Axatilimab Abrogates Inflammatory Cytokines and Chemokines and Interrupts the Differentiation of Monocytes to Macrophages, a Pathogenic Driver of Inflammation and Fibrosis in Cghvd

Introduction: The pathophysiology of chronic graft-versus-host disease (cGVHD) involves at least 3 distinct overlapping processes: tissue damage and inflammation, dysregulation of adaptive immunity leading to chronic inflammation, and fibrosis and end-organ damage. The role of monocyte-derived macrophages as the pathogenic driver of fibrosis and inflammation in cGVHD has been previously described. Colony-stimulating factor 1 receptor (CSF-1R) is a member of the receptor protein tyrosine kinase family of growth factor receptors that has been shown to be the primary pathway regulating the development, differentiation, and function of monocytes and macrophages. Axatilimab is a high-affinity, humanized immunoglobulin G4 monoclonal antibody that targets CSF-1R, thereby blocking CSF-1 signaling, that is under clinical investigation for the treatment of cGVHD. Results from 2 clinical trials (NCT03604692; NCT04710576) have demonstrated the efficacy and tolerability of axatilimab for cGVHD, reducing the symptom burden for most patients, including those with fibrotic cGVHD manifestations. However, the effects of axatilimab on monocytes and macrophages need further investigation.

Methods: The mechanism of action of axatilimab was characterized using in vitro assays with primary human monocytes and macrophages, as well as a human CSF-1R knock-in mouse model. Expression of CSF-1R and monocyte chemoattractant protein (MCP-1) secretion (downstream of CSF-1R) were evaluated. Next, the effects of axatilimab on monocyte-to-macrophage differentiation and on monocyte-derived macrophages were assessed for inflammatory chemokines and cytokine secretion. Purified monocytes (CD14⁺) were cultured with macrophage CSF (M-CSF) and treated with axatilimab for 24 and 72 hours. To assess the effect of axatilimab on monocyte-derived macrophages, monocytes were differentiated into macrophages with M-CSF for 5 days and treated with axatilimab for 72 hours with appropriate controls. Supernatants from these cultures were evaluated with an 80-plex Luminex assay. To evaluate the in vivo effects of axatilimab on circulating blood monocytes, human CSF-1R knock-in mice were treated with a range of axatilimab doses (0.3-30 mg/kg) for different time intervals. A comprehensive flow cytometry panel was used to assess monocytes/macrophages in a human CSF-1R knock-in mouse model at different time points after axatilimab treatment.

Results: Axatilimab treatment of human monocytes for 72 hours resulted in impaired cell survival. Shorter treatment for 24 hours, which did not impair monocyte viability, resulted in inhibition of MCP-1 secretion, a key mediator of immune cell chemotaxis and fibrosis. In addition, axatilimab treatment led to dose-dependent inhibition of other inflammatory cytokines. Specifically, significant dose-dependent inhibition of macrophage inflammatory protein (MIP)-1α, MIP-1β, C-X-C motif chemokine ligand (CXCL)1, CXCL6, MCP-3, and MCP-2 secretion was observed in monocytes treated with axatilimab. Axatilimab treatment of monocyte-derived macrophages inhibited MIP-1α, MIP-1β, MIP-2α, interleukin 8 (IL-8), CXCL5, and MCP-1. Notably, MCP-1 and IL-8 are key regulators of tumor growth factor beta (TGF-β) and are all implicated in fibrotic disease progression. Axatilimab treatment of human CSF-1R knock-in mice led to reductions in the number of monocytes (CD45⁺ CD11b⁺) in the blood and peritoneal lavages without reducing the total leukocyte population.

Conclusions: Taken together, these data suggest that blocking CSF-1/CSF-1R signaling by axatilimab reduces proinflammatory signaling in monocytes and monocyte-derived macrophages and specifically reduces the number of monocytes and macrophages in the peripheral blood and peritoneal lavages of human CSF-1R knock-in mice. These data support the observation from clinical studies (phase 1/2 and pivotal AGAVE-201 study) that nonclassical monocytes were depleted in patients with cGVHD treated with axatilimab.

Bajpai:Incyte Corporation: Current Employment, Current holder of stock options in a privately-held company. Solomon:Incyte Corporation: Current Employment, Current holder of stock options in a privately-held company. Volkova:Incyte Corporation: Current Employment, Current holder of stock options in a privately-held company. Parker:Incyte Corporation: Current Employment, Current holder of stock options in a privately-held company. Kearns:Incyte Corporation: Current Employment, Current holder of stock options in a privately-held company. Cosby:Incyte Corporation: Current Employment, Current holder of stock options in a privately-held company. Kanellopoulou:Incyte Corporation: Current Employment, Current holder of stock options in a privately-held company.