A Monovalent Fab Affinity-Capture and Elution Bridging Immunoassay Overcomes Rheumatoid Factor Interference while Accurately Detecting Antidrug Antibodies.

BACKGROUND: Rheumatoid factor (RF) consists of autoantibodies that bind the fragment crystallizable (Fc) region of human immunoglobulin G (IgG) and present in sera of rheumatoid arthritis (RA) patients. Immunoassays to detect antidrug antibodies (ADA) in RA patient samples may experience interference due to RF binding and crosslinking Fc regions of the capture and detection antibody reagents. To overcome this interference, a novel Fab affinity-capture and elution (ACE)-bridging immunoassay (Fab ACE-Bridge) was developed with monovalent-recombinant Fab to avoid RF interference. METHODS: ACE and ACE-Bridge assays were developed to detect ADA against a therapeutic monoclonal antibody using samples from healthy donors, psoriasis patients, and RA patients. The performance of these assays was compared to a novel Fab ACE-Bridge assay, in which monoclonal antibody was replaced with monovalent Fab. RESULTS: High screening signals in the ACE and ACE-Bridge assays were detected in RA patient samples but not in samples from healthy donors or psoriasis patients. The high screening signals in RA samples did not inhibit to the expected extent in the confirmatory assay, a consistent feature of false-positive screening results. Further investigation revealed RF as the interferent affecting assay performance. Modification of the ACE-Bridge assay by using monovalent Fab eliminated RF interference while allowing for sensitive and drug-tolerant detection of authentic ADA. CONCLUSIONS: RF interfered significantly in traditional ACE and ACE-Bridge assays. Implementation of a novel monovalent Fab ACE-Bridge assay overcame RF interference. The use of monovalent Fab is recommended for immunogenicity assays when assessing ADA in RA patient samples.

Application of a novel drug-tolerant target assay for measuring target engagement when only one epitope remains after therapeutic antibodies bind their targets.

The measurement of proteins with a limited number of available non-overlapping epitopes recognizable by antibodies represents a common challenge for the development of drug-tolerant clinical biomarker assays. For target proteins with two dominant epitopes, only one epitope remains when the other is occupied by the therapeutic antibody. Alternative strategies for overcoming this obstacle have been described in the literature; however, these methods have potential limitations. We have developed a novel method for measuring target engagement when only one epitope remains after therapeutic antibodies bind their analytes. The method combines Affinity Capture Elution (ACE) followed by simultaneous capture and detection of the protein of interest. This novel method has been named ACE-Sandwich. The application of this method is not dependent on the immunoglobulin G subclass of the therapeutic antibody, nor does this method require sample pretreatment. Furthermore, the ACE-Sandwich method is highly sensitive, reproducible, and tolerant to high concentrations of therapeutic antibody.

Affinity capture elution bridging assay: A novel immunoassay format for detection of anti-therapeutic protein antibodies.

BACKGROUND: Increased emphasis on the development of biologics has placed a significant focus on anti-drug antibody (ADA) detection. To address this need, several immunoassay formats have been described for use in characterizing potential immune responses. Two commonly utilized methods include the affinity capture elution (ACE) and bridging formats. While these approaches have been effective in supporting many clinical initiatives, both possess potential disadvantages. Here, we compare these standard methods to a novel format that addresses these noted drawbacks. RESULTS: A novel assay format has been designed to incorporate the benefits of the ACE and bridging methods while overcoming the disadvantages incurred with each approach. The described ACE-Bridge format exhibits excellent sensitivity and precision while providing superior drug tolerance when compared to bridging formats. Further, this assay format is not susceptible to the endogenous target interference that can be an issue in the ACE format. CONCLUSIONS: The ACE-Bridge format provides an often superior option as a screening method to monitor patient ADA responses. This method is unique in its ability to measure ADA in the presence of high circulating endogenous target concentrations (>100 ng/mL) while demonstrating very high drug tolerance.

Development and characterization of a novel ELISA based assay for the quantitation of sub-nanomolar levels of neoepitope exposed NITEGE-containing aggrecan fragments.

Osteoarthritis (OA) is associated with the degradation of aggrecan by aggrecanases (e.g. ADAMTS-4, ADAMTS-5) ultimately leading to the reduction of daily physical activity in aged individuals. The cleavage of aggrecan by aggrecanases generates a series of neoepitope exposed fragments (e.g. NITEGE) in both animal models and osteoarthritic patients. These aggrecan fragments can be used for identifying disease associated biomarkers for the purpose of measuring the efficacy of therapeutic agents in vivo. A monoclonal antibody, 681-3 mab was developed which recognizes the C-terminal neoepitope NITEGE following aggrecan cleavage by aggrecanases. The 681-3 mab has a K(D) of 4.03 x 10(-10) M as determined by Biacore analysis. A polyclonal antibody, NEP522 which specifically binds to intact aggrecan was also developed. These antibodies were used to develop a highly sensitive assay with lower detection limits of 125 pM which was capable of detecting NITEGE fragments in ADAMTS-4/5 digested human aggrecan and in IL-1 alpha stimulated bovine nasal cartilage disk cultures. The NITEGE 681-3/NEP522 sandwich ELISA has applications for screening compounds for aggrecanase(s) inhibitory activity, selection of appropriate OA models, the evaluation of compound efficacy in vivo, as well as the potential to stratify patients for clinical trial design.

Interleukin-12 (IL-12) ameliorates the effects of porcine respiratory and reproductive syndrome virus (PRRSV) infection.

Porcine respiratory and reproductive syndrome virus (PRRSV) disease, one of the most economically significant viral diseases in the swine industry, is characterized by miscarriages, premature farrowing, stillborn pigs, and respiratory disease associated with death and chronic poor performance of nursing and weaned pigs. Interleukin-12 (IL-12) is a key component in driving the development of cell-mediated immunity as well as stimulating interferon-gamma (IFN-gamma) production from T cells and natural killer cells. Although some studies have investigated the use of IL-12 as a vaccine adjuvant in swine, little is known about its effectiveness as a treatment against viral diseases in swine. The present study investigated whether recombinant porcine IL-12 (rpIL-12) enhances the immune response and thereby diminishes the effects of PRRSV infection in young pigs. Interestingly, in vitro experiments demonstrated that rpIL-12 is capable of inducing swine pulmonary alveolar macrophages (PAMs), the target cells of PRRSV, to produce IFN-gamma in a dose and time dependent manner. In addition, in vitro studies also revealed that rpIL-12 treatment was capable of significantly reducing PRRSV viral titers in PAMs. In vivo administration of rpIL-12 significantly decreased PRRSV titers in the lungs and blood of infected animals. Furthermore, treatment with rpIL-12 prevented significant growth retardation in PRRSV-infected animals. Finally, in response to viral antigen recall challenge, PAMs isolated from rpIL-12-treated/PRRSV-infected animals produced greater amounts of IFN-gamma and lesser amounts of interleukin-10 than PAMs isolated from non-rpIL-12-treated/PRRSV-infected animals. Taken together our data indicate that treatment with rpIL-12 may provide an effective approach to control or ameliorate PRRSV-induced disease in swine.

Innate immune response to encephalomyocarditis virus infection mediated by CD1d.

CD1d-reactive natural killer T (NKT) cells can rapidly produce T helper type 1 (Th1) and/or Th2 cytokines, can activate antigen-presenting cell (APC) interleukin-12 (IL-12) production, and are implicated in the regulation of adaptive immune responses. The role of the CD1d system was assessed during infection with encephalomyocarditis virus (EMCV-D), a picornavirus that causes acute diabetes, paralysis and myocarditis. EMCV-D resistance depends on IL-12-mediated interferon-gamma (IFN-gamma) production. CD1d-deficient mice, which also lack CD1d-reactive NKT cells, were substantially more sensitive to infection with EMCV-D. Infected CD1d knockout mice had decreased IL-12 levels in vitro and in vivo, and indeed were protected by treatment with exogenous IL-12. IFN-gamma production in CD1d knockout mice was decreased compared with that in wild-type (WT) mice in response to EMCV-D in vitro, although differences were not detected in vivo. Treatment with anti-asialo-GM1 antibody, to deplete NK cells, caused a marked increase in susceptibility of WT mice to EMCV-D infection, whereas CD1d knockout mice were little affected, suggesting that NK-cell-mediated protection is CD1d-dependent. Therefore, these data indicate that CD1d is essential for optimal responses to acute picornaviral infection. We propose that CD1d-reactive T cells respond to early immune signals and function in the innate immune response to a physiological viral infection by rapidly augmenting APC IL-12 production and activating NK cells.