Discovery of first-in-class calr-targeted precision ADCs delivering a CDK9degrader payload for the treatment of calr-mutated MPNs Free

Myeloproliferative neoplasms (MPNs) are driven by clonal hematopoietic stem cells (HSCs), yet current therapies primarily address symptoms like splenomegaly and fatigue without targeting the malignant progenitors underlying disease. As a result, patients remain at risk for disease progression, thrombotic events, and transformation to secondary AML. There is a clear need for therapies that can eradicate the disease-initiating HSCs and deliver true disease modification.

Somatic mutations in JAK2, CALR, and MPL account for over 90% of MPN cases. CALR mutations, seen in 25–35% of patients, are typically exon 9 frameshifts that create a novel, positively charged C-terminus lacking the ER retention signal (KDEL). Mutant CALR binds MPL in the ER and traffics to the cell surface, where it is selectively presented as a neoantigen on malignant cells and induces downstream JAK/STAT signaling. This unique surface expression makes mutant CALR a promising target for monoclonal antibodies and antibody-drug conjugates (ADCs). Recent proof-of-concept data demonstrating clinical efficacy in essential thrombocythemia patients following treatment with a CALR-targeted monoclonal antibody validates the therapeutic potential of targeting this neoantigen.

Here, we report the development of a novel precision antibody-drug conjugate (pADC), designed to selectively bind mutant CALR and deliver a proprietary CDK9 degrader payload. CDK9 plays a critical role in transcriptional elongation and cell survival in hematologic malignancies, including MPNs. While pharmacologic CDK9 inhibition has shown clinical efficacy in myeloid disorders, its use is limited by on-target hematopoietic toxicity with non-targeted systemic delivery. We hypothesized that targeted delivery of a highly potent CDK9 degrader payload via a CALR-directed ADC would enable selective elimination of CALR-mutant HSCs, while sparing healthy hematopoietic cells and progenitors.

An antibody screened for selective binding and internalization in mutant-CALR cells, with no activity in WT CALR cells, was conjugated to a highly potent, picomolar CDK9 degrader using a cleavable linker designed for efficient intracellular release. The resulting CALR x CDK9 pADC selectively bound and internalized in CALR mutant cell lines and patient samples with no detectable uptake in CALR WT or healthy donor cells. Treatment with the CALR × CDK9 pADC led to selective CDK9 degradation and robust cytotoxicity in CALR-mutant cells, achieving excellent selectivity compared to WT CALR and JAK2V617F cells. Importantly, the pADC demonstrated equipotent activity against both Type I (del52) and Type II (ins5) CALR mutant cells. In primary ex vivo assays, treatment with the pADC suppressed proliferation and expansion of CALR-mutant HSCs, while sparing CD34+ HSCs from healthy and JAK2V617F donors. Inhibition of CALR mutant HSC proliferation was accompanied by a reduction in malignant megakaryocytic/platelet differentiation, with no observed effect on megakaryopoiesis of healthy CD34+ progenitors. In colony-forming assays, the pADC significantly reduced colony formation from CALR mutant MPN patient cells while sparing healthy donor cells. Additionally, we observed a reduction in mutant CALR variant allele frequency (VAF) in primary disease samples following treatment, underscoring its potential for disease modification.

In in vivo models, pADC administration resulted in strong suppression of disease burden, reduced splenomegaly, normalization of pro-inflammatory cytokines, and an extended survival benefit. Treatment was well tolerated, with no off-target effects observed in healthy hematopoietic cells.

In summary, we describe a first-in-class CALR-targeted ADC that delivers a novel CDK9 degrader payload, selectively eliminating CALR-mutant MPN progenitors. These results support further development of a mutant CALR x CDK9 degrader pADC as a potential disease-modifying therapeutic for CALR-mutant MPNs.