A Murine Bispecific Antibody Efficiently Redirects T Cells Against <i>Calr</i> Mutated Stem Cells In Vivo

Calreticulin (CALR) mutations are prevalent in 20%-30% of patients with BCR::ABL1-negative myeloproliferative neoplasms (MPN). Mutant calreticulin (mutCALR), presented by the thrombopoietin receptor (MPL, also known as TPOR or CD110) on the surface of the disease-initiating MPN progenitors, represents an ideal target for curative immunotherapies including monoclonal antibodies, bispecific T cell engaging antibodies (TCE), and CAR-T cell therapies. Despite that two clinical TCE candidates have advanced into phase 1 trials in recent 2 years, depletion of mutCALR+ hematopoietic stem cells and normalization of hematopoiesis remained absent in preclinical evaluation. Here, we developed a bispecific T cell engager DX1-2C11 that specifically and efficiently eradicates mutCALR-expressing cells via recruiting polyclonal T cells. DX1-2C11 depleted Ba/F3 cells expressing mutCALR, as well as primary murine myeloid cells in a dose-dependent manner in vitro. In CALRdel52 transgenic mice, a single dose of DX1-2C11 activated CD4⁺ and CD8⁺ T cells in the peripheral blood, spleen and bone marrow within 24 h. Furthermore, a single dose of DX1-2C11 reduced platelet counts in the periphery and decreased mutant stem/progenitor cell populations in the spleen and bone marrow by Day 7 posttreatment. Notably, the reduction of mutant burden was durably maintained in secondary recipient mice. In the disseminated NSG model, DX1-2C11 delivered immediate tumor burden reduction and significantly prolonged the overall survival of mice compared to the control group. Taken together, these data suggest that bispecific T cell engaging antibody targeting mutCALR represents a curative strategy that efficiently eliminates mutant MPN stem cells in vivo.