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.

Aim: Transgenic mice that overexpress human amyloid precursor protein with Swedish or London (APPswe or APPlon) mutations have been widely used for preclinical Alzheimer's disease (AD) drug development. AD patients, however, rarely possess these mutations or overexpress APP. Results: We developed a sensitive ELISA that specifically and accurately measures low levels of endogenous Aβ40 in mouse plasma, brain and CSF. In wild-type mice treated with a bispecific anti-TfR/BACE1 antibody, significant Aβ reductions were observed in the periphery and the brain. APPlon transgenic mice showed a slightly less reduction, whereas APPswe mice did not have any decrease. Conclusion: This sensitive and well-characterized mouse Aβ40 assay enables the use of wild-type mice for preclinical PK/PD and efficacy studies of potential AD therapeutics.

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.

During drug development, measurement of suitable pharmacodynamic biomarkers is key to establishing in vivo drug activity. Binding of monoclonal antibody (mAb) therapeutics to soluble target proteins often results in elevated serum levels of their target antigen, and measuring total (free and bound) concentration of the target antigen can be an important means of demonstrating that the mAb has reached its specific target. However, accurately measuring soluble circulating antigen in preclinical or clinical samples in the presence of a therapeutic mAb presents a bioanalytical challenge. Particularly in the case of low molecular weight and/or multimeric targets, epitopes for capture and detection of the target by reagent antibodies can be obscured by bound therapeutic mAb. Lymphotoxin-alpha (LTα) is a cytokine in the TNF superfamily that has been implicated in the pathophysiology of autoimmune disease, and is a therapeutic target for neutralizing mAb. During preclinical safety studies in cynomolgus macaques, we encountered difficulties in measuring total LTα in serum of dosed animals. When serum LTα trimer was saturated with the anti-LTα mAb, binding of two reagent antibodies, as required for a classic sandwich ELISA, was not feasible, and dissociation methods were also found to be unsuitable. We therefore developed an approach in which excess anti-LTα mAb was added to the in vitro assay system to fully saturate all binding sites, and an anti-idiotypic antibody was used to detect bound therapeutic antibody. Using this method, total LTα could be accurately measured in cynomolgus macaque serum, and was observed to increase with increasing anti-LTα therapeutic mAb dose. Additional in vitro studies demonstrated that the method worked equally well in human serum. This assay strategy will be useful for quantifying total concentrations of other small and/or multimeric target proteins in the presence of a therapeutic antibody.

Anti–factor D (AFD; FCFD4514S, lampalizumab) is a humanized IgG Fab fragment directed against factor D (fD), a rate-limiting serine protease in the alternative complement pathway (AP). Evaluation of AFD as a potential intravitreal (IVT) therapeutic for dry age-related macular degeneration patients with geographic atrophy (GA) is ongoing. However, it is unclear whether IVT administration of AFD can affect systemic AP activation and potentially compromise host-immune responses. We characterized the pharmacologic properties of AFD and assessed the effects of AFD administered IVT (2 or 20 mg) or intravenous (0.2, 2, or 20 mg) on systemic complement activity in cynomolgus monkeys. For the IVT groups, serum AP activity was reduced for the 20 mg dose group between 2 and 6 hours postinjection. For the intravenous groups, AFD inhibited systemic AP activity for periods of time ranging from 5 minutes (0.2 mg group) to 3 hours (20 mg group). Interestingly, the concentrations of total serum fD increased up to 10-fold relative to predose levels following administration of AFD. Furthermore, AFD was found to inhibit systemic AP activity only when the molar concentration of AFD exceeded that of fD. This occurred in cynomolgus monkeys at serum AFD levels ≥2 µg/ml, a concentration 8-fold greater than the maximum serum concentration observed following a single 10 mg IVT dose in a clinical investigation in patients with GA. Based on these findings, the low levels of serum AFD resulting from IVT administration of a clinically relevant dose are not expected to appreciably affect systemic AP activity.

Abstract Pro-inflammatory cytokines may be useful biomarkers for certain therapies in relevant patient samples. However, detection of low abundance cytokines in patient serum has been difficult for some cytokines, thus there is a need to develop a more sensitive assay than the traditional ELISA. Herein we developed an immuno-PCR assay that is in the low to sub pg/ml range to detect levels of a pro-inflammatory cytokine in human serum samples. Immuno-PCR combines the amplification power of PCR with the versatility of ELISA, resulting in improved sensitivity. Biotinylated anti-analyte bound to Streptavidin-coated tubes was used to capture analyte. The captured analyte was detected by anti-analyte conjugated to DNA, in combination with real-time PCR. This immuno-PCR method was found to be at least ~17-fold more sensitive than ELISA, with a lower limit of quantification of ~2 pg/ml in human serum. While this is a promising improvement, a greater fold-improvement over the ELISA detection limit may still be possible. Therefore, we are currently investigating conditions and variables to further improve the sensitivity of this immuno-PCR assay. This assay strategy may be helpful, not only for biomarker quantification, but also in numerous applications where a very sensitive assay is needed.

Miniaturization of immunoassays has numerous potential advantages over traditional ELISAs. Here we present a novel approach using patterned planar plates (PPPs). These 'wall-less' plates consist of a 16 × 24 array of 2 mm diameter hydrophilic regions surrounded by a hydrophobic polytetrafluoroethylene (PTFE) coating. Assays are performed by adding 2 μL droplets to the hydrophilic areas. These droplets are overlaid with an immiscible mixture of perfluorocarbon liquid (PFCL) that essentially eliminates evaporation. During wash steps, a thin film of PFCL covers the hydrophobic coating and prevents its wetting by wash buffer; as a result, the hydrophilic wells remain intact and inter-well cross-contamination is prevented. We compared the performance of three immunoassays using PPPs versus traditional 384-well ELISA plates. These included assays for soluble FcRH5 in human serum, SDF-1 in mouse serum, and human IgG in mouse plasma. The results show that the PPP assays were closely comparable to the ELISAs in terms of sensitivity, linearity of dilution, and sample quantitation. Moreover, the PPP assays were rapid to perform, easily adapted from ELISA protocols, and used 10- to 50-fold less sample and reagent volume as compared to 384- or 96-well plate ELISAs. As an additional advantage, PPPs conform to established microplate dimensional standards making them compatible with pre-existing equipment and workflows. PPPs therefore represent an attractive and broadly applicable approach to flexible miniaturization of plate-based immunochemical assays.

Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation induced cytidine deaminase (AID). We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest switch, Sϵ, is replaced with prominent recombination hotspot Sμ (SμKI). Inserted Sμ out-competed both endogenous Sγ1 and Sϵ to produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. Intra-switch recombination (ISR) within endogenous Sμ is also diminished in SμKI mice, although the insertion itself exhibits no ISR. Furthermore, reciprocal translocations between c-myc and endogenous Sμ are dramatically lower in SμKI mice, suggesting the knocked-in switch sequesters critical factors. We propose competition among S regions influences CSR, ISR, and chromosomal translocation.

The p110δ isoform of PI3K is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which PI3K mediates inflammation are poorly understood. Here we describe a novel highly selective p110δ small molecule inhibitor, G-286, that allowed dissecting the contribution specifically of p110δ to cellular pathways that are implicated in arthritis disease pathogenesis in human and mice. Using G-286, we demonstrate that p110δ regulates B cell- but not myeloid-cell dependent inflammatory arthritis in mice. G-286 blocks B cell receptor dependent proliferation and reduces autoantibody levels in collagen-induced arthritis abrogating disease. However, G-286 does not significantly inhibit FcγR-mediated inflammatory cytokine production in murine macrophages. Accordingly, selective p110δ inhibition did not significantly affect disease progression in FcγR- and myeloid cell-dependent autoantibody-induced arthritis. Importantly, we provide evidence that human and mice diverge in their relative dependency on p110δ to regulate FcγR responses. In addition to B cells and myeloid cells, T cells and osteoclasts contribute to rheumatoid arthritis pathogenesis. Utilizing G-286, we dissect the relative impact of p110δ inhibition on human and mouse T cell and osteoclast function. These results provide new insight in the role of p110δ in arthritis disease processes and offer a compelling rationale for targeting p110δ for the treatment of human rheumatoid arthritis.

Antibody interactions with Fcγ receptors (FcγRs), like FcγRIIIA, play a critical role in mediating antibody effector functions and thereby contribute significantly to the biologic and therapeutic activity of antibodies. Over the past decade, considerable work has been directed towards production of antibodies with altered binding affinity to FcγRs and evaluation of how the alterations modulate their therapeutic activity. This has been achieved by altering glycosylation status at N297 or by engineering modifications in the crystallizable fragment (Fc) region. While the effects of these modifications on biologic activity and efficacy have been examined, few studies have been conducted to understand their effect on antibody pharmacokinetics (PK). We present here a retrospective analysis in which we characterize the PK of three antibody variants with decreased FcγR binding affinity caused by amino acid substitutions in the Fc region (N297A, N297G, and L234A/L235A) and three antibody variants with increased FcγRIIIA binding affinity caused by afucosylation at N297, and compare their PK to corresponding wild type antibody PK in cynomolgus monkeys. For all antibodies, PK was examined at a dose that was known to be in the linear range. Since production of the N297A and N297G variants in Chinese hamster ovary cells results in aglycosylated antibodies that do not bind to FcγRs, we also examined the effect of expression of an aglycosylated antibody, without sequence change(s), in E. coli. All the variants demonstrated similar PK compared with that of the wild type antibodies, suggesting that, for the six antibodies presented here, altered FcγR binding affinity does not affect PK.

Significance Switch (S) regions are repetitive DNA sequences. During an immune response, one of several S regions recombine with a donor switch (Sμ) that is constitutively “on,” resulting in the production of antibodies with new functions. Donor Sμ is large and very repeat-rich, while another switch, Sε, is less than half its size with a low density of repeats. We replaced Sε with Sμ in mice. These mice switch to Sε more effectively and produce high levels of IgE antibodies implicated in asthma, making this a useful model to study disease. In addition, placing Sμ outside of its native context revealed insights into how switches work. Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation-induced cytidine deaminase. We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest S region, Sε, is replaced with prominent recombination hotspot Sμ. These mice produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. The insertion enhances ε germ-line transcript levels, shows a preference for direct vs. sequential switching, and reduces intraswitch recombination events at native Sμ. These results suggest that the sufficiency of Sμ to mediate IgH rearrangements may be influenced by context-dependent cues.

BACKGROUND:In evaluating the serum concentrations in mice of a Sema3E IgG1 Fc fusion protein, a possible antitumor agent, two ELISAs were developed: a generic assay detecting only the Fc portion of the therapeutic and a specific receptor-binding assay detecting intact protein.RESULTS:An unexpected discrepancy was observed in the measured in vivo serum concentrations, with the generic ELISA yielding higher concentrations than the specific ELISA. Size-exclusion HPLC and SDS-PAGE analysis of in vitro serum stability samples revealed extensive aggregation of Sema3E-Fc. The generic assay recovered more Sema3E-Fc in the presence of aggregates than the specific assay.CONCLUSION:Biophysical characterization combined with immunochemical analysis was key to elucidating not only the nature of the protein instability, but also the cause for the assay discrepancy.

Lymphotoxin α (LTα) is a TNF superfamily cytokine secreted from activated lymphocytes as a homotrimer (LTα3) or complexed on the cell-surface with LTβ as heterotrimers. Compelling evidence indicates that LTα is involved in the pathophysiology of autoimmune diseases. Given that blocking the activity of LTα holds promise as a treatment option, a neutralizing monoclonal antibody (mAb) is under clinical development as a potential therapeutic. In preclinical studies of mAb therapeutics, measuring the level of the target antigen is often of interest as a pharmacodynamic marker. We found that the presence of anti-LTα caused a dose-dependent decrease in ability to detect LTα by immunoassay due to the inability of detection antibodies to bind in the presence of bound therapeutic mAb. To circumvent this problem, an alternate assay strategy was devised to accurately measure total LTα3 (free LTα and LTα/anti-LTα complexes). Specifically, excess anti-LTα was added to fully saturate all binding sites on LTα3. Bound anti-LTα was detected using an anti-idiotype mAb. Using this approach, total LTα was measured in serum of cynomolgus monkeys dosed with anti-LTα. Circulating LTα concentrations showed dose related increases up to 8,000-fold that correlated with anti-LTα PK. This assay strategy may be useful in other situations where low molecular weight and/or multimeric structure of a target antigen makes accurate detection in the presence of therapeutic mAb problematic. KEY WORDS LTα mAb PD

Purpose.: To determine if the progression of age-related macular degeneration (AMD) is associated with complement activation in the eye. Methods.: Immunohistochemistry and ELISAs were used to determine the distribution, concentration, and activation of the alternative pathway complement proteases factor B (FB) and factor D (FD) and the central complement protein C3 in genotyped human postmortem donor eyes graded as having no or minimal drusen (category 1; controls), large drusen (category 3), and large drusen with advanced AMD (category 4). Results.: C3, FB, and FD were present in vitreous and Bruch's membrane choroid (BM/C) interface of the macula of eyes in all tested AMD severity categories (n = 100). C3, FB, and FD were predominantly located to the choroidal vasculature and Bruch's membrane and, together with the serum proteins transferrin and albumin, elevated in BM/C extracts of category 4 eyes (n = 23) compared with category 1 eyes (n = 24). A significant increase in FB activation was found only in vitreous of category 4 eyes (n = 23) compared with category 1 eyes (n = 25). Genetic variants of complement factor H (CFH), C3, C2, and FB associated with increased risk of AMD were correlated with alternative pathway complement activation in vitreous, but not with complement proteins in BM/C protein extracts Conclusions.: Increased activation of the alternative complement pathway in vitreous was controlled by disease stage and genetic variation in the complement pathway, supporting a role for complement activation in AMD disease pathogenesis.

The neonatal Fc receptor (FcRn) plays an important and well-known role in immunoglobulin G (IgG) catabolism; however, its role in the disposition of IgG after subcutaneous (SC) administration, including bioavailability, is relatively unknown. To examine the potential effect of FcRn on IgG SC bioavailability, we engineered three anti-amyloid β monoclonal antibody (mAb) reverse chimeric mouse IgG2a (mIgG2a) Fc variants (I253A.H435A, N434H and N434Y) with different binding affinities to mouse FcRn (mFcRn) and compared their SC bioavailability to that of the wild-type (WT) mAb in mice. Our results indicated that the SC bioavailability of mIgG2a was affected by mFcRn-binding affinity. Variant I253A.H435A, which did not bind to mFcRn at either pH 6.0 or pH 7.4, had the lowest bioavailability (41.8%). Variant N434Y, which had the greatest increase in binding affinity at both pH 6.0 and pH 7.4, had comparable bioavailability to the WT antibody (86.1% vs. 76.3%), whereas Variant N434H, which had modestly increased binding affinity at pH 6.0 to mFcRn and affinity comparable to the WT antibody at pH 7.4, had the highest bioavailability (94.7%). A semi-mechanism-based pharmacokinetic model, which described well the observed data with the WT antibody and variant I253A.H435A, is consistent with the hypothesis that the decreased bioavailability of variant I253A.H435A was due to loss of the FcRn-mediated protection from catabolism at the absorption site. Together, these data demonstrate that FcRn plays an important role in SC bioavailability of therapeutic IgG antibodies.