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Updated: Oct 3, 2025
Engineering Oncogenic Heterozygous Gain-of-Function Mutations in Human Hematopoietic Stem and Progenitor Cells
Published on: March 10, 2023
Targeting human CALR-mutated MPN progenitors with a neoepitope-directed monoclonal antibody
Denis Tvorogov1, Chloe A L Thompson-Peach2,3, Johannes Foßelteder4
1Centre for Cancer Biology, SA Pathology and University of South Australia, Adelaide, SA, Australia.
Abstract:
Calreticulin (CALR) is recurrently mutated in myelofibrosis via a frameshift that removes an endoplasmic reticulum retention signal, creating a neoepitope potentially targetable by immunotherapeutic approaches. We developed a specific rat monoclonal IgG2α antibody, 4D7, directed against the common sequence encoded by both insertion and deletion mutations. 4D7 selectively bound to cells co-expressing mutant CALR and thrombopoietin receptor (TpoR) and blocked JAK-STAT signalling, TPO-independent proliferation and megakaryocyte differentiation of mutant CALR myelofibrosis progenitors by disrupting the binding of CALR dimers to TpoR. Importantly, 4D7 inhibited proliferation of patient samples with both insertion and deletion CALR mutations but not JAK2 V617F and prolonged survival in xenografted bone marrow models of mutant CALR-dependent myeloproliferation. Together, our data demonstrate a novel therapeutic approach to target a problematic disease driven by a recurrent somatic mutation that would normally be considered undruggable.
Insights
A novel antibody, 4D7, targets mutated Calreticulin (CALR) in myelofibrosis by blocking TpoR signaling. This approach inhibits cancer cell proliferation and improves survival in preclinical models.
Area of Science:
- Oncology
- Immunology
- Hematology
Background:
- Calreticulin (CALR) mutations are common in myelofibrosis, creating neoepitopes.
- These mutations lead to thrombopoietin receptor (TpoR) signaling and uncontrolled cell growth.
Purpose of the Study:
- To develop and evaluate a targeted antibody therapy against mutant CALR in myelofibrosis.
- To investigate the therapeutic potential of antibody 4D7 in preclinical models.
Main Methods:
- Development of a rat monoclonal antibody (4D7) against mutant CALR neoepitopes.
- Assessment of 4D7 binding, JAK-STAT signaling inhibition, and anti-proliferative effects in cell lines and patient samples.
- Evaluation of 4D7 efficacy in xenografted bone marrow models.
Main Results:
- Antibody 4D7 selectively targets cells with mutant CALR and TpoR.
- 4D7 inhibits TPO-independent proliferation and megakaryocyte differentiation by disrupting CALR dimer binding to TpoR.
- 4D7 demonstrated efficacy in patient samples with CALR mutations and prolonged survival in preclinical models.
Conclusions:
- Antibody 4D7 represents a novel therapeutic strategy for CALR-mutated myelofibrosis.
- This approach targets a previously undruggable mutation, offering a new avenue for treating myelofibrosis.
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