A novel immunoassay to measure total serum lymphotoxin-α levels in the presence of an anti-LTα therapeutic antibody.

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.

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.

A case study on Sema3E-Fc aggregation and assay-dependent differences in quantitation.

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.

Specific Btk inhibition suppresses B cell- and myeloid cell-mediated arthritis.

Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1ß and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.

Increased targeting of donor switch region and IgE in Sgamma1-deficient B cells.

Ab class switch recombination involves a recombination between two repetitive DNA sequences known as switch (S) regions that vary in length, content, and density of the repeats. Abs expressed by B cells are diversified by somatic hypermutation and class switch recombination. Both class switch recombination and somatic hypermutation are initiated by activation-induced cytidine deaminase (AID), which preferentially recognizes certain hot spots that are far more enriched in the S regions. We found that removal of the largest S region, Sgamma1 (10 kb), in mice can result in the accumulation of mutations and short-range intra-S recombination in the donor Smu region. Furthermore, elevated levels of IgE were detected in trinitrophenol-OVA-immunized mice and in anti-CD40 plus IL-4-stimulated B cells in vitro. We propose that AID availability and targeting in part might be regulated by its DNA substrate. Thus, prominently transcribed S regions, such as Sgamma1, might provide a sufficient sink for AID protein to titrate away AID from other accessible sites within or outside the Ig locus.

Lymphotoxin-alphabeta heterotrimers are cleaved by metalloproteinases and contribute to synovitis in rheumatoid arthritis.

Tumor necrosis factor-superfamily (TNF-SF) members, lymphotoxin (LT)-alpha and LTbeta, are proinflammatory cytokines associated with pathology in rheumatoid arthritis. LTalpha3 homotrimers are secreted, whereas LTalpha(1)beta(2) heterotrimers are expressed on the surface of activated lymphocytes. As many TNF-SF members are actively cleaved from cell membranes, we determined whether LTalphabeta heterotrimers are also cleaved, and are biologically active in rheumatoid arthritis (RA) patients. LTalphabeta heterotrimers were detected in culture supernatants from activated human T-helper (Th) 0, Th1, and Th17 cells, together with LTalpha3 and TNFalpha. The heterotimers were actively cleaved from the cell surface by ADAM17 metalloproteinase (MMP) and MMP-8, and cleavage was inhibited by TAPI-1, a TNF-alpha converting enzyme (TACE) inhibitor. Soluble LTalphabeta was detected in serum from both normal donors and RA patients, and was elevated in synovial fluid from RA patients compared to osteoarthritis (OA) patients. Levels of LTalphabeta in RA patient synovial fluid correlated with increased TNFalpha, IL-8, IL-12, IL-1beta, IFN-gamma, and IL-6 cytokines. Moreover, recombinant LTalpha1beta2-induced CXCL1, CXCL2, IL-6, IL-8, VCAM-1, and ICAM-1 from primary synovial fibroblasts isolated from RA patients. Therefore, soluble LTalphabeta in synovial fluid is associated with a proinflammatory cytokine milieu that contributes to synovitis in RA.

Pharmacokinetics of humanized monoclonal anti-tumor necrosis factor-{alpha} antibody and its neonatal Fc receptor variants in mice and cynomolgus monkeys.

The neonatal Fc receptor (FcRn) plays a critical role in maintaining homeostasis of IgG antibodies. Recent studies have shown that the FcRn-IgG interaction can be modulated to alter the pharmacokinetics of the antibody. This has been achieved by altering amino acid residues in the FcRn-binding domain of the antibody, resulting in a change in the pH-dependent binding affinity of the antibody to FcRn. The purpose of this study was to examine the impact of the pH-dependent FcRn binding affinity on the pharmacokinetics of the antibody with changes in the Asn434 residue. Two anti-tumor necrosis factor-alpha monoclonal antibody (mAb) FcRn variants (N434A and N434H) were engineered, and pharmacokinetic studies of the two FcRn variants together with the wild type (WT) were conducted in mice and cynomolgus monkeys. N434A, which had binding properties to murine FcRn similar to those of the WT, had the same pharmacokinetic profile as the WT in mice. N434H, with the highest binding affinity to murine FcRn at pH 7.4, had a faster clearance (16.1 ml/day/kg) and a lower bioavailability (61.3%) compared with the WT (5.07 ml/day/kg, 73.2%) and N434A (5.90 ml/day/kg, 72.4%) in mice. N434A and N434H, which had higher binding affinity at pH 6.0 to monkey FcRn with comparable affinity at pH 7.4, had significantly higher areas under the serum concentration-time curve from time 0 to day 7 than the WT (749 +/- 71.9 and 819 +/- 81.5 versus 592 +/- 56.8 microg/ml . day) in monkeys. Thus, increasing the binding affinity of mAbs to FcRn at pH 6.0 while keeping a low binding affinity at pH 7.4 improves the pharmacokinetics of these molecules.

Improving therapeutic efficacy of a complement receptor by structure-based affinity maturation.

CRIg is a recently discovered complement C3 receptor expressed on a subpopulation of tissue-resident macrophages. The extracellular IgV domain of CRIg (CRIg-ECD) holds considerable promise as a potential therapeutic because it selectively inhibits the alternative pathway of complement by binding to C3b and inhibiting proteolytic activation of C3 and C5. However, CRIg binds weakly to the convertase subunit C3b (K(D) = 1.1 microm), and thus a relatively high concentration of protein is required to reach nearly complete complement inhibition. To improve therapeutic efficacy while minimizing risk of immunogenicity, we devised a phage display strategy to evolve a high affinity CRIg-ECD variant with a minimal number of mutations. Using the crystal structure of CRIg in complex with C3b as a guide for library design, we isolated a CRIg-ECD double mutant (Q64R/M86Y, CRIg-v27) that showed increased binding affinity and improved complement inhibitory activity relative to CRIg-ECD. In a mouse model of arthritis, treatment with a Fc fusion of CRIg-v27 resulted in a significant reduction in clinical scores compared with treatment with an Fc fusion of CRIg-ECD. This study clearly illustrates how phage display technology and structural information can be combined to generate proteins with nearly natural sequences that act as potent complement inhibitors with greatly improved therapeutic efficacy.

Technology comparisons for anti-therapeutic antibody and neutralizing antibody assays in the context of an anti-TNF pharmacokinetic study.

A single-dose cynomolgus monkey pharmacokinetic study was performed comparing two monoclonal anti-TNF antibodies (mAbs), GNExTNFvF and Humira. Normal pharmacokinetic profiles were observed over the first week of the study, followed by a rapid drop in serum mAb levels after day 8. In order to determine whether an anti-therapeutic antibody (ATA) response led to the abnormal clearance of antibody in this study, ATA assays were developed using two electrochemiluminescent technologies, BioVeris and Meso Scale Discovery (MSD). Characterization of the assays demonstrated that the two platforms gave similar sensitivities and tolerance to the presence of therapeutic antibody. Analysis of the cynomolgus monkey serum samples revealed that all animals developed significant ATA titers with log titer values of 2-4, with the BioVeris and MSD technologies giving very similar results. Immunodepletion studies confirmed the CDR-specificity of the ATA response for the GNExTNFvF-dosed cynos, although the Humira-dosed cynos showed both CDR-specific and human IgG1 framework-specific ATAs. To further characterize the ATA response, neutralizing antibody (NAb) assays were developed using two different approaches, flow cytometry and MSD. Flow cytometry and MSD cell-binding assays used Jurkat cells transfected with noncleavable TNF (huTNF(NC)). Neutralizing activity was assessed by the ability of ATA-positive serum samples to block the binding of biotinylated anti-TNF to huTNF(NC) Jurkat cells, showing that all but one animal developed neutralizing antibodies. Although both technologies displayed similar trends, the MSD approach showed greater differentiation between samples and could detect a broader range of neutralizing activities.

Anti-BR3 antibodies: a new class of B-cell immunotherapy combining cellular depletion and survival blockade.

Removal of pathogenic B lymphocytes by depletion of monoclonal antibodies (mAbs) or deprivation of B-cell survival factors has demonstrated clinical benefit in both oncologic and immunologic diseases. Partial clinical responses and emerging data demonstrating incomplete B-cell depletion after immunotherapy fuels the need for improved therapeutic modalities. Lessons from the first generation of therapeutics directed against B-cell-specific antigens (CD20, CD22) are being applied to develop novel antibodies with additional functional attributes. We describe the generation of a novel class of B-cell-directed therapy (anti-BR3 mAbs) that combines the depleting capacity of a therapeutic mAb and blockade of B-cell-activating factor (BAFF)-BR3 B-cell survival. In mice, treatment with antagonistic anti-BR3 antibodies results in quantitatively greater reduction in some B-cell subsets and qualitatively different effects on bone marrow plasma cells compared with BR3-Fc BAFF blockade or with anti-CD20 treatment. Comparative analysis of BR3-Fc and anti-BR3 mAb reveals a lower B-cell dependence for BAFF-mediated survival in nonhuman primates than in mice. This novel class of B-cell-targeted therapies shows species characteristics in mice and primates that will guide translation to treatment of human disease.

Species-dependent serum interference in a sandwich ELISA for Apo2L/TRAIL.

To support pre-clinical studies of Apo2L/TRAIL in rodents and non-human primates, a sandwich ELISA was developed using two mouse monoclonal anti-Apo2L/TRAIL antibodies. Mouse, rat, cynomolgus monkey, and chimpanzee serum at concentrations of > or =1% were found to interfere with accurate quantitation of Apo2L/TRAIL. Moreover, the characteristics of the serum interference for each species were different. In order to resolve the observed serum effect, studies were performed in which salts, detergents, and blocking proteins were added to the sample diluent, and optimized sample diluents that eliminated serum interference were developed for mouse, cynomolgus monkey, and chimpanzee serum. These buffers consisted of a base assay diluent (PBS/0.5% BSA/0.05% Tween-20/10 ppm ProClin 300) supplemented with: NaCl (mouse serum); NaCl, EDTA, CHAPS, bovine gamma globulin (BGG), and human IgG (cynomolgus monkey serum); and NaCl and EDTA (chimpanzee serum). Full characterization studies were performed for the "buffer" ELISA run in base assay diluent (intended for non-serum samples) as well as the assays optimized for mouse serum and cynomolgus monkey serum. Precision, accuracy, linearity, and specificity were found to be satisfactory. With the availability of a rabbit polyclonal antibody against Apo2L/TRAIL, a new pAb/mAb ELISA was developed. This assay was not only more sensitive by > or =6-fold, but it was also much less subject to serum interference.

Synthetic anti-BR3 antibodies that mimic BAFF binding and target both human and murine B cells.

BR3, which is expressed on all mature B cells, is a specific receptor for the B-cell survival and maturation factor BAFF (B-cell-activating factor belonging to the tumor necrosis factor [TNF] family). In order to investigate the consequences of targeting BR3 in murine models and to assess the potential of BR3 antibodies as human therapeutics, synthetic antibody phage libraries were employed to identify BAFF-blocking antibodies cross-reactive to murine and human BR3, which share 52% identity in their extracellular domains. We found an antibody, CB1, which exhibits muM affinity for murine BR3 and very weak affinity for the human receptor. CB3s, an affinity-matured variant of CB1, has sub-nM affinity for BR3 from both species. Alanine scanning and crystallographic structural analysis of the CB3s/BR3 complex reveal that CB3s mimics BAFF by interacting with a similar region of the BR3 surface. Despite this similarity in binding epitopes, CB1 variants antagonize BAFF-dependent human B-cell proliferation in vitro and are effective at reducing murine B-cell populations in vivo, showing significant promise as therapeutics for human B-cell-mediated diseases.

Receptor-selective mutants of apoptosis-inducing ligand 2/tumor necrosis factor-related apoptosis-inducing ligand reveal a greater contribution of death receptor (DR) 5 than DR4 to apoptosis signaling.

Apoptosis-inducing ligand 2 (Apo2L), also called tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), triggers programmed cell death in various types of cancer cells but not in most normal cells. Apo2L/TRAIL is a homotrimeric protein that interacts with five receptors: death receptor 4 (DR4) and DR5 mediate apoptosis activation, whereas decoy receptor 1 (DcR1), DcR2, and osteoprotegerin counteract this function. Many cancer cell lines express both DR4 and DR5, and each of these receptors can initiate apoptosis independently of the other. However, the relative contribution of DR4 and DR5 to ligand-induced apoptosis is unknown. To investigate this question, we generated death receptor-selective Apo2L/TRAIL variants using a novel approach that enables phage display of mutated trimeric proteins. Selective binding to DR4 or DR5 was achieved with three to six-ligand amino acid substitutions. The DR4-selective Apo2L/TRAIL variants examined in this study showed a markedly reduced ability to trigger apoptosis, whereas the DR5-selective variants had minimally decreased or slightly increased apoptosis-inducing activity. These results suggest that DR5 may contribute more than DR4 to Apo2L/TRAIL-induced apoptosis in cancer cells that express both death receptors.

Design, construction, and in vitro analyses of multivalent antibodies.

Some Abs are more efficacious after being cross-linked to form dimers or multimers, presumably as a result of binding to and clustering more surface target to either amplify or diversify cellular signaling. To improve the therapeutic potency of these types of Abs, we designed and generated Abs that express tandem Fab repeats with the aim of mimicking cross-linked Abs. The versatile design of the system enables the creation of a series of multivalent human IgG Ab forms including tetravalent IgG1, tetravalent F(ab')2, and linear Fab multimers with either three or four consecutively linked Fabs. The multimerized Abs target the cell surface receptors HER2, death receptor 5, and CD20, and are more efficacious than their parent mAbs in triggering antitumor cellular responses, indicating they could be useful both as reagents for study as well as novel therapeutics.