Pharmacodynamic analysis of AGAVE-201 indicates changes in CSF-1R–expressing cells and associated biomarkers potentially contributing to chronic graft-versus-host disease resolution

Abstract Introduction: Chronic graft-versus-host disease (cGVHD) is a major cause of morbidity and nonrelapse mortality among patients who undergo allogeneic hematopoietic stem cell transplantation. In patients with cGVHD, colony-stimulating factor 1 receptor (CSF-1R)–dependent monocytes and macrophages potentiate inflammation and fibrosis, which contribute to multiorgan damage. Treatment with axatilimab (AXA), a high-affinity monoclonal antibody targeting CSF-1R, depletes CSF-1R–dependent monocytes and macrophages. Based on findings from the pivotal phase 2 AGAVE-201 study (NCT04710576), AXA dosed at 0.3 mg/kg every 2 weeks (Q2W) was approved by the US Food and Drug Administration for the treatment of cGVHD after failure of ≥2 prior lines of therapy. In AGAVE-201, an overall response (95% CI) occurred in 74% (63%–83%) of patients receiving AXA 0.3 mg/kg Q2W in the first 6 treatment cycles (primary endpoint), and AXA was generally well tolerated. Although higher doses of AXA evaluated in AGAVE-201 also achieved the primary endpoint of the study, the 0.3 mg/kg dose demonstrated a superior safety profile. Aims: To assess the pharmacodynamic (PD) changes in circulating monocytes and serum proteome of patients with cGVHD treated with AXA in AGAVE-201. Methods: AGAVE-201 enrolled 241 patients (0.3 mg/kg Q2W, n=80; 1 mg/kg Q2W, n=81; 3 mg/kg every 4 weeks, n=80). Whole blood and serum were collected at specific time points before and after infusion. Monocytes were analyzed by a qualified flow cytometry assay and were defined as CD45+ → CD14+ HLA-DR+, with monocyte subtypes defined as classical (CD14high/CD16low), nonclassical (NC; CD14low/CD16high), and intermediate (CD14high/CD16high). The CSF-1R ligands CSF-1 and IL-34 were quantitated by commercially available enzyme-linked immunosorbent assays. Broad proteomic analysis was performed using OLINK Explore HT consisting of >5000 proteins. Differentially expressed proteins (DEPs) in the 1 mg/kg and 3 mg/kg cohorts were identified at Cycle 1 Day 8 (C1D8) and Cycle 2 Day 1 (C2D1) and compared with baseline based on a fold change ≥1.5 and P≤0.05. Results: CSF-1 and IL-34 serum levels increased, and NC monocytes decreased in a time- and dose-dependent manner in patients treated with AXA. Interpretation of PD effects at the 0.3 mg/kg dose was limited due to infrequent sampling; however, consistent with prior studies, transient reductions in NC monocytes were observed at Day 3–4 after dose, returning to baseline by Day 8 in a healthy volunteer study conducted in Japan. Changes across the dose range were consistent with the extent of AXA exposure and clearance. Based on proteomic analysis of AGAVE-201 serum samples, DEPs were identified at 1 mg/kg (C1D8, 193 DEPs; C2D1, 47 DEPs) and 3 mg/kg (C1D8, 504 DEPs; C2D1, 304 DEPs) relative to baseline. Proteins impacted by AXA included soluble cellular markers expressed by CSF-1R positive cells, including monocytes, macrophages, and dendritic cells (DCs). CLEC10A (a C-type lectin family member implicated in immune surveillance, primarily found on immature myeloid DCs and alternatively activated macrophages) was significantly downregulated in a dose-dependent manner. Cell surface markers expressed by monocytes, including CD163 (a scavenger receptor) and CD300E (an activating receptor), were also significantly reduced by AXA. In addition, EBI3 (a subunit of IL-27 expressed by Kupffer cells, NC monocytes, and intermediate monocytes), SIGLEC-10 (an inhibitory receptor expressed on immune cells, including macrophages), and ECM1 (a product of fibroblasts and macrophages associated with inflammation and fibrosis), were all significantly reduced by AXA. Notably, several matrix proteins expressed in skin (keratin 5 and collagens type V and IX) were upregulated by AXA, with inverse correlations to skin tightening clinical scores, suggesting a potential mechanistic link to improvements in skin fibrosis.Conclusion: Proteomic analysis of AGAVE-201 revealed changes in soluble markers associated with CSF-1R–expressing cells and were consistent with a reduction in the number and cellular function of CSF-1R–regulated monocyte lineage, contributing to disease resolution. Biomarkers that correlate with skin tightening improvement may indicate potential involvement in cGVHD skin disease and/or resolution in response to AXA. These findings support the mechanism of action of AXA and provide hemodynamic evidence of target engagement and tissue-specific effects.