# A Murine Bispecific Antibody Efficiently Redirects T Cells Against Calr Mutated Stem Cells In Vivo

## TL;DR

A new bispecific antibody efficiently targets and eliminates mutated stem cells in a mouse model of myeloproliferative neoplasms.

## Contribution

A bispecific T cell engager (DX1-2C11) is developed that effectively targets and depletes calreticulin-mutated stem cells in vivo.

## Key findings

- DX1-2C11 efficiently depletes mutCALR+ cells in vitro and activates T cells in multiple organs within 24 hours.
- A single dose of DX1-2C11 reduces platelet counts and mutant stem/progenitor cells in spleen and bone marrow.
- DX1-2C11 significantly prolongs survival in a disseminated mouse model of MPN.

## Abstract

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.