Paper Details

ABSTRACT

Background: Myeloproliferative neoplasms (MPNs) arise from the acquisition of mutations in JAK2, CALR or MPL, causing oncogenic JAK-STAT activation and megakaryocyte (MK)-biased haematopoiesis. In Essential Thrombocythemia (ET), excess thrombopoiesis dominates, leading to elevated platelet counts. Symptoms include fatigue, headaches and dizziness with significant morbidity and mortality due to vascular events and a risk of progression to myelofibrosis and blast phase MPN. Platelet count control is a?key goal in ET but current therapies (hydroxyurea, interferon) are non-targeted cytoreductive agents offering slow, partial disease control often due to cytopenias. Recent clinical data show that antibodies against mutant CALR protein may safely control platelets, but this approach is irrelevant to the 70-80% of ET patients without a CALR mutation. 

Aim: To develop a mutation-agnostic targeted therapy that can safely control platelet levels and deliver disease modification in ET. 

Methods & Results: To discover MPN-selective targets samples from >100 MPN patients (CALR & JAK2 mutant) and matched healthy donors were analysed using parallel single cell genotyping/transcriptomics (TARGET-seq), droplet scRNAseq (10x) and cell surface proteomics (flow cytometry, CyTOF). Machine learning algorithms were developed to use this data to select surface proteins suitable for antibody targeting of mutant cells. This identified G6B as a mutation-agnostic cell surface antigen, expressed by mutant but not wild-type hematopoietic stem cells (HSCs) and overexpressed in MPN patient MK/platelet lineage cells. We concluded that G6B is an attractive target for disease control in MPNs – with potential for antibody-mediated platelet count control and clearance of fibrosis-driving MKs and mutant HSCs. 

Antibodies against G6B were generated by mouse inoculation, multiple rounds of phage-library selection and cell screening. A specific, high affinity antibody, was identified and humanised to an IgG1 format. Selective binding was shown to human MPN cells, platelets and MKs. To enable in vivo mechanistic studies, a humanised murine model, generated by CRISPR modification of the extra-facial domain of the mouse G6B locus was generated that also showed specific binding to MKs and platelets. 

Next, we tested an array of Fc modifications to identify an attenuation approach that preserved antibody-mediated clearance while avoiding unwanted platelet aggregation and/or activation. In an in vitro assay for antibody-dependent cellular phagocytosis (ADCP), a LALA modification attenuated, but did not completely nullify, the response to G6B expressing cells, indicating that G6B+ cells may still be cleared by ADCP – yielding ATX-011. 

G6B is a regulator of MK maturation and platelet activation, however, ATX-011 did not directly induce activation or aggregation of human platelets, nor affect agonist-induced aggregation. Also, ATX-011 had no impact on MK maturation from induced pluripotent stem cells in vitro. Epitope mapping showed ATX-011’s binding site to be distal to the ligand binding site. Together these data show ATX-011 does not alter G6B function. 

The ability of ATX-011 to induce platelet clearance was tested in non-human primates. A rapid and pronounced platelet reduction was observed, with platelets reduced by 80% at 96 hr (n=2). An inverse correlation between antibody exposure and platelet count was seen with recovery of platelet levels upon antibody clearance. There was no effect on haemoglobin or white cell counts, indicative of platelet specificity. Notably, no changes to inflammatory cytokines, CD40L or D-dimer levels were observed, confirming no induction of platelet activation or coagulopathy in vivo. Instead, the kinetics of response suggest that ATX-011 enhances physiological reticulo-endothelial platelet clearance. Modelling based on primate data predicts that q3w-q4w dosing with ATX-011 will lead to sustained platelet normalisation in patients with ET. 

Summary: The rapid and robust platelet count reduction in the absence of toxicity in NHPs suggests that ATX-011 is a safe therapy that will enhance platelet clearance in patients with ET, regardless of driver mutation. Further, expression of G6B on fibrosis-driving MKs and MPN-initiating stem cells together with preservation of ADCP activity of ATX-011, supports its potential to deliver disease-modifying activity. Regulatory enabling studies are underway, and a Phase 1/2A study is planned for 2027.