Molecular characterization of patients (pts) with myeloproliferative neoplasms treated with INCA033989 demonstrates selective targeting of CALR mutant hematopoietic cells

Abstract Background: Essential thrombocythemia (ET) and myelofibrosis (MF) are driven by mutations in calreticulin (mutCALR) in ~30% of patients (pts) and result in substantial morbidity and mortality. INCA033989 (‘989‘), a novel, fully human, Fc-silenced, IgG1 monoclonal antibody, selectively inhibits oncogenic signaling and proliferation of cells expressing mutCALR and thrombopoietin receptor. This mechanism of action has the potential to selectively eliminate malignant cells and restore healthy hematopoiesis. 989 is currently being studied in 2 phase 1 first-in-human trials (NCT05936359; NCT06034002) in pts with ET or MF. This analysis describes the baseline molecular characteristics and preliminary molecular responses to 989. Methods: Comprehensive clinicogenomics and histological analyses were undertaken using 3 approaches. A targeted NGS panel (Archer® VariantPlex® Core Myeloid panel) containing 37 genes associated with myeloid malignancies was used to analyze whole blood and bone marrow aspirate samples at enrollment and longitudinally to characterize the somatic mutation landscape and molecular dynamics following treatment. Molecular response (MR) was defined as a 20% reduction (best overall response) in mutCALR VAF between baseline and post-treatment. In addition, single-cell multi-omic analysis (TapestriTM) was conducted in a subset of pts on peripheral blood mononuclear cells using a custom panel to assess 47 cell surface proteins and 37 genes. Immunohistochemistry (IHC) using an antibody specific for mutCALR was performed on bone marrow biopsies at enrollment and post-treatment. Semi-automated annotation of mutCALR-positive and negative megakaryocytes (MKs) was performed, and counts were normalized to the total MK population determined by nuclei count. Data cutoff was based on a biospecimen collection date of May 2025. Results: 110 enrolled pts had evaluable genomic data, 51 and 59 with ET and MF, respectively. The frequencies of pts with mutCALR Type-1, Type-2, Type-other were 55%, 29%, 16% (ET) and 58%, 24%, 19% (MF), respectively. The mean (range) of mutCALR VAF at enrollment for ET was 0.33 (0.12-0.75) and MF was 0.38 (0.30-0.85). Only 5 pts (5%) had a VAF >0.5 suggesting loss of heterozygosity, and all had Type-2 mutations. A strong correlation was noted between mutCALR VAF of bone marrow and peripheral blood samples (R2=0.97). Co-occurring (nondriver) somatic variants were identified in 33% of ET pts and 83% of MF pts. The range for co-occurring variants per pt was 0-3 for ET and 0-9 for MF. The most frequently mutated gene in ET pts was TET2 (16%), while ASXL1 (42%), TET2 (31%), and EZH2 (12%) were frequent in MF. In ET, a hematologic response at any time occurred in 41/51 pts (80%) with longitudinal VAF data available for 39 of the 41 patients. Of these 39 patients, 37 (95%) had a reduction of mutCALR VAF (range 2%-91%) and 22 (56%) achieved MR. 16/41 (39%) achieved an IWG/ELN-defined durable peripheral blood count remission. Of these 16 patients, 14 (88%) achieved MR and their baseline VAF ranged from 0.22 to 0.34. In MF, 27 pts had evaluable spleen response and longitudinal VAF data available. Of these 27 pts, 10 (37%) had at least a 25% spleen volume reduction at week 24; all 10 pts had a VAF reduction (range 6.9%-33%) and 5 achieved MR. 21 MF pts had evaluable anemia response and longitudinal VAF data. Of these 21 pts, 12 (57%) had a major or minor anemia response; all 12 pts had a VAF reduction (range 5%-27%) and 4 (33%) had MR. Single-cell analysis in a subset of ET and MF pts revealed selective reductions in mutCALR+ hemopoietic stem/progenitor cells (HSPCs) by the end of cycle 3. Bone marrow IHC analysis of ET and MF pts showed a decrease in the density of total MKs including a decrease in the percentage of mutCALR+ MKs and a concomitant increase in the percentage of mutCALR-negative MKs.Conclusions: Pts with MF had higher mutCALR VAF than pts with ET and were more likely to have co-occurring somatic mutations at enrollment. Correlation between clinical response and post-treatment VAF reduction was observed in both ET and MF, with more frequent MRs achieved in ET. Post-treatment MR occurred over a range of baseline VAFs, which included patients with co-occurring variant(s). A rapid and selective reduction in mutCALR+ HSPCs and MKs was observed within 4 months of 989 treatment and correlated with clinical responses, suggestive of potential disease modification and recovery of normal hematopoiesis.