Remibrutinib (LOU064) inhibits neuroinflammation driven by B cells and myeloid cells in preclinical models of multiple sclerosis

Abstract

Background: Bruton's tyrosine kinase (BTK) is a key signaling node in B cell receptor (BCR) and Fc receptor (FcR) signaling. BTK inhibitors (BTKi) are an emerging oral treatment option for patients suffering from multiple sclerosis (MS). Remibrutinib (LOU064) is a potent, highly selective covalent BTKi with a promising preclinical and clinical profile for MS and other autoimmune or autoallergic indications.

Methods: The efficacy and mechanism of action of remibrutinib was assessed in two different experimental autoimmune encephalomyelitis (EAE) mouse models for MS. The impact of remibrutinib on B cell-driven EAE pathology was determined after immunization with human myelin oligodendrocyte glycoprotein (HuMOG). The efficacy on myeloid cell and microglia driven neuroinflammation was determined in the RatMOG EAE. In addition, we assessed the relationship of efficacy to BTK occupancy in tissue, ex vivo T cell response, as well as single cell RNA-sequencing (scRNA-seq) in brain and spinal cord tissue.

Results: Remibrutinib inhibited B cell-dependent HuMOG EAE in dose-dependent manner and strongly reduced neurological symptoms. At the efficacious oral dose of 30 mg/kg, remibrutinib showed strong BTK occupancy in the peripheral immune organs and in the brain of EAE mice. Ex vivo MOG-specific T cell recall response was reduced, but not polyclonal T cell response, indicating absence of non-specific T cell inhibition. Remibrutinib also inhibited RatMOG EAE, suggesting that myeloid cell and microglia inhibition contribute to its efficacy in EAE. Remibrutinib did not reduce B cells, total Ig levels nor MOG-specific antibody response. In brain and spinal cord tissue a clear anti-inflammatory effect in microglia was detected by scRNA-seq. Finally, remibrutinib showed potent inhibition of in vitro immune complex-driven inflammatory response in human microglia.

Conclusion: Remibrutinib inhibited EAE models by a two-pronged mechanism based on inhibition of pathogenic B cell autoreactivity, as well as direct anti-inflammatory effects in microglia. Remibrutinib showed efficacy in both models in absence of direct B cell depletion, broad T cell inhibition or reduction of total Ig levels. These findings support the view that remibrutinib may represent a novel treatment option for patients with MS.

Keywords: Autoimmunity; BTK; LOU064; Multiple sclerosis; Neuroinflammation; Remibrutinib.

Fig. 1

Remibrutinib inhibits HuMOG EAE. Dosing was started on day of immunization and continued to study end. a The 30 mg/kg dose showed statistically significant efficacy from day 11 onward (Kruskal–Wallis with Dunn’s test, n = 8–10 per group, means with standard errors). Peak BTK occupancy was assessed in b spleen, c lymph nodes and d brain homogenates 1, 5 and 8 h after the last dose. Shown are the BTK occupancy levels of individual animals and the group means with standard deviations as whiskers. Statistical significance of the 30 mg/kg dose group reached p < 0.05 from day 11 to study end (*) and p < 0.01 between days 13 and 23 (**) vs vehicle treatment (ANOVA followed by Dunnett’s test, mean ± , SEM, n = 5)

Fig. 2

Remibrutinib inhibits huMOG specific, but not polyclonal T cell recall response. T cell recall responses were assessed 8 days after immunization with HuMOG antigen and remibrutinib dosing. Isolated splenocytes and draining lymph node cells were incubated in vitro with HuMOG for 48 h. Antigen-specific proliferation was determined by [3H]-thymidine incorporation. Remibrutinib-treated animals showed a dose-dependent significant reduction in HuMOG-induced proliferation of a spleen and b lymph node cells. c Polyclonal stimulation with anti-CD3/CD28 was not significantly affected by remibrutinib treatment. Statistical analysis vs vehicle treatment (ANOVA followed by Dunnett’s test (mean ± , SEM, n = 5, *** for p < 0.001, ** for p < 0.01 and * for p < 0.05)

Fig. 3

Remibrutinib does not deplete B cells in HuMOG EAE, but shows trend for reduced Th17 T cell frequencies. Mice immunized with HumanMOG showed no significant changes in the frequencies of a CD19 B cells or b CD4 T cells analyzed in the total lymphocyte gate in spleen, blood or lymphnodes (LN). In contrast, c PMA/ionomycin induced intracellular IL-17 staining revealed a trend for reduced Th17 CD4 T cell frequencies with a p value of 0.057 and 0.072 for 3 and 30 mg/kg dose, respectively. (Mean ± , n = 5, two way ANOVA followed by Dunnett’s test.)

Fig. 4

Remibrutinib inhibits RatMOG EAE. a Dosing of 30 mg/kg p.o. b.i.d. remibrutinib significantly reduced RatMOG EAE scores from day 12 onward (p < 0.05). Group sizes n = 10 per treatment. Statistical significance determined using Kruskal–Wallis with Dunn’s test (means with standard errors). b Trough BTK occupancy was assessed 16 h after the last dose in spleen, blood and brain homogenate (p < 0.001). Statistical significance determined by ANOVA and Sidak’s test (LOU  remibrutinib, VEH vehicle)

Fig. 5

Remibrutinib reduced serum NfL levels in RatMOG EAE. Treatment with remibrutinib showed a trend for reduced serum NfL levels compared to the vehicle treatment group (means with standard errors, p = 0.172, t-test)

Fig. 6

RatMOG EAE brain and spinal cord single cell gene expression and effects of remibrutinib on microglia. a Cell populations detected by scRNA-seq. The scRNA-seq profiling of brains and spinal cords of RatMOG EAE mice showed 13 different cell types, including stromal cells (fibroblasts, endothelial cells), all major immune cell types recruited in the CNS (B cells, T cells, DCs, monocytes and macrophages), and resident cells of the CNS: neurons, neuroepithelial cells, astrocytes, oligodendrocytes and microglia, which we further classified in HM and DAM. b The UMAP representation of the identified cell types in brains and spinal cords of EAE mice showed that BTK was mostly expressed in microglia, myeloid cells and B cells. c The analysis of the neuroinflammatory gene signature in homeostatic and disease-associated microglia showed significant effects of remibrutinib in brain and spinal cord at both timepoints (p < 0.001, one-tailed Mann–Whitney U test)

Fig. 7

Remibrutinib inhibits IC-induced TNFα secretion from human iMicroglia in vitro. Four different iMicroglia batches were tested independently and showed varying levels of IC-induced TNFα (batches are color coded). Batch response was normalized to maximal TNFα secretion and concentration response curves to remibrutinib were calculated using a four-parameter logistic regression. Remibrutinib inhibited TNFα secretion with an IC₅₀ of 1.1 nM