TY - JOUR
AU - Long, Andrew J
AB - Abstract Objectives: Bruton’s tyrosine kinase (BTK) is a non-receptor tyrosine kinase required for intracellular signaling downstream of multiple immunoreceptors. We evaluated ABBV-105, a covalent BTK inhibitor, using in vitro and in vivo assays to determine potency, selectivity, and efficacy to validate the therapeutic potential of ABBV-105 in inflammatory disease. Methods: ABBV-105 potency and selectivity were evaluated in enzymatic and cellular assays. The impact of ABBV-105 on B cell function in vivo was assessed using mechanistic models of antibody production. Efficacy of ABBV-105 in chronic inflammatory disease was evaluated in animal models of arthritis and lupus. Measurement of BTK occupancy was employed as a target engagement biomarker. Results: ABBV-105 irreversibly inhibits BTK, demonstrating superior kinome selectivity and is potent in B cell receptor, Fc receptor, and TLR-9-dependent cellular assays. Oral administration resulted in rapid clearance in plasma, but maintenance of BTK splenic occupancy. ABBV-105 inhibited antibody responses to thymus-independent and thymus-dependent antigens, paw swelling and bone destruction in rat collagen induced arthritis, and reduced disease in an IFNα-accelerated lupus nephritis model. BTK occupancy in disease models correlated with in vivo efficacy. Conclusion: ABBV-105, a selective BTK inhibitor, demonstrates compelling efficacy in pre-clinical mechanistic models of antibody production and in models of rheumatoid arthritis and lupus. Bruton’s tyrosine kinase (BTK);, rheumatoid arthritis;, lupus;, covalent inhibitor Introduction Bruton’s tyrosine kinase (BTK), a member of the TEC kinase family, is a non-receptor tyrosine kinase expressed in multiple cells of hematopoietic lineage including B cells, neutrophils, monocytes/macrophages, osteoclasts, and platelets [1]. BTK is a critical part of the downstream signaling cascade of multiple immunoreceptors including the B cell receptor (BCR), activating Fc receptors, and Toll-like receptor-9 [2–4]. For example, in B cells following antigen binding to the BCR, receptor aggregation leads to the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAM) and subsequent activation of the kinases Lyn and Syk [3]. In concert with BLNK, these kinases then phosphorylate and activate BTK, which subsequently activates phospholipase C (PLC)γ2 [5–7]. This leads to the release of intracellular calcium stores and drives the continuation of the signaling cascade, culminating in NFκB activation and proliferation, survival, or differentiation of the activated B cell [7,8]. Mutations in BTK in both humans and mice have significant consequences to the overall immunological phenotype of affected individuals. In humans, a variety of different mutations in BTK can result in the primary immunodeficiency disorder X-linked agammaglobulinemia (XLA), characterized by a loss of circulating B cells, a severe reduction in serum IgG, and a significantly increased risk of bacterial infections, most notably respiratory infections [9–11]. In addition to the B cell defect, XLA patients also display defects in macrophage phagocytosis and Toll-like receptor 9 (TLR-9) signaling [12,13]. BTK knockout mice, as well as mice containing the naturally occurring xid mutation display a similar, although milder, phenotype to XLA patients [14,15]. These BTK deficient mice have significant B cell defects resulting in reduction in peripheral B cell numbers, reduced IgM and IgG3 serum concentrations, and reduced in vivo responses to certain antigens [15]. In addition to BCR-related deficiencies, BTK deficient mice also display defects in FcεRI-dependent activation of mast cells, TLR-9-mediated cytokine production, and the impaired maturation and function of neutrophils [4,16,17]. BTK plays a role in normal hematopoietic cell development and function but is also implicated in several autoimmune disease pathologies including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). CD19+ peripheral blood B cells from RA patients have increased levels of BTK phosphorylation compared to healthy controls [18]. B cell-restricted overexpression of BTK leads to a lupus-like phenotype characterized by spontaneous germinal center formation, increased plasma cells, and autoantibody production [19]. Activation of Fcγ receptors by immune complex containing autoantibodies leads to subsequent engagement of downstream inflammatory pathways, playing a key role in the pathology of both RA and SLE [20,21]. BTK has a significant role in Fc-mediated phagocytosis and immune complex signaling due to its function in the FcγR signaling pathway and, therefore, provides further evidence that BTK-dependent signaling may be a critical part of disease responses in RA and SLE patients [12,22]. Small molecule inhibition of BTK has demonstrated efficacy in several preclinical models of both RA and SLE, although these molecules carry activity toward other Tec family kinases in addition to BTK [23,24]. Due to both its involvement in various pro-inflammatory pathways and its restriction to the hematopoietic lineage, BTK is an attractive target for pharmacological inhibition in immunological disease [1]. We report here our work on ABBV-105, an irreversible, highly selective, and potent inhibitor of BTK. ABBV-105 demonstrated inhibition across several in vitro assays with impact on several different BTK mediated signaling pathways. Oral dosing of ABBV-105 yielded efficacy in vivo in both a rat model of collagen induced arthritis (CIA) and a mouse model of lupus nephritis. Efficacy in both models correlates with occupancy of the BTK enzyme by ABBV-105, a pharmacodynamic measure of target engagement. Finally, ABBV-105 demonstrates a reduction in antibody responses both in the context of a lupus model as well as in regards to thymus-independent and thymus-dependent antigen stimulation. Methods Animals Female Lewis rats age 6-8 weeks were purchased from Charles River Labs (Portage, MI). NZB/W F1 mice were purchased from Jackson Laboratories (Bar Harbor, ME). C57/BL6 mice were purchased from Taconic (Hudson, NY). All animals were acclimated in the facility for at least seven days prior to use with rats were housed three per cage and mice housed five per cage on a 12 hours light dark cycle with food and water provided ad libitum. All animal studies were performed in accordance with and under protocols approved by the AbbVie Bioresearch Center Institutional Animal Care and Use Committee (IACUC) in accordance with the Principles of Laboratory Animal Care and all applicable national and local laws. Biochemical assays Human BTK (aa 393–659) was expressed in SF9 cells with an N-terminal His6 tag. Activity was measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) based detection of phosphorylated peptide (Biotin-(Ahx)-GAEEEIYAAFFA-COOH). Final concentrations: 9 nM enzyme (BTK), 0.2 μM peptide, 50 mM MOPSO pH 6.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.01% BSA, 0.1 mM Na3VO4, and 0.01 mM ATP. Reaction was quenched at 60 minutes by with 100 mM EDTA, followed by 30 mM HEPES pH 7.0, 0.06% BSA, 0.006% Tween-20, 0.24 M KF, 80 ng/mL PT66K (Cisbio, Bedford, MA), and 0.6 μg/mL SAXL (Prozyme, Hayward, CA). TR-FRET counts were measured after 60 minutes on a Rubystar (BMG). Reversibility of BTK Binding was measured by incubating 400 nM BTK with inhibitors at their IC90 for 30 minutes. Activity was measured as above after a 400-fold dilution into reaction buffer. Additional kinases assays were measured as above at ATP equivalent to its KM. IgE-mediated basophil degranulation Heparinized human whole blood was incubated with inhibitor for 30 minutes at 37 °C, stimulated with anti-IgE (Beckman Coulter, Brea, CA), and incubated for 30 minutes at 37 °C. After holding on ice for 10 minutes, samples centrifuged for 10 minutes at 1000 rpm at 4 °C. Supernatants were analyzed for histamine by Homogeneous Time Resolved Fluorescence (HTRF) (Cisbio, Bedford, MA). IgM-mediated human B-cell proliferation Primary human B cells (Biological Specialty Corporation, Colmar, PA) were thawed, re-suspended in media (RPMI supplemented with 10% FBS, 2 mM l-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, and 1% Penicillin-Streptomycin), and plated at 1 × 105 cells/well in 100 μL. Inhibitor in media/1% DMSO was added to cells and incubated for 30 minutes. Cells were then stimulated with anti-human IgM (Jackson ImmunoResearch, West Grove, PA), incubated for 48 hours, pulsed with 1 μCi/25μL/well [CH3-3H] thymidine, and incubated 16–18 hours. Cells were harvested using the UniFilter-96 Cell Harvester (PerkinElmer, Waltham, MA). Samples were washed with 1× PBS, dried, and counted TopCount NXT HTS (PerkinElmer, Waltham, MA) after addition of 50 μL/well scintillant. IgG-mediated macrophage IL-6 production Primary human monocytes (Biological Specialty Corporation, Colmar, PA) were added to inhibitor as 37 °C for 30 minutes (2 × 105 cells/well, in RPMI with 10% FBS, 1% Penicillin-Streptomycin, 1% l-glutamine, and 0.4% DMSO). Cells were added to plates coated with 40 ng/μL human serum IgG (Sigma-Aldrich, St. Louis, MO), incubated overnight, centrifuged, and supernatants were collected to evaluate IL-6 (Meso-Scale Discovery, Rockville, MD). TLR-stimulated human PBMC TNF production Primary human PBMCs (Biological Specialty Corporation, Colmar, PA) were thawed, re-suspended in media (RPMI supplemented with 2% FBS and 1% Penicillin-Streptomycin), plated at 2 × 105 cells/well, and incubated with inhibitor for 30 minutes. Cells were stimulated with either CpG (2.5 μM; InvivoGen, San Diego, CA), LPS (100 ng/ml; Sigma-Aldrich, St. Louis, MO), or R848 (3 μg/ml; InvivoGen, San Diego, CA). After 24 hours, plates were centrifuged, and supernatant was collected for TNFα (Meso-Scale Discovery, Rockville, MD). Immunization of mice with thymus-dependent and -independent antigens To assess antibody responses to thymus independent antigens, female C57/BL6 were immunized IP with either 20 μg NP-LPS or NP-Ficoll (Biosearch Technologies, Petaluma, CA) in PBS. Mice were dosed orally with ABBV-105 once a day (QD) or twice a day (BID) beginning one day prior to immunization. After seven days, mice were sacrificed and plasma was collected for anti-NP antibody analysis. To assess antibody responses to Prevnar-13 vaccine, female C57/BL6 mice were immunized IP with 1 μg Prevnar-13 (Hanna Pharmaceutical Supply, Wilmington, DE) in PBS. Mice were dosed orally QD with ABBV-105 for 14 days or IP three times per week with cyclophosphamide beginning on the day of immunization. Plasma was collected on day 14 to evaluate antibody titers. For evaluation of the antibody response to the thymus dependent antigen, female C57/BL6 mice were immunized IP with 100 μg NP(23)-KLH (Biosearch Technologies, Petaluma, CA) in PBS with Imject Alum Thermo Scientific, IL). After 35 days, mice were boosted with 100 μg NP(23)-KLH in PBS. Mice were dosed orally QD or BID with 10mpk ABBV-105. Treatment started on day –1 continuing through day 42 or started on day 35 prior to boost continuing through day 42. Mice were bled, and plasma collected on days 7, 14, 21, 35, and 42. Measurement of anti-NP and anti-Prevnar antibodies by ELISA Ninety-six-well ELISA plates were coated with NP(15)-BSA (Biosearch Technologies, Petaluma, CA) at 10 μg/ml in carbonate/bicarbonate buffer (Sigma-Aldrich, St. Louis, MO) and blocked with 3% BSA/0.02% sodium azide. Samples were five-fold serially diluted starting at a 1:100 dilution and added to plates in duplicate. Antibodies were detected using HRP-conjugated goat anti-mouse IgM or IgG3 (1:5000; Southern Biotech, AL). Anti-NP titers were reported as the EC50 of the serial dilution curve using a four-parameter logistic model curve fit calculation in Softmax Pro (Molecular Devices, Sunnyvale, CA). To evaluate anti-polysaccharide IgM and IgG antibodies following immunization with Prevnar, a commercially available ELISA for polysaccharide antigens in the Prevnar-13 vaccine was completed following manufacturer’s instructions (Alpha Diagnostics International, San Antonio, TX). Samples were serially diluted from 1:100 to 1:3200 and data are reported as Relative Dilution Factor (RDF) defined as the dilution that elicited an optical density reading of 1.0 in the ELISA assay. Induction of collagen-induced arthritis Lewis rats were immunized with 600 µg bovine type II collagen in incomplete Freund’s adjuvant (IFA) intradermally on day 0 on the base of tail, left flank, and right flank and boosted with collagen in IFA near the same locations on day 6. Paw volume was measured using a micro-controlled volume meter plethysmograph (Ugo Basile, Gemonio VA, Italy) with left and right paw volumes averaged for analysis. Paw volume baseline was assessed on day 8 (baseline), 11, 13, 15, and 18. Rats were dosed orally QD for seven days beginning on day 11 post-immunization with ABBV-105. Data are presented as a pooled analysis of two independent experiments. On day 18, plasma was collected to measure drug concentrations from three animals per group at 0.25, 0.5, 1, 2, 4, 6, 12, and 24 hours post-dose. For BTK occupancy analysis spleens were collected and frozen in liquid nitrogen from three animals per group at 2, 12, and 24 hours post-dose. Assessment of bone destruction in rat CIA by micro-computed tomography Hind paws from were removed at the middle of the tibia/fibula and stored in 10% neutral-buffered formalin. Paws were imaged using a Scanco µCT40 (Scanco Medical AG, Brüttisellen, Switzerland). A cylindrical contour was manually drawn around the proximal junction of the calcaneous and navicular bone and extending into the tarsals. Three-dimensional quantitative evaluation was performed by Scanco AG analytical software for bone volume (mm3) and surface area to volumetric ratio, giving an approximation of tarsal surface roughness (mm−1). Statistical analyses were performed using 1-way ANOVA with Dunnett’s post-test when compared to vehicle. Evaluation of BTK enzyme occupancy Assessment of BTK covalent occupancy by ABBV-105 was similar to published work [23]. Briefly, snap frozen spleens were homogenized using a Dounce tissue homogenizer (VWR, Radnor, PA) in the presence of protease inhibitors (EMD Millipore, Burlington, MA). Spleen homogenate was mixed with a covalent occupancy probe and incubated on a plate shaker two hours at room temperature. Samples were transferred to a streptavidin-coated 96-well plate (RnD Systems, Minneapolis, MN) and incubated on a plate shaker for one hour at room temperature. After washing, BTK was detected using rabbit anti-mouse BTK (1:1000, Cell Signaling Technology) followed by goat anti-rabbit HRP-conjugated antibody (1:5000, Life Technologies). Percent BTK occupancy was calculated as a proportion of free BTK in a vehicle-treated spleen compared to a compound-treated spleen. IFN-α accelerated lupus nephritis NZBWF1 mice (24 per group) were injected with 5 × 109 IFNα expressing adenovirus particles (Welgen, Worcester, MA) in PBS. Mice were dosed with ABBV-105 orally QD or BID for 56 days beginning on day 7 post-adenovirus immunization. Urine protein levels and body weights were recorded starting on Day 6 and weekly thereafter. Proteinuria was evaluated using Albustix Reagent Strips for Urinalysis (VWR, Radnor, PA). Urine protein levels were graded visually as 0 = Trace, 1 = 30 mg/dL, 3 = 100 mg/dL, 5 = 300 mg/dL, and 7 = 2000+ mg/dL. Mice were considered to have severe proteinuria based on either maintaining two consecutive weeks of urine protein >300 mg/dL prior to Death or Euthanasia. Moribund animals were euthanized according to IACUC guidelines and the dates recorded. For proteinuria and survival measurements statistical significance evaluated by Log-Rank (Mantel-Cox) survival analysis. Compound exposure was evaluated on days 7, 35, and 62 at 0.25, 1, 2, 6, 12, and 24 hours post-dose. On day 43, nine animals per group were sacrificed and spleens were collected and frozen in liquid nitrogen for evaluation of BTK occupancy. Measurement of anti-dsDNA antibodies in lupus nephritis model Plasma anti-dsDNA antibody levels were determined using an ELISA assay. 96 well ELISA plates were coated with poly-D-Lysine (Sigma-Aldrich, St. Louis, MO) overnight at 4 °C followed by coating with calf thymus DNA (Sigma-Aldrich, St. Louis, MO) in overnight at 4 °C. Plates were blocked and then plasma samples were serially diluted 5-fold starting at 1:50 and added to the plates. IgG was detected using HRP-linked goat anti-mouse IgG (1:2000, Jackson ImmunoResearch, West Grove, PA) and developed using TMB Solution (Invitrogen, Carlsbad, CA) Anti-dsDNA titers were reported as the EC50 of the serial dilution curve using a four-parameter logistic model curve fit calculation in Softmax Pro (Molecular Devices, Sunnyvale, CA). Statistical analysis All in vitro data are reported as average with standard error of the mean. In vivo data are reported as average with standard error of the mean and specific statistical analyses are discussed in the methodology for each experiment. Results ABBV-105: An irreversible, selective and potent inhibitor of BTK ABBV-105 was optimized to irreversibly inhibit BTK using an acrylamide moiety as an electrophile to covalently modify the active site cysteine 481. A similar strategy has been shown to successfully provide durable BTK inhibition and a favorable selectivity profile against the majority of protein kinases, which lack an active site cysteine [24]. ABBV-105 inhibits activity of the catalytic domain of BTK in a time-dependent manner, decreasing BTK enzymatic activity with longer pre-incubation of BTK with drug (data not shown). As ABBV-105 has an irreversible interaction (see below), this IC50 is not an equilibrium estimate of binding affinity, but rather a surrogate for a reaction rate constant. Here we define the IC50 based on the degree of inhibition observed under the defined condition of a one hour enzymatic assay without pre-incubating drug and enzyme, and the resulting IC50 of ABBV-105 for BTK catalytic domain is 0.18 μM (Figure 1(A), Table 1). Mutating Cys481 to serine results in a BTK catalytic domain with a similar specific activity and ATP KM as the wild-type construct (not shown, manuscript in preparation). ABBV-105 inhibits BTK (C481S) with an IC50 of 2.6 μM, indicating a significant loss in potency upon exchanging the targeted thiol nucleophile with an alcohol, suggesting Cys481 is important in the manner in which ABBV-105 inhibits BTK. Figure 1. Open in new tabDownload slide ABBV-105 irreversibly inhibits BTK enzyme activity and blocks BTK-dependent cellular activation. (A) Inhibition by ABBV-105 of recombinant human catalytic domain BTK (aa 393–659) enzymatic activity (blue) compared to the C481S mutant of the same construct (red). (B) Selectivity of ABBV-105 compared to CC-292 against the 10 other protein kinases containing a cysteine in the analogous position, 1× (red), 10× (orange), 100× (yellow), and 1000× (green) fold selectivity over BTK. (C) BTK enzymatic activity is not restored up to 24 hours after diluting drug from an IC90 to an IC10 concentration of ABBV-105 (red), compared to a DMSO control (blue). (D) Inhibition by ABBV-105 of activity in multiple in vitro cellular assays. Figure 1. Open in new tabDownload slide ABBV-105 irreversibly inhibits BTK enzyme activity and blocks BTK-dependent cellular activation. (A) Inhibition by ABBV-105 of recombinant human catalytic domain BTK (aa 393–659) enzymatic activity (blue) compared to the C481S mutant of the same construct (red). (B) Selectivity of ABBV-105 compared to CC-292 against the 10 other protein kinases containing a cysteine in the analogous position, 1× (red), 10× (orange), 100× (yellow), and 1000× (green) fold selectivity over BTK. (C) BTK enzymatic activity is not restored up to 24 hours after diluting drug from an IC90 to an IC10 concentration of ABBV-105 (red), compared to a DMSO control (blue). (D) Inhibition by ABBV-105 of activity in multiple in vitro cellular assays. The kinome selectivity of a covalent kinase inhibitor like ABBV-105 needs to be addressed in terms of two distinct aspects: its potential covalent reactivity toward the ten other protein kinases with a cysteine in the analogous position (a time-dependent phenomenon) and its binding affinity toward the remainder of the kinome without an active site nucleophile (an equilibrium measurement). We generated activity assays for each of these 10 other kinases with an active site cysteine, maintaining the same time parameters as for the BTK method, and generated IC50 values for ABBV-105 with each. Selectivity ratios for ABBV-105 (relative to BTK) range from 33 to >280 for these ten kinases (Figure 1(B)). This experiment also revealed that for many kinases, ABBV-105’s selectivity was superior to a previously described BTK covalent inhibitor CC-292 also run in our panel [23]. Relative to CC-292, ABBV-105 showed improvements in selectivity within the TEC family with increased ITK, ETK/BMX, TEC, and TXK selectivity ratios. ABBV-105 was also tested in the DiscoverX KINOMEscan® panel consisting of 456 kinases, which uses a ligand competition method. We evaluated kinome selectivity at a concentration that achieves 80% inhibition, the threshold we used in our PK/PD modeling to determine the efficacious exposure. This was found to occur at 0.015 μM ABBV-105 in a dose-response experiment in the DiscoverX BTK assay. Kinome profiling at 0.015 μM ABBV-105 found that only significant inhibition on BTK (Supplemental Table 1). The lower BTK biochemical IC50 in the DiscoverX panel (3.1 nM, not shown) is likely due to a longer incubation time than used to generate Figure 1(A), highlighting the importance of controlling for the time variable in comparing biochemical IC50 estimates for a time-dependent inhibitor. Table 1. Potency of ABBV-105 across different in vitro assays detailed in Figure 1(D). Cell type Stimulation Signaling pathway Readout IC50 (μM) Recombinant BTK Enzyme – – 0.180 Human basophils Anti-IgE FCεR Histamine 0.062 Human B cells Anti-IgM BCR Proliferation 0.008 Human monocytes Plate bound IgG FCγR IL-6 0.006 Human PBMCs CpG DNA TLR9 TNF 0.003 Human PBMCs R848 TLR7 TNF >1 Human PBMCs LPS TLR4 TNF >1 Cell type Stimulation Signaling pathway Readout IC50 (μM) Recombinant BTK Enzyme – – 0.180 Human basophils Anti-IgE FCεR Histamine 0.062 Human B cells Anti-IgM BCR Proliferation 0.008 Human monocytes Plate bound IgG FCγR IL-6 0.006 Human PBMCs CpG DNA TLR9 TNF 0.003 Human PBMCs R848 TLR7 TNF >1 Human PBMCs LPS TLR4 TNF >1 Open in new tab Table 1. Potency of ABBV-105 across different in vitro assays detailed in Figure 1(D). Cell type Stimulation Signaling pathway Readout IC50 (μM) Recombinant BTK Enzyme – – 0.180 Human basophils Anti-IgE FCεR Histamine 0.062 Human B cells Anti-IgM BCR Proliferation 0.008 Human monocytes Plate bound IgG FCγR IL-6 0.006 Human PBMCs CpG DNA TLR9 TNF 0.003 Human PBMCs R848 TLR7 TNF >1 Human PBMCs LPS TLR4 TNF >1 Cell type Stimulation Signaling pathway Readout IC50 (μM) Recombinant BTK Enzyme – – 0.180 Human basophils Anti-IgE FCεR Histamine 0.062 Human B cells Anti-IgM BCR Proliferation 0.008 Human monocytes Plate bound IgG FCγR IL-6 0.006 Human PBMCs CpG DNA TLR9 TNF 0.003 Human PBMCs R848 TLR7 TNF >1 Human PBMCs LPS TLR4 TNF >1 Open in new tab To confirm that ABBV-105 interacts irreversibly with BTK, we evaluated the dissociation kinetics of ABBV-105. ABBV-105, at the IC90 was mixed and incubated with BTK for 30 minutes to allow for complete association. This mixture was then diluted 400-fold to less than the IC10, and the enzymatic activity was measured by detection of the phosphorylated product by TR-FRET. The rate of recovery of kinase activity was measured relative to BTK that had not been exposed to inhibitor. Pre-treatment of BTK with ABBV-105 results in a lack of any measurable BTK activity once the compound is diluted, suggesting that the inhibitor is irreversibly bound to BTK for greater than 24 hours (Figure 1(C)). Evaluation of ABBV-105 in multiple cellular signaling pathways As BTK is downstream of ITAM-coupled signaling receptors in several different immunological cell types, we evaluated ABBV-105 in cell-based assays to assess the specificity and mechanism of action of ABBV-105 (Figure 1(D); Table 1). ABBV-105 inhibited histamine release from IgE-stimulated basophils and IL-6 release from IgG-stimulated monocytes, which utilize Fcε and Fcγ receptors respectively. ABBV-105 inhibited IgM-mediated B cell proliferation, which is dependent on signaling through the BCR. ABBV-105 also inhibited TNF-release from CpG-DNA stimulated PBMCs, which signals through TLR9, although it did not inhibit the function of TLRs that do not use ITAM motifs, namely, TNF release from PBMCs stimulated either through TLR4 (with LPS) or through TLR7/8 (with R848). Evaluation of ABBV-105 in thymus-independent and thymus-dependent antibody responses XLA patients demonstrate a lack of specific antibody production and an increased risk of bacterial infections [11]. Similarly, BTK deficient mice fail to elicit normal antibody responses to both thymus-independent and thymus-dependent antigens [15]. Since ABBV-105 has significant impacts on IgM-mediated B cell proliferation in vitro there is a possibility that clinical use of a BTK inhibitor could reduce or eliminate the ability of patients to mount antibody responses to antigenic challenges. To better understand the effect of ABBV-105 on antibody responses to different antigens, we examined the responses to the thymus-independent antigens NP-LPS and NP-Ficoll, the pneumococcal vaccine Prevnar, or to the thymus-dependent antigen NP-KLH. We evaluated the anti-NP IgM and IgG3 responses in mice on day 7 following immunization with the thymus-independent antigens NP-LPS and NP-Ficoll. Mice were treated with ABBV-105 10mg/kg QD or BID starting one day prior to immunization. ABBV-105 did not significantly inhibit the anti-NP IgM or IgG3 response to NP-LPS (Figure 2(A)). In contrast, when mice were immunized with NP-Ficoll, the anti-NP IgM and IgG3 responses were both significantly inhibited (Figure 2(B)). BID dosing of ABBV-105 did not provide a significant increase in response compared to QD dosing. As a consequence of the covalent and irreversible nature of the binding of ABBV-105 to BTK, target engagement can be quantitated in vivo by measuring BTK occupancy using a biotinylated probe derived from a related covalent BTK inhibitor. Splenic BTK occupancy was measured 24 hours after the last dose of ABBV-105 for mice dosed QD and 12 hours for mice dosed BID. Splenic occupancy was similar between mice immunized with NP-LPS (10 mg/kg QD = 64.3% ± 8.1%; BID = 77.8% ± 1.5%) or NP-Ficoll (10 mg/kg QD = 53.1% ± 8.1%; BID = 65.6% ± 3.0%). Figure 2. Open in new tabDownload slide ABBV-105 inhibits antibody responses to NP-Ficoll and NP-KLH, but not to NP-LPS or Prevnar-13. Female C57/BL6 mice were immunized with NP-LPS (n = 10), NP-Ficoll (n = 10), Prevnar-13 (n = 20), or NP-KLH (n = 10). For Prevnar-13 immunization studies, untreated naïve animals (n = 4) were used as a control. Mice were dosed orally with ABBV-105 once a day (QD) or twice a day (BID) beginning one day prior to immunization with all antigens and for NP-KLH also one day prior to boost. A/B) anti-NP IgM and IgG3 in mice immunized with NP-LPS (A) or NP-Ficoll (B, C) anti-CPS13 IgM and IgG in mice immunized with Prevnar-13 D/E) Anti-KLH IgM (D) or anti-KLH IgG (E) responses in mice immunized with NP(23)-KLH in alum. Statistical significance determined by one-way ANOVA (A/B/C) or two-way ANOVA (D/E). *p < .05, **p < .01, ***p < .001. Figure 2. Open in new tabDownload slide ABBV-105 inhibits antibody responses to NP-Ficoll and NP-KLH, but not to NP-LPS or Prevnar-13. Female C57/BL6 mice were immunized with NP-LPS (n = 10), NP-Ficoll (n = 10), Prevnar-13 (n = 20), or NP-KLH (n = 10). For Prevnar-13 immunization studies, untreated naïve animals (n = 4) were used as a control. Mice were dosed orally with ABBV-105 once a day (QD) or twice a day (BID) beginning one day prior to immunization with all antigens and for NP-KLH also one day prior to boost. A/B) anti-NP IgM and IgG3 in mice immunized with NP-LPS (A) or NP-Ficoll (B, C) anti-CPS13 IgM and IgG in mice immunized with Prevnar-13 D/E) Anti-KLH IgM (D) or anti-KLH IgG (E) responses in mice immunized with NP(23)-KLH in alum. Statistical significance determined by one-way ANOVA (A/B/C) or two-way ANOVA (D/E). *p < .05, **p < .01, ***p < .001. To evaluate the potential effect of ABBV-105 on responses to vaccines, we evaluated the antibody response to the pneumococcal vaccine Prevnar. Treatment with a dose response of ABBV-105 began on the day of immunization with Prevnar and IgM and IgG antibody responses to the 13 polysaccharides contained in the vaccine were measured 14 days later. ABBV-105 did not significantly inhibit either the anti-pneumococcal IgM or IgG response, whereas the positive control cyclophosphamide completely abrogated the antibody response (Figure 2(C)). Splenic BTK occupancy was measured 24 hours after the last dose of ABBV-105 and demonstrated a dose dependent increase in occupancy (10 mg/kg = 46.8% ± 3.4%; 3 mg/kg = 40.2% ± 3.2%; 1 mg/kg = 34.3% ± 4.1%). To further understand the effect of BTK inhibition on antibody responses, we evaluated the primary and secondary anti-NP IgM and IgG1 responses to the thymus-dependent antigen NP-KLH. ABBV-105 did not impact anti-NP IgM responses prior to the boost on day 35 (Figure 2(D)). However, following the boost on day 35, ABBV-105 treatment significantly reduced anti-NP IgM antibodies, and this occurred both in mice that were treated from the beginning of the study as well as in those where treatment began just prior to the boost. No significant difference was observed between QD and BID treatment with ABBV-105. ABBV-105 did not significantly impact anti-NP IgG1 responses on days 7, 14, or 21 (Figure 2(E)). Treatment with 10 mg/kg ABBV-105 significantly inhibited the anti-NP IgG1 response on day 35, but the other treatment groups were not significantly different from the vehicle control. Splenic BTK occupancy was measured two hours after the last dose of ABBV-105 and demonstrated comparable occupancy across all groups (10 mg/kg QDD1–42 = 99.0% ± 0.2%; 10 mg/kg BIDD1–42 = 97.2% ± 0.5%; 10 mg/kg QDD34–42 = 98.9% ± 0.2%; 10 mg/kg BIDD34–42 = 97.7% ± 0.6%). Efficacy of ABBV-105 in CIA and the relationship to BTK occupancy In RA, pathogenic antibodies activate downstream inflammatory processes in part through FcγR-mediated signaling [20,21]. Since we had previously demonstrated that ABBV-105 inhibits IgG-stimulated cytokine production (Figure 1(D)), we next wanted to know if inhibition of BTK was sufficient to inhibit inflammation in a rodent model of arthritis known to have a significant contribution from FcγR signaling. Therefore, we evaluated ABBV-105 in a rat CIA model with therapeutic treatment, beginning at the first signs of inflammation. Daily, oral treatment of rats with ABBV-105 resulted in dose-dependent inhibition of paw swelling throughout the course of disease (Figure 3(A); n = 9 per group). Whole blood samples were collected from three animals per group to measure drug concentration at the end of the study and the area under the curve (AUC) drug concentration values ± SEM were used to evaluate the exposure response-relationship. Paw swelling data from two independent CIA experiments were used for the pharmacokinetic/pharmacodynamics (PKPD) modeling. We used a direct Emax model with drug concentration AUC selected as the exposure parameter for PKPD evaluation. ABBV-105 demonstrates exposure-dependent inhibition of increases in paw volume (Figure 3(B)). The exposures that provide 50% and 80% inhibition of paw swelling on the last day were calculated (AUC50, 0–24 = 4.5 ± 1.9 ng × hour/mL; AUC80, 0–24 = 19 ± 8 ng × hour/mL). Figure 3. Open in new tabDownload slide ABBV-105 inhibits paw swelling and bone destruction in a rat collagen-induced arthritis model. Lewis rats (n = 9 per experiment) were immunized with collagen in IFA locations on day 6. Rats were dosed orally QD for seven days beginning on day 11 post-immunization with ABBV-105. Data are presented as a pooled analysis of two independent experiments. (A) Paw swelling. (B) Percent inhibition of paw swelling on day 18. (C) Hind paws collected on day 18 and analyzed by µCT to assess bone volume (mm3). Statistical significance versus vehicle determined by one-way ANOVA. *p < .05, **p < .01, ***p < .001. (D) Day 18 plasma drug concentrations and splenic BTK occupancy. (E) Day 18 BTK occupancy by dose. (F) Correlation between percent inhibition of paw swelling in rat CIA and percent BTK occupancy at 2 or 12 hours post day 18 dose. Correlation assessed by linear regression analysis and r2 value noted. Data are presented as pooled data from two independent experiments. Figure 3. Open in new tabDownload slide ABBV-105 inhibits paw swelling and bone destruction in a rat collagen-induced arthritis model. Lewis rats (n = 9 per experiment) were immunized with collagen in IFA locations on day 6. Rats were dosed orally QD for seven days beginning on day 11 post-immunization with ABBV-105. Data are presented as a pooled analysis of two independent experiments. (A) Paw swelling. (B) Percent inhibition of paw swelling on day 18. (C) Hind paws collected on day 18 and analyzed by µCT to assess bone volume (mm3). Statistical significance versus vehicle determined by one-way ANOVA. *p < .05, **p < .01, ***p < .001. (D) Day 18 plasma drug concentrations and splenic BTK occupancy. (E) Day 18 BTK occupancy by dose. (F) Correlation between percent inhibition of paw swelling in rat CIA and percent BTK occupancy at 2 or 12 hours post day 18 dose. Correlation assessed by linear regression analysis and r2 value noted. Data are presented as pooled data from two independent experiments. Within the rat CIA model, inflammatory processes result in bone destruction in the ankle joint mediated by osteoclast activity [25]. In addition, studies in mice have demonstrated that BTK plays a significant role in osteoclastogenesis [26]. To assess the disease modifying effect of ABBV-105 on bone, we analyzed ankles by micro-computed tomography (μCT) at the termination of the studies. Bone erosions can be quantified by three-dimensional evaluation of the ankle joint to calculate total bone volume. Compared to vehicle control-treated animals, ABBV-105 significantly inhibited bone volume loss in a dose dependent manner consistent with the observed anti-inflammatory effects (Figure 3(C)). To understand the association between target engagement and efficacy in the CIA model, we evaluated splenic BTK occupancy to assess the relationship between it and inhibition of paw swelling. Following administration, there was a rapid clearance of ABBV-105 in plasma while occupancy of BTK by ABBV-105 in homogenized spleen samples is maintained for an extended period of time (Figure 3(D)). BTK occupancy increased in a dose-dependent manner, reaching a maximum at two hours post-dose and decreasing over time (Figure 3(E)). At 2 and 12 hours post-dose, there is a strong, positive correlation between BTK occupancy and inhibition of paw swelling (Figure 3(F)). Efficacy of ABBV-105 in an IFNα accelerated lupus nephritis model Through its role in BCR signaling, BTK plays a key role in the development of pathogenic autoantibodies. This has been demonstrated in murine lupus models as demonstrated by studies that crossed the BTK deficient xid strain onto the lupus prone MRL/lpr background resulting in a reduction of autoantibody production [27]. In addition, BTK inhibitors have demonstrated efficacy across both the MRL/lpr and NZB/W models of lupus nephritis [24,28]. We therefore evaluated ABBV-105 in an IFN-α-accelerated model of lupus in NZB/W F1 mice. Typically, female mice of the F1 generation of NZB x NZW crosses develop proteinuria leading to increased mortality by 12 months of age. Administration of an IFN-α expressing adenovirus to pre-diseased mice can lead to a rapid onset of proteinuria and mortality, thereby creating a shorter model to characterize novel therapeutics [29]. We evaluated ABBV-105 treatment in the IFN-α accelerated model, initiating QD or BID treatment seven days after the injection of the IFN-α adenovirus, but prior to the onset of proteinuria. ABBV-105 significantly prevented the onset of proteinuria and prolonged survival at the 10 mg/kg QD and BID doses, while lower doses did not significantly inhibit these endpoints (Figure 4(A,B); n = 20 per group). BID dosing with ABBV-105 did not provide any greater effect on survival or proteinuria than QD dosing. We also evaluated the effect of ABBV-105 on the production of anti-dsDNA autoantibodies on day 14 and 28 following IFN-α adenovirus administration. Both the 10 mg/kg QD and BID doses of ABBV-105 significantly reduced plasma levels of anti-dsDNA IgG antibodies on day 28 (Figure 4(C)). The anti-dsDNA IgG antibody levels were not significantly different between the 10 mg/kg BID and QD doses. Figure 4. Open in new tabDownload slide ABBV-105 reduces anti-dsDNA autoantibodies, reduces proteinuria, and prolongs survival in an IFNα accelerated model of lupus nephritis. NZBWF1 mice (n = 24) were injected with IFNα expressing adenovirus. Mice were dosed with ABBV-105 orally QD or BID for 56 days beginning on day 7 post-adenovirus immunization. (A) Proteinuria levels in NZBWF1 mice at day 56. Statistical significance evaluated by Log-Rank (Mantel-Cox) survival analysis. (B) Mouse survival (C) Day 43 splenic BTK occupancy. (D) Plasma anti-dsDNA IgG antibodies on days 14 and 28. Statistical significance determined by two-way ANOVA. *p < .05, **p < .01, ***p < .001. Figure 4. Open in new tabDownload slide ABBV-105 reduces anti-dsDNA autoantibodies, reduces proteinuria, and prolongs survival in an IFNα accelerated model of lupus nephritis. NZBWF1 mice (n = 24) were injected with IFNα expressing adenovirus. Mice were dosed with ABBV-105 orally QD or BID for 56 days beginning on day 7 post-adenovirus immunization. (A) Proteinuria levels in NZBWF1 mice at day 56. Statistical significance evaluated by Log-Rank (Mantel-Cox) survival analysis. (B) Mouse survival (C) Day 43 splenic BTK occupancy. (D) Plasma anti-dsDNA IgG antibodies on days 14 and 28. Statistical significance determined by two-way ANOVA. *p < .05, **p < .01, ***p < .001. As a marker of target engagement, we measured splenic BTK occupancy as was done in the rat CIA model. Spleens were harvested on day 43 following IFN-α adenovirus injection from three animals per group at both 2 or 24 hours post-dose with ABBV-105. BTK occupancy was increased in a dose-dependent manner at both time points. BTK occupancy was not significantly different between the 10mg/kg QD and BID doses at either time point, but there was a trend towards increased occupancy at the 24-hour time point in the mice dosed BID (Figure 4(D)). Discussion The identification and development of BTK inhibitors has enabled the evaluation of the role of BTK within multiple models of inflammation and autoimmunity [23,24]. Although there is significant evidence linking BTK to the pathogenesis of different autoimmune diseases, clinical proof of concept has yet to be established. We report here the characterization of ABBV-105, a novel irreversible, highly selective, and potent inhibitor of BTK in multiple models of autoimmune disease. Covalent, irreversible kinase inhibitors provide an advantageous strategy to enable improvements in the selectivity and potency of inhibitors targeting a particular kinase [30]. This approach also allows for specific, durable interaction with the target of interest while minimizing excess circulating drug which may limit systemic off-target toxicities. ABBV-105 is an acrylamide-containing inhibitor that targets Cys-481 in the active site, which leads to irreversible inhibition of BTK. ABBV-105 is a potent inhibitor of BTK with a measured IC50 of 180 nM against the enzyme. It demonstrates an excellent selectivity profile against other protein kinases with improved selectivity relative to a previously described covalent BTK inhibitor (CC-292; [23]) for all protein kinases carrying a cysteine in the active site position analogous to Cys481 (Figure 1(B)). Furthermore, in comparison to published kinase selectivity data for Ibrutinib [24], ABBV-105 brings improved BTK selectivity ratios for BLK, ERBB2, ETK/BMX, and ITK. We also note that ABBV-105’s overall selectivity in these Cysteine-containing kinases rivals or exceeds that published for a reversible BTK inhibitor [31]. Thus, ABBV-105 offers high selectivity for BTK inhibition in a molecule designed to bind irreversibly and utilize the same favorable extended duration of action as other less selective covalent inhibitors. ABBV-105 is highly potent at inhibiting cellular responses with known BTK involvement but has no impact in TLR4- and TLR7/8-dependent cellular responses in which BTK is not present in the pathway (Table 1). ABBV-105 was highly potent in BCR, FcεR, FcγR, and TLR-9 mediated assays, all of which are mediated by BTK dependent signaling pathways [2,3]. In contrast, ABBV-105 had no effect within TLR4-or TLR7-mediated cellular assays, neither of which involves BTK in its signaling pathway [32]. A notable observation is that the IC50 of ABBV-105 in the cellular setting is greater than what is observed within the BTK enzymatic assay. This may be due to the time-dependent nature of the inhibition and the different incubation times of ABBV-105 with its target. Consistent with this argument, ABBV-105 potency in the one hour anti-IgE-stimulated basophil histamine assay (62 nM) is much closer to the BTK enzyme IC50 (180 nM) than it is to the potency in the other cellular assays (3–8 nM), all of which involve drug incubation for at least 18 hours. Although this interaction is highly specific for the broader kinome, there are 10 other protein kinases with an analogous cysteine adjacent to the hinge region in the ATP site, for which cross-reactivity might be anticipated. An example of one with a defined undesirable phenotype is EGFR whose inhibition leads to a skin rash observed in oncology clinical trials [33]. ABBV-105 was greater than 30-fold selective over all 10 of these kinases with a cysteine in active site (Table 1). In addition, ABBV-105 was uniquely active against BTK in a full kinome panel, at a concentration relevant to our targeted EC80 response. Both XLA patients and BTK-deficient mice have defects in antibody responses to injected antigens [11,15]. BTK inhibitors also demonstrate inhibition of antibody responses in both the context of disease models as well as in mechanistic antibody-response models [28,34]. Consistent with this in vivo inhibition, ABBV-105 also significantly inhibits IgM-induced B cell proliferation, an early step in the cascade that leads to antibody production (Figure 1(D)). Because of the involvement of BTK in antibody production, we evaluated the impact of ABBV-105 on antibody responses to both thymus-dependent and independent antigens. ABBV-105 did not significantly impact antibody production to the thymus-independent type 1 (TI-1) antigen NP-LPS, but significantly inhibited both the anti-NP IgM and IgG3 response to the thymus-independent type 2 (TI-2) antigen NP-Ficoll (Figures 2(A,B)). These data are consistent with the effects observed in BTK deficient mice, as well as with the mechanisms involved in the B cell response to each antigen type. The TI-1 and TI-2 responses differ in the mechanisms involved in B cell activation. The TI-1 response requires secondary B cell activation signals derived from BTK-independent TLR ligands to enhance BCR signaling, while the TI-2 response requires repetitive polysaccharide structures that result in B cell activation through extensive BCR crosslinking, a highly BTK dependent process [35]. To further explore the effect of BTK inhibition on thymus-independent antibody responses, we evaluated the production of antibodies to 13 polysaccharide antigens (anti-CPS13) following immunization with Prevnar, a vaccine of the capsular polysaccharides of Streptococcus pneumoniae. We had anticipated that ABBV-105 would inhibit the response, due to the polysaccharide nature of the antigens involved. However, ABBV-105 inhibition did not affect anti-CPS13 antibodies at any dose tested (Figure 2(C)). Since pneumococcal vaccines contain TLR ligands in addition to the polysaccharide antigens, TLR activation may be required to drive the antibody responses to these antigens [36]. Thus, rather than a pure TI-2 response similar to the anti-NP-Ficoll response that is entirely BTK-dependent, antibody generation to Prevnar is a mixed TI-1/TI-2 response that is not uniquely-dependent on BTK. We also examined the impact of ABBV-105 on the antibody response to the thymus dependent antigen NP-KLH. ABBV-105 did not have any impact on the primary response to NP-KLH, but significantly inhibited the anti-NP IgM response following a secondary challenge and had minimal impacts on anti-NP IgG1. This is consistent with the published literature in BTK KO and xid mice that shows that IgM, but not IgG1 secondary responses to thymus-dependent antigens are reduced [15]. It is also in line with studies where BTK inhibitors did not impact IgG1 memory B cells [34]. For the anti-NP IgM response, there is a trend towards a greater reduction in antibody production by mice that were treated throughout the study starting prior to the primary immunization compared to mice that were only treated before the secondary antigen challenge. This suggests that, although BTK inhibition does not significantly impact the primary antibody response, it may affect the development of IgM + memory B cells during the primary antibody response. Pathogenic antibodies play a critical role in RA and SLE; both in the activation of immune cells via FcγR mediated signaling and on osteoclast activity and function [37,38]. ABBV-105 impacts both B cell proliferation in vitro and antibody production in vivo, suggesting that ABBV-105 may limit pathogenic antibody production. ABBV-105 also inhibits FcγR mediated signaling and may, therefore, suppress the Fc-mediated pathogenic antibody signaling that can enhance autoimmune responses. In rat CIA, the development of autoantibodies to collagen is required for disease induction, which is mediated by effector cells following the formation of anti-collagen immune complexes and subsequent FcγR mediated signaling [39]. We selected a time point to initiate treatment in this model where anti-collagen antibodies were already generated in order to examine the effect of ABBV-105 on immune activation downstream of immune complex formation as opposed to inhibiting anti-collagen antibody formation [39]. Using this treatment mode, we determined that ABBV-105, in a dose and drug concentration-dependent manner, fully inhibited paw swelling in the CIA model to a degree comparable to the glucocorticoid modulator prednisone. In a separate study, ABBV-105 did not significantly inhibit anti-collagen antibody levels when dosed either prophylactically or therapeutically, despite demonstrating full inhibition of paw swelling (data not shown). This suggests that BTK inhibition significantly impacts the downstream mechanisms of immune activation irrespective of any impact on autoantibody production, consistent with the demonstrated effect on inhibiting FcγR mediated signaling. The covalent nature of ABBV-105 allows one to directly measure target engagement of BTK. This feature can be useful as a pre-clinical or clinical biomarker to assess the pharmacodynamics and pharmacokinetic response of BTK inhibitors [23]. Following oral dosing of ABBV-105 in CIA animals, we observed that the concentration of ABBV-105 was rapidly cleared, whereas BTK occupancy was maintained at high levels for at least 12 hours post-dose, demonstrating an uncoupling of the pharmacokinetics of ABBV-105 from the pharmacodynamic target engagement (Figure 3). This uncoupling is one of the key advantages of covalent inhibitors as it serves to maintain the in vivo selectivity of ABBV-105. That is, non-covalent, off-target interactions are only likely to occur at sustained drug levels and most off-target impacts will be short lived due to the rapid clearance of ABBV-105 [40]. To understand the relationship between target engagement and efficacy in rat CIA, we measured BTK occupancy in the spleen at several time points following ABBV-105 dosing. We observed a positive correlation between efficacy and BTK occupancy at 2 and 12 hours post-ABBV105 dose (Figure 3(F)). At the dose of ABBV-105 that completely inhibited paw swelling (10mg/kg), BTK occupancy was 90% at 2 hours and 80% at 12 hours. By 24 hours post dose the occupancy significantly dropped (<40%), suggesting that high levels of BTK occupancy do not need to be maintained for 24 hours in order to achieve full efficacy in the rat CIA model. Therefore, it is possible that maintenance of a lower, but sustained level of occupancy across a 24-hour period is capable of inhibiting paw swelling comparable to achieving high levels of occupancy following a single compound dose, potentially avoiding any undesirable effects associated with higher doses. In addition to assessing the impact of ABBV-105 on paw swelling in rat CIA, we also evaluated the impact on bone destruction and demonstrated a dose-dependent protection of bone volume that correlated with the impact on paw inflammation. Although it is possible that bone protection is secondary to an anti-inflammatory effect of ABBV-105, published literature suggests that BTK inhibition may have a direct impact on osteoclasts, thereby impacting bone loss irrespective of effects on inflammation. BTK is known to be involved in the maturation of osteoclasts, as BTK-deficient bone marrow-derived macrophages from xid mice show decreased RANKL-dependent osteoclast formation due to impaired pre-osteoclast fusion [41]. In addition, the BTK inhibitor Ibrutinib inhibits the differentiation of osteoclasts in vitro and protects against RANKL-induced bone loss in vivo [42]. Examining the effect of the more selective ABBV-105 on osteoclastogenesis as well as within in vivo bone loss models could more clearly address the involvement of BTK in osteoclast formation and function. ABBV-105 can significantly inhibit certain antibody responses, so we examined the impact of ABBV-105 on autoantibody responses in the context of a chronic disease model. We chose to evaluate ABBV-105 in a model of lupus nephritis due to the role of BTK in B cell activation, antibody production, immune complex mediated signaling, and other mechanisms that play a role in the development of lupus nephritis. BTK is required for the breach of tolerance that leads to the production of pathogenic autoantibodies to DNA that lead to systemic inflammation [43]. Furthermore, BTK-dependent FcγR mediated activation of TLR9 is an important immune activation mechanism leading to the production of IFNα, a key contributor to inflammation in SLE [44]. ABBV-105 significantly inhibited the development of anti-dsDNA antibodies, delayed the onset of proteinuria, and prolonged survival in an IFN-α accelerated model of lupus nephritis. Although there is a significant delay in the onset of proteinuria, a significant proportion of mice still develop proteinuria, indicating that underlying disease has not been fully controlled. Likewise, although anti-dsDNA antibodies are reduced in ABBV-105 treated mice, they are not completely abrogated and increase over time. This is in contrast to what was observed in rat CIA, where paw swelling could be completely inhibited back to baseline levels even in the absence of any impact on autoantibody production. There are multiple explanations for the distinctions in the efficacy profiles of ABBV-105 in arthritis, antibody production, and lupus models. One possible explanation for this disconnect in the extent of efficacy in the chronic disease models is that the lupus model requires more prolonged target coverage than the CIA model. However, we evaluated twice-daily dosing, thereby maintaining increased target engagement over a 24-hour period but did not observe further inhibition of disease compared to once daily dosing. This suggests that maximum efficacy for targeting this mechanism within the lupus model was achieved. BTK is also required for clearance of apoptotic cells, a process that limits TLR exposure to endogenous pro-inflammatory ligands [12,45]. Diseased lupus mice already have a reduction in phagocytic efficiency, so pharmacological inhibition of BTK could further reduce the efficiency of apoptotic cell phagocytosis, leading to the greater availability of pro-inflammatory TLR ligands and the maintenance of an inflammatory state even if BTK inhibition blocks other inflammatory pathways (i.e. FcγR signaling) [46]. To eliminate this explanation, future studies could evaluate apoptotic cell phagocytosis within ABBV-105-treated lupus mice to determine if clearance is impaired. A final possibility is that inhibiting BTK alone is insufficient to fully inhibit the signaling pathways involved in some rodent species. Interestingly, there is evidence of redundant mechanisms for BTK in mice. BTK deficient mice, although they demonstrate defective B cell development and reduced antibody responses to certain antigens, have a milder phenotype compared to XLA patients [15]. TEC kinase has been shown to be a compensatory kinase for BTK in that mice lacking both TEC and BTK show a much more severe block in B cell development and a deficit in both primary and secondary antibody production compared with mice deficient in either kinase alone [47]. This redundancy is not limited to BTK signaling in B cells as TEC kinase is also able to compensate for GPVI-mediated activation and aggregation of platelets, suggesting this compensation is found across multiple pathways involving BTK [48]. To better understand if this hypothesis is sufficient to explain the difference between the rat CIA and mouse lupus models, further evaluation of BTK inhibition in rat antibody-response mechanistic models is needed. If the hypothesis is correct and TEC compensation occurs in mice, but not rats, antibody responses to thymus-dependent antibodies should be fully-inhibited following immunization in rats treated with ABBV-105. The observed redundancy of TEC kinase with BTK in mice suggests that assessing a selective BTK inhibitor in mouse models of inflammation or disease may underestimate the potential efficacy in humans where TEC does not play the same redundant role. This may be important when we consider the thymus dependent/independent antibody response data we generated above. Understanding antibody responses in patients treated with ABBV-105 will be important as BTK inhibition could have a significantly greater effect on antibody responses to antigens compared to what we have observed here in mice, an important consideration for patients treated with ABBV-105 in regards both to infection risk and vaccinations. We have demonstrated that ABBV-105 is a covalent, irreversible, potent, and highly selective inhibitor of BTK that specifically inhibits BTK dependent cellular functions, including both BCR and FcγR mediated signaling. ABBV-105 is active across multiple in vivo assays demonstrating efficacy in rodent models of arthritis and lupus and also demonstrating efficacy consistent with BTK deficient mice in mechanistic models of antibody production. ABBV-105 is a highly selective and potent inhibitor that could be used to evaluate the contribution of BTK signaling to human autoimmune disease, which has not been explored clinically, and may provide a new therapy for RA and lupus patients. Acknowledgements The authors like to acknowledge the technical contributions of Dan Shi and Sage Foley for in vitro assay work and Shaughn Bryant for providing μCT analysis. Conflict of interest The design and study conduct for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Funding The financial support for this research was provided by AbbVie. References Mohamed AJ , Yu L, Bäckesjö C-M, Vargas L, Faryal R, Aints A, et al. Bruton’s tyrosine kinase (BTK): function, regulation, and transformation with special emphasis on the PH domain . Immunol Rev . 2009 ; 228 ( 1 ): 58 – 73 . 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Please see Erratum http://dx.doi.org/10.1080/10.1080/14397595.2018.1507513 Supplemental data for this article can be accessed here. © 2018 Japan College of Rheumatology This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - ABBV-105, a selective and irreversible inhibitor of Bruton’s tyrosine kinase, is efficacious in multiple preclinical models of inflammation
JF - Modern Rheumatology
DO - 10.1080/14397595.2018.1484269
DA - 2019-05-04
UR - https://www.deepdyve.com/lp/oxford-university-press/abbv-105-a-selective-and-irreversible-inhibitor-of-bruton-s-tyrosine-mOnoN17CYv
SP - 510
EP - 522
VL - 29
IS - 3
DP - DeepDyve
ER -