Evaluating the Effect of BTK Inhibitor Tolebrutinib in Human Tri-culture
Ross Gruber1, Anna Blazier2, Lan Lee2, Sean Ryan2, Agnes Cheong2, Evis Havari2, Timothy Turner2, Dimitry Ofengeim2
1Sanofi Genzyme, 2Sanofi
Objective:
Characterize pharmacological properties of the CNS-penetrant Bruton’s tyrosine kinase (BTK) inhibitor tolebrutinib in human neural cells by using a human tri-culture system.
Background:

 Neuroinflammation in the brain and spinal cord, a process driven largely by CNS-resident microglia, has been proposed as a significant contributor to disability accumulation in patients with multiple sclerosis (MS). BTK is expressed in microglia, as well as B lymphocytes and monocytes/macrophages found in the periphery. Previous immunohistochemistry studies and single-nucleus RNA sequencing (snRNAseq) from tissues derived from autopsy samples demonstrated that BTK was expressed in B cells and in microglial cells, with increased levels in MS lesion samples. BTK inhibition may provide therapeutic benefit within the CNS by targeting innate immunity associated with MS disease progression.

Design/Methods:

Immunofluorescence staining and RNAseq in human tri-culture cells. Cytokine secretion assays were performed using enzyme-linked immunosorbent assay (ELISA).

Results:
Transcriptome analysis and ELISA were used to identify a BTK-dependent transcriptional signature and secreted cytokine signature in human induced pluripotent stem cell (iPSC)-derived tri-culture consisting of human neurons, astrocytes, and microglia. RNAseq data illustrated a tolebrutinib-specific transcriptomic signature in human tri-culture cells and a complex culture of neurons, astrocytes, and microglia, indicating that modulation of BTK in these cells can impact neuroinflammatory pathways relevant to MS. To that end, we examined RNAseq data of human iPSC-derived tri-culture and found that tolebrutinib can modulate microglial genes and have non-cell autonomous impacts on neurons and astrocytes during stimulation of fragment crystallizable gamma receptor (FcγR).
Conclusions:

We extend our previous findings on the role of BTK in microglia to show that BTK-dependent inflammatory signalling in human microglia and tri-cultures can be modulated using tolebrutinib in vitro. Our work contributes to improved understanding of BTK signalling in neuroinflammation and how BTK inhibitors could target the neuroinflammation thought to contribute to MS disability progression.