Clinical Trial Support in Myeloproliferative Neoplasms (MPNs) Therapy Development

Myeloproliferative neoplasms (MPNs) driven by somatic CALR exon 9 frameshifts represent approximately 25–35% of essential thrombocythemia (ET) and myelofibrosis (MF) cases. These mutations generate a neomorphic C-terminal neoepitope that aberrantly activates the thrombopoietin receptor (MPL/TPOR), driving megakaryocytic hyperproliferation. INCA033989 is a first-in-class monoclonal antibody, developed by Incyte Corporation, that selectively binds this mutant CALR neoepitope at the cell surface, sparing wild-type hematopoietic cells. A central challenge in characterizing the mechanism of action for INCA033989 was confirming that the therapy selectively eliminates mutCALR-expressing hematopoietic cells while preserving normal hematopoiesis in the same patient samples. Because no legacy assay, including bulk variant allele frequency (VAF) quantification, conventional flow cytometry, or standard next generation sequencing, can answer this question sufficiently, Mission Bio’s Tapestri® Platform provided this co-measurement capability through a purpose-built single-cell DNA and protein assay. A custom single-cell genotyping panel and a multiplexed cell-surface protein (antibody-oligo conjugate, AOC) immunophenotyping panel profiled each cell’s CALR mutation status and lineage identity simultaneously in peripheral blood mononuclear cells (PBMCs). The resulting data localized the mutCALR clone to the stem/progenitor and myeloid compartments, confirmed selective depletion of mutCALR+ cells in clinical responders with a concomitant shift toward wild-type hematopoiesis, and revealed subclonal co-mutation dynamics invisible to legacy method approaches. The INCA033989 molecule is now in clinical trials thanks to the initial single-cell multiomic characterization done on Tapestri. Learn more about how the therapy was characterized using the purpose-built single-cell assay developed by Mission Bio.

Audience and Use Case

This resource is designed for translational researchers and pharmaceutical developers evaluating therapeutic mechanisms of action, on-target selectivity, and clonal resistance profiles in myeloid malignancy clinical programs.

What You Get

Download the complete technical note to access full verification methodologies, assay design parameters, and lineage-resolved clinical trial data plots. Complete the brief form below to download the PDF asset.

FAQ

Q: Why are legacy bulk sequencing and conventional flow cytometry insufficient for evaluating myeloproliferative neoplasm mutant CALR-targeted therapies?

A: Bulk sequencing averages genetic signals across thousands of cells, masking how mutations are distributed across distinct lineages. Conventional flow cytometry identifies cell phenotypes but cannot read their genetic status. Only single-cell multiomics pairs genotype with immunophenotype in the exact same cell to confirm if mutant cells are specifically targeted while healthy cells are preserved.

Q: How does the single-cell assay architecture overcome the sequencing and coverage challenges associated with complex calreticulin indels seen in myeloproliferative neoplasm?

A: Large insertions and deletions within calreticulin exon 9 frequently cause coverage drops or dropouts in standard sequencing assays, compromising single-cell data quality. The purpose-built Tapestri platform overcomes this by utilizing a redesigned exon 9 amplicon specifically optimized to handle these complex frameshift variants. In performance validation, this targeted approach achieved highly robust genotyping efficiency for both Type 1 (52-bp deletion) and Type 2 (5-bp insertion) variants across profiled cells. This high coverage reliability ensures definitive cell-by-cell classification, allowing researchers to confidently differentiate mutant clones from wild-type backgrounds and map compound clones co-harboring additional high-risk somatic variants.

Q: Can this single-cell multiomic framework be applied to other myeloproliferative neoplasm targets?

A: Yes. The assay architecture is completely modular, allowing researchers to reconfigure the targeted genomic and cell-surface protein panels to match any driver mutation, high-risk co-mutation, or lineage hierarchy of interest across myeloid disease programs.