Btk-specific inhibition blocks pathogenic plasma cell signatures and myeloid cell-associated damage in IFNα-driven lupus nephritis.

Systemic lupus erythematosus (SLE) is often associated with exaggerated B cell activation promoting plasma cell generation, immune-complex deposition in the kidney, renal infiltration of myeloid cells, and glomerular nephritis. Type-I IFNs amplify these autoimmune processes and promote severe disease. Bruton's tyrosine kinase (Btk) inhibitors are considered novel therapies for SLE. We describe the characterization of a highly selective reversible Btk inhibitor, G-744. G-744 is efficacious, and superior to blocking BAFF and Syk, in ameliorating severe lupus nephritis in both spontaneous and IFNα-accelerated lupus in NZB/W_F1 mice in therapeutic regimens. Selective Btk inhibition ablated plasmablast generation, reduced autoantibodies, and - similar to cyclophosphamide - improved renal pathology in IFNα-accelerated lupus. Employing global transcriptional profiling of spleen and kidney coupled with cross-species human modular repertoire analyses, we identify similarities in the inflammatory process between mice and humans, and we demonstrate that G-744 reduced gene expression signatures essential for splenic B cell terminal differentiation, particularly the secretory pathway, as well as renal transcriptional profiles coupled with myeloid cell-mediated pathology and glomerular plus tubulointerstitial disease in human glomerulonephritis patients. These findings reveal the mechanism through which a selective Btk inhibitor blocks murine autoimmune kidney disease, highlighting pathway activity that may translate to human SLE.

Dual B cell immunotherapy is superior to individual anti-CD20 depletion or BAFF blockade in murine models of spontaneous or accelerated lupus.

OBJECTIVE: To determine whether a combination of B cell depletion and BAFF blockade is more effective than monotherapy in treating models of spontaneous or accelerated systemic lupus erythematosus (SLE) in (NZB × NZW)F1 mice. METHODS: Clinical parameters such as disease progression-free survival, proteinuria, and renal injury were assessed in models of spontaneous, interferon-α (IFNα)-accelerated, or pristane-accelerated lupus in (NZB × NZW)F1 mice. Treatment arms included anti-CD20 (B cell depletion), B lymphocyte stimulator receptor 3 fusion protein (BR-3-Fc) (BAFF blockade), the combination of anti-CD20 and BR-3-Fc, isotype control, or cyclophosphamide. In models of spontaneous, IFNα-accelerated, or pristane-accelerated lupus, mice were treated for 24 weeks, 8 weeks, or 12 weeks, respectively. Peripheral and resident B cell subsets and various autoantibodies were examined. RESULTS: Compared to B cell depletion or BAFF blockade alone, combined therapy significantly improved disease manifestations in all 3 lupus models. In addition, marginal zone B cells, plasmablasts, and circulating and tissue plasma cells were decreased more effectively. Dual B cell immunotherapy also reduced multiple classes of pathogenic autoantibodies, consistent with its observed effectiveness in reducing immune complex-mediated renal injury. CONCLUSION: Dual immunotherapy via B cell depletion and BAFF blockade is more efficacious than single agent immunotherapy in murine SLE models, and this combination treatment is predicted to be an effective strategy for immunotherapy in human SLE.

Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration.

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.

Troubleshooting PEG-hGH detection supporting pharmacokinetic evaluation in growth hormone deficient patients.

INTRODUCTION: The ability to quantify systemic concentrations of protein therapeutics is complicated by the presence of endogenous analyte, specific binding proteins, and nonspecific matrix components in biological matrices (Lee & Ma, 2007). Further complications can be introduced following pegylation whereby polyethylene glycol (PEG) impedes epitope recognition. Due to substantial interference from high affinity binding proteins and the inability to measure systemic drug concentrations under normal conditions, acid dissociation was implemented to facilitate the pharmacokinetic evaluation of clinical samples containing pegylated human growth hormone (PEG-hGH). METHODS: A sandwich electrochemiluminescent immunosorbent assay (ECLA) was employed using an anti-PEG capture, anti-hGH detection format, thereby eliminating cross-reactivity with endogenous compound. Samples were acid treated with glycine buffered hydrochloric acid (approximately pH 2.0) to dissociate PEG-hGH from serum resident growth hormone binding protein (GHBP; 3). After neutralization with a HEPES-based neutralizing buffer, samples were diluted in a casein-based assay buffer containing 0.2% I-block, 0.1% Tween-20, 2M NaCl, and 10% normal mouse serum to eliminate nonspecific matrix effects. Meso Scale Discovery (MSD) technology was employed to achieve sensitivity requirements. RESULTS: The drug detection assay was validated in the presence and absence of acid dissociation. Validation parameters included: intra- and inter-assay accuracy and precision, selectivity (>60 lots of normal and growth hormone deficient human serum), cross-reactivity/specificity (Genotropin, hemoglobin, lipid, and bilirubin), dilutional linearity and stability (DeSilva et al., 2003; Shah et al., 1992; Smolec et al., 2005 Viswanathan et al., 2007; Food and Drug Administration, 2001). A 10-fold molar excess of GHBP was found to decrease PEG-hGH detection by >90% while acid dissociation was shown to recover >80% of the analyte. PEG-hGH clinical pharmacokinetic samples were analyzed with and without acid treatment. Only 38% of the mid-dose samples were quantifiable without acid treatment while 92% were quantifiable after acid dissociation. DISCUSSION: The implementation of acid dissociation was found to substantially increase the number of quantifiable pharmacokinetic samples over the drug exposure time course and contributed significantly to the robustness of pharmacokinetic evaluation.