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BTK inhibition prevents anti-FVIII memory response
tion of FVIII-specific memory B cells, as suggested from experiments in FVIII-deficient mice.5 ITI is, however, pro- hibitively costly, requires extreme compliance from the patients and their families, and is successful in only 60- 80% of cases.6–8 Direct depletion of B cells with the anti- CD20 antibody rituximab (Mabthera®) is also used, although with limited success and unpredictable conse- quences in the long-term in populations of pediatric patients.9
The development of FVIII inhibitors results from the engagement of a classical T-cell-dependent immune response10 as evidenced by the presence of class-switched, high affinity anti-FVIII antibodies. B cells play key roles in primary T-cell-dependent immune responses, by forming and sustaining germinal centers, by differentiating into antibody-secreting plasma cells and possibly, as recently suggested, as antigen-presenting marginal zone (MZ) B cells involved in the initial stages of activation of immune effectors.11 During recall responses, memory B cells can be reactivated upon antigen encounter and differentiate into plasma cells, replenish the memory B-cell pool or partici- pate as key professional antigen-presenting cells owing to a higher prevalence of the cells and to the expression of a higher affinity antigen-specific B-cell receptor (BCR).12,13 Antigen-specific B cells are thus potential targets to pre- vent primary or recall antigen-specific immune responses.
Engagement of a surface-exposed BCR by its cognate antigen triggers the formation of an intracellular signaling complex which enhances downstream signaling through the phosphorylation and ubiquitination of proteins. Bruton tyrosine kinase (BTK) is a key proximal and rate- limiting component of the signaling cascade critical for B- cell activation, proliferation and survival.14 This cytosolic Tec kinase is activated only when BCR signaling promotes its recruitment at the inner cell membrane. Activated BTK in turn phosphorylates the phospholipase Cγ2, which leads to the downstream production of inositol triphos- phate and diacylglycerol, resulting in calcium flux and finally to the activation of the NF-κB and NFAT-dependent pathways.16 BTK is a strategic therapeutic target for B-cell malignancies that require BTK signaling for cell survival, and for autoimmune diseases associated with the pres- ence of pathogenic autoantibodies such as rheumatoid arthritis15 or lupus.16 Several small-molecule inhibitors of BTK have been developed.17 (R)-5-amino-1-(1- cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxyl] phenyl)- 1H pyrazole-4-carboxamide, or PF-06250112, is a selective potent, orally bioavailable, small-molecule inhibitor of BTK. PF-06250112 forms a covalent but reversible adduct with BTK upon binding to the Cys481 residue that is proximal to the ATP-binding pocket.18 Using a mouse model of severe hemophilia A, we evaluated the therapeu- tic potential of BTK inhibition by PF-06250112 on the development of a primary anti-FVIII immune response and on the FVIII-specific memory B-cell recall response.
Methods
PF-06250112 formulation
For in vitro studies, PF-06250112 (Pfizer, New York, NY, USA) was solubilized at 1 mg/mL in dimethylsulfoxide (Sigma-Aldrich, St Louis, MO, USA). For per os administration, PF-06250112 was prepared in 0.5% methylcellulose, 0.5% hydroxypropylmethyl- cellulose acetate succinate H grade and 20 mM Tris at pH 7.4.
Flow cytometry
BTK inhibition with PF-06250112 was evaluated on splenocytes labeled with anti-CD86-FITC (BD Pharmingen, San Jose, CA, USA), and anti-B220-PE (BioLegend, San Diego, CA, USA). Phenotypic analyses were performed using anti-CD4-Alexa 700, anti-CD19-Pacific blue (Biolegend), anti-CD45-APC (eBiosciences, San Diego, CA, USA); anti-CD3e-FITC, anti-CD11b-PE (BD Pharmingen), anti-CD21-APC Cy7, anti-CD23-PE Cy7 and anti- GL7 Alexa 488 (Biolegend). Fluorescence activated cell sorting analysis was done on live cells using BD LSRII and FACSDiva soft- ware.
Treatment of factor VIII-deficient mice
Seven to 11 week-old exon 16 FVIII-deficient mice on a C57Bl/6 background (a gift from Prof. H.H. Kazazian, Department of Genetics, University of Pennsylvania School of Medicine, PA, USA) were handled in agreement with ethical authority guidelines (experimentation on mice was approved by the Animal Ethics Committee, authorization #02058.04 granted by the Direction Générale de la Recherche et de l’Innovation). PF-06250112 and analogs have half-lives of about 7 h.18,19 For preventive treatment, mice were fed for 5 consecutive days per week, during 4 weeks, with PF-06250112 (15 mg/kg) or vehicle in order to cover about 10 FVIII half-lives.20 Mice were injected with human recombinant B domain-deleted FVIII (0.5 mg BDD-FVIII, Refacto, Pfizer) once a week, 2 h after the second feed of the week. Mice were bled 5 days after the fourth FVIII injection. To investigate the effect of PF- 06250112 on recall immune responses to FVIII, mice were injected intravenously with 0.5 mg of BDD-FVIII or phosphate-buffered saline (PBS) once a week for 4 weeks. Ninety days after the last FVIII injection, FVIII-sensitized mice with anti-FVIII circulating antibodies were fed for 5 days a week, during 2 weeks, with PF- 06250112 (15 mg/kg) or vehicle. Only one challenge with FVIII was performed with 1 mg of BDD-FVIII, 2 h after the second day of the first week of feeding. Mice were bled before and 7 and 14 days after FVIII challenge.
Evaluation of anti-factor VIII immune responses
Anti-FVIII IgG in mouse serum were measured by enzyme- linked immunosorbent assay (ELISA) and FVIII inhibitory titers were measured with a chromogenic assay (Siemens, Marburg, Germany).21 Proliferation of splenocytes was assessed in 96-well plates (0.25x106 cells/well) with concanavalin A (Sigma) or BDD- FVIII for 72 h.22 Cell proliferation was measured by incorporation of [3H]-thymidine (0.5 mCi/well) for an additional 18 h, using a β- counter (Microbeta 1450, Perkin Elmer).
Stimulation of adoptively transferred memory B cells
Spleens from FVIII-sensitized mice that had developed detectable anti-FVIII IgG (typically 80% of the mice) were collect- ed 7 days after the fourth injection with 1 mg of BDD-FVIII. After lysis of erythrocytes, splenocytes were pooled and plasma cells were depleted using a goat anti-mouse CD138 polyclonal anti- body (BD Pharmingen), conjugated to anti-goat IgG-coated Dynabeads (Thermo Fisher, Waltham, MA, USA).23 FVIII-naïve FVIII-deficient mice were then injected intravenously with 107 CD138-depleted splenocytes. Feeding of mice with PF-06250112 or vehicle was initiated 24 h after adoptive transfer for 5 consecu- tive days a week during 2 weeks and 1 mg of BDD-FVIII was injected intravenously only once 2 h after the first feed. Mice were bled 14 days after the FVIII injection.
Ex vivo memory B-cell differentiation assay
Pooled splenocytes from five FVIII-sensitized mice having developed anti-FVIII IgG were cultured at 1.5x106 cells/mL in
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