Targeted T Cells Against Oncogenic Calreticulin Mutants

Blood cancer are caused by acquired genetic alterations of blood cells that lead to their increased production which often can evolve to a stage with life-threatening complications (blood clotting, organ damage, bleeding). This proposal is aimed at those blood cancers that are caused by calreticulin (CALR) gene mutations, namely myeloproliferative neoplasms. The nature of the mutation and the mechanisms how CALR oncogenes work make it an ideal target for immunotherapy. This proposal aims to engineer T lymphocytes (immune cells capable of cancer cell killing) that can selectively find, connect to and kill cancerous blood cells and leave the healthy cells unharmed. We will also study the mechanisms how cancer cells could escape T cell killing. The long-term aim of the project is to lay the foundations for immunotherapy for this type of blood cancer.

Myeloproliferative neoplasms (MPN) are chronic blood cancers characterized by increased blood cell production, thromboembolic complications and evolution to acute leukemia. MPN oncogenesis is driven by acquired hematopoietic stem cell mutations in three genes (JAK2, CALR and MPL), which all trigger constitutive activation of cytokine receptors and downstream signaling. In this project, we focused on MPN driven by CALR mutations that translate into an oncoprotein with unique antigenic properties. CALR mutations enable unique interactions of mutant with the thrombopoietin receptor (MPL). The activated mutCALR-MPL complex is transported to the cell surface of the mutated cells. Hence, it constitutes a cancer specific antigen. In this proposal, we aimed to exploit the presence of mutant CALR on the surface of malignant cells and egineered immune cells (T lymphocytes) specifically recognizing mutant CALR via a chimeric antigen receptor (CAR). These CAR-T cells specifically killed CALR mutated blood cells isolated from MPN patients as well as CALR mutated mouse blood cells. To validate the potential clinical utility of these CAR-T cells, we treated mouse models of CALR mutated MPN. Despite the effective killing of cancer cells in vitro (in cell cultures) we did not observe any killing when the CAR-T cells were administered to mice. We subsequently found out that the CAR-T cells were inactivated by the secreted mutant CALR produced by the cancer cells. We also detected the secretion of mutant CALR in MPN patients. To overcome the inhibition by secreted CALR, we designed a antibody that binds both the T cells and the CALR mutated cancer cells. These bis-specific antibodies deliver T cell dependent cancer cell killing in vitro and in vivo in mice. Our results demonstrate, that T cell redirection by bi-specific anti-CALR/anti-CD3e antibodies, rather than genetically engineered anti-CALR CAR-T cells, is the preferred immunotherapy approach in CALR mutated MPN. Our results hold promise for specific immunotherapy of CALR mutated blood cancers applicable in a clinical setting.