding and ultimately death. Further, 10-20% of MF patients will subsequently develop Acute Myeloid Leukaemia which is an urgent, life-threatening condition. Currently, there are no satisfactory treatments for MF. The only curative therapeutic approach is an allogeneic stem cell transplant (allo-SCT), which is only suitable for younger, medically fit patients, but even in this setting, it has a particularly poor outcome. Myelofibrosis is a neglected area with unmet medical need.

A T-cell is a specialised immune cell whose job is to seek out and kill virus infected cells with great specificity. By applying gene engineering techniques in the laboratory, it is now possible to redirect patient T-cells to recognise and kill their cancer cells with the same potency as they would naturally attack a viral infection. This is done using by inserting an artificial protein called a chimeric antigen receptor (CAR) into patient T-cells to create CAR T-cells. CAR T-cells represent a 'living drug', forming part of the patient's immune system, preventing recurrence. CAR T-cells are highly effective against blood cancers that do not respond to conventional therapies: in studies of patients with B-cell leukaemia, complete responses to CAR T-cell therapy can be observed in over 90% of cases and over 50% of children/young adults with leukaemia are 'cured' by a single dose.

The focus of this project is to develop CAR T-cells for MF, targeting the mutated cell surface protein mutCALR (mutated Calreticulin) on the malignant cells. mutCALR is found in 25% of all cases of MF and represents an excellent potential target for CAR T-cells. Our approach will be to develop an antibody targeting mutCALR using known laboratory techniques and to engineer this antibody into CAR format. We will then proceed to test the mutCALR CAR in a series of in vitro and in vivo settings to define how effective and safe it may be as a future therapy for MF.

We believe that CAR-T therapy can be a very good treatment option for patients with Myelofibrosis. The chronic nature of the disease means that there is ample time to manufacture personalised CAR-T cells which is a main problem in other malignancies that progress quickly. Additionally, it appears that in 25% of cases, the disease is driven by mutCALR on the surface of the malignant cells, such that targeted immunotherapy using CAR-T cells may eliminate the disease completely, whilst providing lasting tumour immunity.

We wish to develop a chimeric antigen receptor-T cell (CAR-T) therapy for patients with Myelofibrosis (MF). MF is a bone marrow disorder caused by a disordered megakaryocyte progenitor cell clone and results in early death and acute myeloid leukaemia. It is an unmet clinical need as the only available curative option is a stem cell transplant which carries high mortality and it is unfeasible for most patients.

Mutations in Calreticulin (CALR) have been identified as a disease driving mutation with oncogenic properties in 25% of MF cases. CALR mutations mutations cause a +1bp frame shift which results in an alternative reading frame translated into a novel C-terminus which includes the sequence 'SPARPRTSCREACLQGWTEA'. Also, the endoplasmic reticulum signal (KDEL motif) is no longer present which allows mutCALR to escape ER. This mutated CALR is translocated to the cell surface, where it leads to constitutive JAK2/STAT5 signaling via activation of the Thrombopoietin receptor (MPL/TpoR). Therefore, CALR may be an excellent target for CAR-T therapy.

Initially, we will develop an array of monoclonal antibodies against the novel sequence found in mutCALR by initiating a rat vaccination campaign. We will then validate its surface expression in HSCs and progenitor cells and measure its serum concentration. Once a successful monoclonal antibody has been developed, we will incorporate it into a CAR-T construct.

The optimal CAR-T cell construct in terms of the co-stimulation and other cytokine release will be determined by performing in vitro experiments and testing their efficacy against mutCALR MF HSCs. Successful CAR-T constructs will then be tested in vivo in MISTRG murine models who will be transduced with mutCALR HSCs from MF patients.