Author Of 5 Presentations
P0070 - Effect of evobrutinib, a BTK inhibitor, on immune cell and immunoglobulin levels in relapsing MS: an open-label extension to a phase II study (ID 1683)
Abstract
Background
Evobrutinib (EVO), a highly selective Bruton’s tyrosine kinase (BTK) inhibitor, has a dual mode of action on B cells and myeloid cells involved in multiple sclerosis (MS) pathogenesis. A Phase II randomized study (NCT02975349) investigated the effect of EVO on immune cells and immunoglobulins (Ig). After a 48-week randomized, double-blind period (DBP), relapsing MS (RMS) patients treated with EVO showed no evidence of B cell depletion or clinically relevant changes in memory or mature-naïve B cell subsets. IgG levels remained stable and slight elevations and reductions, respectively, in IgA and IgM levels were observed.
Objectives
To investigate the long-term effects of EVO on B cells (total, mature-naïve and memory subsets), T cells (total, helper and cytotoxic subsets), NK cells, and Ig levels after 48 additional weeks in the ongoing open-label extension (OLE).
Methods
Adults with RMS were randomized double-blind to EVO 25 mg QD, 75 mg QD, 75 mg BID, or placebo (PBO). PBO patients switched to EVO 25mg QD at Week 24. At Week 48, all patients were OLE-eligible, and received EVO 75 mg QD (median ≈48 weeks), then 75 mg BID. Safety of EVO, including assessment of total B cell counts and Ig levels, was a secondary endpoint; effects on B cell subsets, T cells, and NK cells were exploratory. Immune cell counts were assessed at OLE Week 48 relative to DBP baseline, and Ig levels at OLE Weeks 24 and 48.
Results
Of 213 patients receiving EVO during the DBP, 164 (77%) entered the OLE and 148 (90%) completed ≥60 additional treatment weeks. Investigation of total CD19+ B cells and B cell subsets revealed a decrease in CD19+ B cells and in mature-naïve B cells in all groups originally randomized to EVO. The decrease in mature-naïve B cells was consistent with that observed for CD19+ B cell counts, however no evidence of a change in the memory B cell levels was observed. No relevant changes in IgG levels relative to DBP baseline were observed. Mean IgA and IgM levels remained increased and decreased, respectively, but mean values were within normal ranges. Furthermore, there was no evidence of a change in T or NK cell parameters. Overall, EVO treatment was not associated with an increased risk of infections.
Conclusions
Immune cell numbers and Ig levels seen in patients receiving EVO for 48 weeks of the OLE were consistent with those in the DBP. The results suggest a gradual decline of B cells over time with consistent BTK inhibition, however the clinical meaningfulness of these changes remains to be determined. The observed changes in B cells, IgA and IgM levels were not associated with an enhanced risk of infections. These findings suggest that the continuous pharmacological inhibition of BTK over 96 weeks with EVO does not lead to substantial B cell reductions or changes in Ig levels.
P0334 - Evobrutinib, a highly selective BTK inhibitor, prevents antigen-activation of B cells and ameliorates experimental autoimmune encephalomyelitis (ID 1125)
Abstract
Background
Background: B cells are key mediators of inflammatory processes in multiple sclerosis, a notion substantiated by the success of B-cell depletion therapies; however, overall depletion does not only target pathogenic B cells but can also affect regulatory B-cell properties. An alternative strategy may be the specific inhibition of Bruton’s tyrosine kinase (BTK), which is centrally involved in B-cell receptor (BCR) signaling and subsequently mediates B-cell activation and differentiation. BTK inhibitors therefore hold the promise to control pathogenic functions such as antigen presentation and cytokine release.
Objectives
Objectives: To evaluate the BTK inhibitor evobrutinib in a mouse model of experimental autoimmune encephalomyelitis (EAE).
Methods
Methods: C57Bl/6 mice received oral evobrutinib or vehicle starting 7 days before immunization with conformational MOG1-117 protein (a B cell–mediated model of EAE). EAE severity was assessed for 60 days using a standard scale. B-cell maturation and activation markers on B and T cells were analyzed by flow cytometry on day 12 post immunization. T cell proliferation and differentiation were assessed after a 3-day co-culture with BTKi-treated B cells. Intracellular calcium flux was analyzed using calcium-sensitive dyes and BCR or T cell receptor (TCR) stimulation. BTK expression and phosphorylation as well as cytokine production were assessed on healthy human B cells via PhosFlow protocols or ELISA, respectively.
Results
Results: Evobrutinib showed a dose-dependent amelioration of EAE severity throughout the 60-day observation period. Evobrutinib led to an accumulation of follicular type (FO) II B cells and a corresponding reduction in FO I B cells, a BTK-dependent transition. Expression of CD86, CD69, and major histocompatibility complex class II on B cells, and CD25 and CD69 on T cells, was reduced. Evobrutinib inhibited the B cell-mediated proliferation and proinflammatory differentiation of T cells. BCR-mediated mobilization of excitatory calcium was reduced by evobrutinib, while TCR signaling remained unaffected. In human B cells, BTK expression and phosphorylation were depending on the maturation of B cells, while the overall cytokine release was inhibited by evobrutinib.
Conclusions
Conclusion: Evobrutinib efficiently reduces BTK-dependent signaling after BCR stimulation, preventing B-cell activation, proinflammatory differentiation, and function. This translates into reduced CNS inflammation and clinical amelioration in a B cell–mediated EAE model.
P0403 - T-bet+ B-cell development in MS: Association with Bruton’s Tyrosine Kinase activity and targeting by evobrutinib (ID 1104)
Abstract
Background
B-cell depletion is an efficacious treatment in relapsing and progressive multiple sclerosis (MS). A phase II trial (NCT02975349) showed promising results for Bruton’s tyrosine kinase (BTK) inhibitor evobrutinib in the treatment of MS. Previously, we found that T-bet+ B cells preferentially infiltrate the central nervous system and are induced under IFN-γ- and TLR9-stimulating, germinal center-like conditions in MS.
Objectives
We aimed to elucidate how BTK is expressed and activated in distinct ex vivo B-cell subsets during the course of MS. Moreover, the relation between BTK activity and T-bet+ B-cell differentiation was assessed both ex vivo and in vitro.
Methods
We determined BTK and phosphorylated BTK (pBTK) levels in transitional, naive mature, class-switched and non class-switched B cells in blood from both treatment-naive patients with CIS, RRMS, SPMS and PPMS and healthy controls (HC; n=30 per group), as well as clinical MS responders and non-responders to natalizumab (pre- vs 1y post-treatment) using flow cytometry. Purified naive mature and memory B cells were cultured under several IL-21/CD40L-inducing conditions with and without evobrutinib.
Results
BTK was mainly expressed in non-class-switched memory B cells, while pBTK levels were high in both class-switched and unswitched memory B cells. In contrast to BTK, pBTK was significantly higher in ex vivo memory B cells of RRMS and SPMS compared to CIS, PPMS and HC groups. In both RRMS and SPMS, pBTK was also less induced after a-IgM stimulation. BTK and pBTK levels were elevated in blood B cells from clinical responders, but not in non-responders to natalizumab. These levels correlated positively with CXCR3 and VLA-4 expression. No correlation was seen for CXCR4, CXCR5, CD40 and HLA-DR. In vitro experiments revealed that pBTK in B cells was particularly triggered by IFN-γ and TLR9 induction. Evobrutinib attenuated class-switching during in vitro cultures of naive B cells, while it interfered with plasmablast formation in memory B-cell cultures. T-bet and T-bet-related markers (CD21, CD11c) were only affected by evobrutinib in IFN-γ- and TLR9-stimulating naive B-cell cultures.
Conclusions
These data demonstrate that BTK is more activated in memory B cells from RRMS and SPMS patients and functionally related to pathogenic T-bet+ B-cell development. This study provides new mechanistic insights into how evobrutinib intervenes in human B-cell differentiation and can modulate the clinical course of MS.
P0404 - The Bruton’s tyrosine kinase inhibitor evobrutinib ameliorates meningeal inflammation in experimental autoimmune encephalomyelitis (ID 1354)
Abstract
Background
Leptomeningeal inflammation in multiple sclerosis (MS) is associated with worse clinical outcomes and greater cortical pathology. Both B cells and myeloid cells are found in areas of meningeal inflammation. We previously demonstrated that, in the relapsing–remitting encephalomyelitis (EAE) model in SJL mice, ultra-high field contrast-enhanced magnetic resonance imaging (MRI) could identify and track areas of meningeal inflammation. Bruton’s tyrosine kinase (BTK) mediates signaling through B cell receptor and Fc receptor pathways and leads to B cell and myeloid cell activation. We therefore hypothesized that a BTK inhibitor could target meningeal inflammation in EAE.
Objectives
To test the effect of evobrutinib, a highly selective BTK inhibitor, as a potential therapy targeting meningeal inflammation in a mouse model of MS.
Methods
We immunized 7- to 8-week-old female SJL/J mice with proteolipid protein 139–151 peptide and complete Freund’s adjuvant to induce EAE. Animals were weighed and disease severity was scored starting at 7 days post-immunization; at 6 weeks they underwent Gadolinium-enhanced MRI. Mice demonstrating the presence of meningeal contrast enhancement were randomized to receive daily oral doses by gavage of evobrutinib (10 mg/kg) or vehicle control between Weeks 6–10 post-immunization. MRI was repeated at Weeks 8 and 10 to assess meningeal inflammation, and brain tissues were collected for histopathological analysis.
Results
At baseline, both vehicle (n=16) and evobrutinib (n=19) groups had a similar number of areas of meningeal contrast enhancement (median: 10.5 vs 11; p=0.25). Following treatment, a greater reduction in the number of areas of meningeal contrast enhancement was identified in the evobrutinib group vs the vehicle group (median change: -3 vs 0.5; p=0.003). A significant decrease in B cells in areas of meningeal inflammation in the evobrutinib group compared to the vehicle group was noted. Also, astrocytosis in the adjacent cortex was reduced in the evobrutinib group compared with vehicle.
Conclusions
An amelioration of established meningeal inflammation, as assessed by imaging and pathological measures, in a relapsing–remitting EAE model was observed with evobrutinib treatment, suggesting the potential utility of this agent to target this phenomenon in MS patients.
P0962 - Expression of Bruton’s tyrosine kinase in B cell-rich meningeal infiltrates in two models of progressive MS (ID 1391)
Abstract
Background
The success of anti-CD20 therapies has emphasized the important contribution of B cells to the disease process in MS. Indeed, B cells sequestered in the central nervous system (CNS), including the ones forming organized leptomeningeal aggregates, are suspected to be a source of underlying progressive disease activity in MS. Their presence, when associated with adjacent subpial cortical pathology, is predictive of an aggressive disease course with rapid, unrelenting progression of disability. Yet CNS B cells are protected from the direct effects of anti-CD20 therapies. An alternative approach is to target the inflammatory process in the CNS by inhibiting Bruton’s tyrosine kinase (BTK), an enzyme critically involved in B cell activation and survival, but also expressed by macrophages and microglia. BTK inhibitors are small molecules potentially able to enter the CNS, that have the capacity to restrict peripheral and CNS-compartmentalized B cell responses.
Objectives
Here, we investigated the expression pattern of BTK in brain tissue from a spontaneous naturally-occurring canine neuroinflammatory disease (granulomatous meningoencephalomyelitis, GME), and from a murine experimental autoimmune encephalomyelitis (EAE) model, with both recapitulating determining aspects of progressive MS pathology.
Methods
To this end, we studied CNS samples from GME (n=9) and EAE (n=6) cases by immunofluorescent/confocal analysis of leptomeningeal and parenchymal areas affected by the neuroinflammatory process.
Results
Our analysis reveals both models are characterized by prominent B cell infiltrates in the leptomeninges that associate with submeningeal injury in the underlying parenchymal tissue. These injurious changes are reminiscent of the subpial neuropathology characteristic of MS. In both models, we found robust BTK expression within CD20+ B cell-rich leptomeningeal infiltrates. Meningeal B cell infiltration extended into the parenchymal tissue via the Virchow Robin spaces of penetrating vessels. BTK expression was also detected in B cells within these spaces and in cells moving from the perivascular space into the parenchymal tissue. In GME, BTK was also detected in microglia/macrophages in leptomeningeal infiltrates and in the neighboring parenchyma.
Conclusions
The prominent expression of BTK in leptomeningeal and perivascular B-cell rich infiltrates underscores its value as a potential candidate to target CNS compartmentalized immune responses thought to drive the progression of disability seen in MS.