Preparation,characterization and application of anti-human OX40 ligand (OX40L) monoclonal antibodies and establishment of a sandwich ELISA for autoimmune diseases detection

OX40L (CD252, TNFSF4), a type II transmembrane protein which like other tumor necrosis factor ligands, involved in the costimulation and differentiation of T cells, functions as a positive signal in immune response. To investigate the biological function of soluble OX40L (sOX40L), three functional anti-OX40L monoclonal antibodies (mAbs) 3D2, 3F7 and 2H3 were obtained by hybridoma technology. Besides, specificity of the mAbs was further demonstrated by ELISA, Western blot and Immunofluorescence experiments. We also developed a novel enzyme-linked immunosorbent assay (ELISA) based on two anti-human OX40L antibodies 3D2 and 3F7 with different epitopes. Using the ELISA system, we found that sOX40L in the sera of healthy donors increases in an age-dependent manner and that enhanced sOX40L expression in some autoimmune diseases especially in rheumatoid arthritis (RA) patients, suggesting the potential diagnostic significance of sOX40L in the autoimmune diseases. Together, these data demonstrate that the existence of circulating sOX40L in human sera might play an important role in immunoregulation.     

A Sensitive,High-Volume,Colorimetric Assay for Protein Phosphatases

Protein phosphatases are intimately involved in a variety of cellular processes, many of which are of interest to the pharmaceutical industry. Phosphatase assays generally employ radioisotopes, making them tedious to perform, costly, and hazardous, while other procedures require antibodies and/or are unsuitable for mass screening efforts. To facilitate screening for inhibitors of the CD45 protein tyrosine phosphatase (PTPase), we have developed a sensitive colorimetric assay, using small volumes in 96-well microtiter plates and read on a standard ELISA plate reader. The assay was sensitive down to 0.5 nmol of released phosphate and can be easily run by robotics to assay thousands of compounds in a day. The assay is sparing of reagents and has been successfully used with all classes of phosphatases. The reagents are nonradioactive, readily obtainable, and minimal in cost. This assay should facilitate the search for specific inhibitors of phosphatases.     

Development of a miniaturized assay for the high-throughput screening program for poly(ADP-ribose) polymerase-1

Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, and neurodegenerative disease. With these emerging medicinal interests, various screening programs have identified small molecules that inhibit PARP with reasonable potencies. However, the increasing numbers of diverse small molecules generated through combinatorial chemistry necessitate the use of robust assays with good sensitivity and specificity for use as a high-throughput screening (HTS) program. Here, we report the development and the validation of a nonisotopic PARP-1 assay suitable for HTS by converting a biotinylated NAD-based colorimetric assay to a miniaturized 384-well plate format. Comparing with the conventional methods, this miniaturized PARP-1 inhibition assay was equally sensitive with excellent reproducibility and cost-effectiveness. Because nonisotopic PARP-1 inhibition assays are widely used, the methodology described in this article can expand the feasibility of this assay as a high-throughput assay for screening of PARP-1 inhibitors from a random chemical library.     

ELISA on a microchip with a photodiode for detection of amphetamine in plasma and urine

A rapid and sensitive assay was developed for the detection of amphetamine in plasma and urine. The method relies on the principle of competitive ELISA (enzyme-linked immunosorbent assay). A flow microchip with a total volume of 7 microL was used for the development of a chemiluminescent ELISA technique. Solutions, samples, and the chemiluminescence substrate were injected by a flow system, and a photodiode detector was used to measure the light intensity. The incubation time of the competitor (competition phase) was reduced to 10 min. Calibration curves corresponding to analyte concentrations ranging from 40 to 1,000 microg/L in urine samples and from 6 to 96 microg/L in plasma samples were obtained. The detection limits were in the region of 20 and 6 microg/L in urine and plasma, respectively. The main focus of the work was on speed, reliability, reproducibility, and operational stability of the assay. This method was proven readily adaptable to automation and provided reproducible results.     

Evaluation of Acoustic Membrane Microparticle (AMMP) Technology for a Sensitive Ligand Binding Assay to Support Pharmacokinetic Determinations of a Biotherapeutic

Achieving the required sensitivity can be a challenge in the development of ligand binding assays for pharmacokinetic (PK) determinations of biotherapeutics. To address this need, BioScale’s Acoustic Membrane Microparticle (AMMP) technology was evaluated for the quantification of a PEGylated domain antibody (dAb) biotherapeutic. Previous uses of this technology had shown utility in biomarker and process development applications and this is the first application, to our knowledge, for PK determinations. In this evaluation, AMMP was capable of delivering a sensitivity of 0.750 ng/mL, which surpasses the sensitivity requirements for the majority of assays to support PK determinations. This evaluation demonstrates that this emerging technology has the ability to produce the required sensitivity, reproducibility, and selectivity needed to meet the industry’s standards for PK analysis.     

HSS体外生物学活性测定的MTT比色法优化及改进

筛选测定HSS体外生物学活性的MTT比色法的最佳实验条件.分析比较不同条件下MTT显色反应的特点,确定适用于人肝癌细胞株SMMC7721的最佳实验条件,并采用优化条件检测不同剂量下HSS的体外生物学活性.结果表明SMMC7721细胞数量与MTT比色法的吸光度成正相关,线性良好.正交实验优化条件为5×104cells/ml细胞密度,6h后加入HSS,最适浓度为100μg/ml,作用36h后加入MTT溶液.MTT用量50μg,孵育时间6h,DMSO溶解反应产物Formazan,于570nm处测定吸光值.在优化条件下测定HSS体外生物学活性,灵敏度明显高于已发表的方法.一定剂量范围内,活性随剂量增加而增大,超过一定剂量后活性反而下降.应用优化的MTT比色法,以SMMC7221细胞株,检测HSS体外生物学活性是一种灵敏而稳定的方法.     

Therapeutic strategies for the costimulatory molecule OX40 in T-cell-mediated immunity

The T cell co-stimulatory molecule OX40 and its cognate ligand OX40L have attracted broad research interest as a therapeutic target in T cell-mediated diseases. Accumulating preclinical evidence highlights the therapeutic efficacy of both agonist and blockade of the OX40–OX40L interaction. Despite this progress, many questions about the immuno-modulator roles of OX40 on T cell function remain unanswered. In this review we summarize the impact of the OX40–OX40L interaction on T cell subsets, including Th1, Th2, Th9, Th17, Th22, Treg, Tfh, and CD8⁺ T cells, to gain a comprehensive understanding of anti-OX40 mAb-based therapies. The potential therapeutic application of the OX40–OX40L interaction in autoimmunity diseases and cancer immunotherapy are further discussed; OX40–OX40L blockade may ameliorate autoantigen-specific T cell responses and reduce immune activity in autoimmunity diseases. We also explore the rationale of targeting OX40–OX40L interactions in cancer immunotherapy. Ligation of OX40 with targeted agonist anti-OX40 mAbs conveys activating signals to T cells. When combined with other therapeutic treatments, such as anti-PD-1 or anti-CTLA-4 blockade, cytokines, chemotherapy, or radiotherapy, the anti-tumor activity of agonist anti-OX40 treatment will be further enhanced. These data collectively suggest great potential for OX40-mediated therapies.     

OX40-OX40L相互作用对C57BL/6小鼠淋巴细胞活性的影响

目的探讨OX40-OX40L相互作用对C57BL/6小鼠脾淋巴细胞增殖及细胞因子表达的影响。方法应用MTT法检测小鼠淋巴细胞增殖;ELISA法检测淋巴细胞表达细胞因子IL-4、IL-2及INF-γ表达。结果与对照组比较,Anti-OX40特异性刺激OX40-OX40L轴后,小鼠淋巴细胞增殖增强,分泌IL-2和INF-γ含量明显增加,48h细胞增殖最佳;IL-4含量无明显变化。应用特异性anti-OX40L抗体阻断OX40-OX40L轴后,淋巴细胞增殖及分泌细胞因子IL-2和INF-γ含量明显受到抑制。结论 OX40-OX40L相互作用能够调控C57BL/6小鼠脾淋巴细胞增殖及促炎性细胞因子表达。     

Systematic Verification of Bioanalytical Similarity Between a Biosimilar and a Reference Biotherapeutic: Committee Recommendations for the Development and Validation of a Single Ligand-Binding Assay to Support Pharmacokinetic Assessments

For biosimilar drug development, it is critical to demonstrate similar physiochemical characteristics, efficacy, and safety of the biosimilar product compared to the reference product. Therefore, pharmacokinetic (PK) and immunogenicity (antidrug antibody, ADA) assays that allow for the demonstration of biosimilarity are critical. Under the auspices of the American Association of Pharmaceutical Scientists (AAPS) Ligand-Binding Assay Bioanalytical Focus Group (LBABFG), a Biosimilars Action Program Committee (APC) was formed in 2011. The goals of this Biosimilars APC were to provide a forum for in-depth discussions on issues surrounding the development and validation of PK and immunogenicity assays in support of biosimilar drug development and to make recommendations thereof. The Biosimilars APC’s recommendations for the development and validation of ligand-binding assays (LBAs) to support the PK assessments for biosimilar drug development are presented here. Analytical recommendations for the development and validation of LBAs to support immunogenicity assessments will be the subject of a separate white paper.

Electronic supplementary material

The online version of this article (doi:10.1208/s12248-014-9669-5) contains supplementary material, which is available to authorized users.KEY WORDS: bioanalytical method validation, biological product, biosimilar, ligand-binding assay, pharmacokinetic     

Understanding and Reducing the Experimental Variability of In Vitro Plasma Protein Binding Measurements

The experimental measurement of plasma protein binding is a useful in vitro Absorption Distribution Metabolism and Excretion(ADME) assay currently conducted in both screening and definitive early development candidate modes. The fraction unbound is utilized to calculate important pharmacokinetic (PK) parameters such as unbound clearance and unbound volume of distribution in animals that can be used to make human PK and dose predictions and estimate clinically relevant drug–drug interaction potential. Although these types of assays have been executed for decades, a rigorous statistical analysis of sources of variability has not been conducted because of the tedious nature of the manual experiment. Automated conduct of the incubations using a 96-well equilibrium dialysis device as well as high-throughput liquid chromatography–mass spectrometry quantitation has now made this level of rigor accessible and useful. Sources of variability were assessed including well position, day-to-day, and site-to-site reproducibility. Optimal pH conditions were determined using a design of experiments method interrogating buffer strength, CO₂% and device preparation conditions. Variability was minimized by implementing an in-well control that is concurrently analyzed with new chemical entity analytes. Data acceptance criteria have been set for both the in-well control and the range of analyte variability, with a sliding scale tied to analyte-binding characteristics.     

Individualized Dosing of Therapeutic Monoclonal Antibodies—a Changing Treatment Paradigm?

The introduction of monoclonal antibodies (mAbs) to the treatment of inflammatory bowel disease (IBD) was an important medical milestone. MAbs have been demonstrated as safe and efficacious treatments of IBD. However, a large percentage of patients either fail to respond initially or lose response to therapy after a period of treatment. Although there are factors associated with poor treatment outcomes in IBD, one cause for treatment failure may be low mAb exposure. Consequently, gastroenterologists have begun using therapeutic drug monitoring (TDM) to guide dose adjustment. However, while beneficial, TDM does not provide sufficient information to effectively adjust doses. The pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs are complex, with numerous factors impacting on mAb PK and PD. The concept of dashboard-guided dosing based on Bayesian PK models allows physicians to combine TDM with factors influencing mAb PK to individualize therapy more effectively. One issue with TDM has been the slow turnaround of assay results, either necessitating an additional clinic visit for a sample or reacting to TDM results at a subsequent, rather than the current, dose. New point-of-care (POC) assays for mAbs are being developed that would potentially allow physicians to determine drug concentration quickly. However, work remains to understand how to determine what target exposure is needed for an individual patient, and whether the combination of POC assays and dashboards presents a safe approach with substantial outcome benefit over the current standard of care.     

The importance of performing in vitro cytotoxicity testing before immunomodulation evaluation

This study uses the well known sensitive and reproducible human peripheral blood lymphocyte (PBL) assay as a model for the possible discrimination between cytotoxic and/or immunomodulative properties of 5 reference compounds: cadmium chloride (CdCl2): didodecyl-dimethyl ammonium bromide (DDAB); cyclosporin (CsA); dexamethasone (DEXA); and theophylline. Two colorimetric cytotoxicity assays were employed, the thiazolyl blue tetrazolium bromide and neutral red, and the H-thymidine DNA incorporation assay on phytohemagglutinin stimulated or non-stimulated PBL was used, as a parameter of cell mediated immunity. This study demonstrated the importance of performing colorimetric or other cytotoxicity assays before any investigation of mechanisms of action of different substances on immunomodulation and relative human risk assessment. This study showed that CdCl2 and DDAB have a cytotoxic rather than immunomodulative effect on PBL and that CsA, DEXA and to a lesser extent theophylline have immunomodulative rather than cytotoxic effects on PBL within the range of the concentrations studied.     

The bioanalysis of the monoclonal antibody trastuzumab by high-performance liquid chromatography with fluorescence detection after immuno-affinity purification from human serum

For the quantification of therapeutic monoclonal antibodies in biological specimens, enzyme-linked immunosorbent assay (ELISA) is the most widely used technique. ELISA's have some limitations and therefore alternative analytical techniques are being explored. In this study we describe the development of a bioanalytical assay using high-performance liquid chromatography (HPLC) coupled with fluorescence detection for the bioanalysis of the monoclonal antibody trastuzumab. Different extraction procedures were explored, like isolation using protein A and protein G. Finally a method using immuno-affinity purification has been developed. Trastuzumab is isolated from human serum using sepharose coupled with anti-trastuzumab idiotype antibodies. After extraction samples are injected onto a Zorbax 300SB C₈ column at 75 °C using the organic solvents isopropanol and acetonitrile with high eluotropic strengths. The assay quantifies trastuzumab from 5 to 40 μg/mL in human serum with accuracies <20%. Samples with concentrations above the upper limit of quantification (>ULOQ; >40 μg/mL) can be diluted 5 times with control human serum prior to sample pre-treatment. The assay can now be used to analyse serum samples of patients treated with trastuzumab. The obtained results are comparable to those obtained using ELISA. This is the first report describing a bioanalytical assay using HPLC and fluorescence detection for the quantification of a monoclonal antibody at the intact protein level in human serum. This unique approach has the advantage compared to ELISA that a HPLC separation step is introduced to improve the selectivity. This method is a potential alternative to ELISA to support pharmacokinetic evaluations. However, for purification of trastuzumab from serum anti-idiotype antibodies are necessary. These anti-idiotype antibodies are also used in ELISA and as ELISA is more sensitive and less labor-intensive, ELISA probably remains the analytical technique of first choise.     

A sensitive chemiluminescent enzyme immunoassay for the bioanalysis of carboxyl-terminal B-chain analogues of human insulin.

Quantification of analogues of human insulin in biological matrices is complicated by differences in their immunoreactivity and the presence of both the analogue and endogenous concentrations of insulin in test samples. To facilitate pharmacokinetic comparisons of carboxyl-terminal B-chain analogues of human insulin, we undertook development of a sensitive ELISA. The ELISA detection method was optimized systematically to permit routine analysis of 10-microl serum samples. Accordingly, a noncompetitive 'sandwich' chemiluminescent ELISA was validated for the quantification of carboxyl-terminal B-chain insulin analogues in human serum over a concentration range from 5 to 3125 pM. The mean bias (RE%) within the validated range varied from -10.3 to 4.3%, with an intermediate precision (inter-assay CV%) from 4.2 to 11.5%. The two-sided 90% expectation tolerance interval for total measurement error was within +/-25% of the nominal concentration for all levels of validation samples. Insulin lispro, human insulin, proinsulin, despentapeptide insulin (DPI) and porcine insulin displayed comparable crossreactivity in the ELISA. Potential utility of the new assay for insulin bioanalysis in nonhuman species was investigated by assessing the pharmacokinetic profile of DPI in rats following administration of a single subcutaneous dose. The sensitive chemiluminescent detection method is simple to perform and should be readily adaptable for ELISAs of other therapeutic proteins.     

Recommendations for Selection and Characterization of Protein Biomarker Assay Calibrator Material

As biomarkers continue to become an integral part of drug development and decision-making, there are increased expectations for reliable and quantitative assays. Protein biomarker assay results are directly influenced by the calibrator material. The selection of calibrator material presents many challenges that impact the relative accuracy and performance of the assay. There is an industry-wide challenge finding reliable and well-characterized calibrator material with good documentation. Several case studies are presented that demonstrate some of the challenges involved in selecting appropriate calibrators along with the resolutions that were ultimately applied. From these experiences, we present here a set of recommendations for selecting and characterizing calibrator material based on the intended purpose of the assay. Finally, we introduce a commutability approach, based on common clinical chemistry practices, which can be used to demonstrate inter-changeability with calibrator materials across multiple lots and technology platforms for all types of protein biomarker assays.     

A novel method for quantitative measurement of a therapeutic monoclonal antibody in the presence of its target protein using enzymatic digestion

Over the past decades, the use of therapeutic monoclonal antibodies (mAbs) has become an important strategy in the treatment of various diseases. To enable pharmacokinetic (PK) assessment, specific immunoassays need to be developed to quantify mAbs in blood. In these assays, the presence of bound target protein can lead to severe underestimation of mAb concentration. Here we describe a novel approach for the quantification of total (free plus bound) human mAb concentration, in human and non-human primate serum, in the presence of a high level of target protein. The method is based on sample digestion with pepsin under optimized conditions to fully digest the target while keeping the mAb in the form of immunoreactive fragments. The quantification of mAb is then performed by ELISA without interference from the target. This method allows accurate quantification of as low as 50 ng/ml mAb in the presence of up to 100-fold target molar excess. Intra- and inter-run precision is better than 10%, and intra- and inter-run accuracy in the range of 89.3–106.7%. In conclusion, this general and simple approach allows the accurate and sensitive measurement of preclinical and clinical samples avoiding target interference.     

Rapid Microfluidic Assay for the Detection of Botulinum Neurotoxin in Animal Sera

Potent Botulinum neurotoxins (BoNTs) represent a threat to public health and safety. Botulism is a disease caused by BoNT intoxication that results in muscle paralysis that can be fatal. Sensitive assays capable of detecting BoNTs from different substrates and settings are essential to limit foodborne contamination and morbidity. In this report, we describe a rapid 96-well microfluidic double sandwich immunoassay for the sensitive detection of BoNT-A from animal sera. This BoNT microfluidic assay requires only 5 μL of serum, provides results in 75 min using a standard fluorescence microplate reader and generates minimal hazardous waste. The assay has a <30 pg·mL⁻¹ limit of detection (LOD) of BoNT-A from spiked human serum. This sensitive microfluidic BoNT-A assay offers a fast and simplified workflow suitable for the detection of BoNT-A from serum samples of limited volume in most laboratory settings.     

Development of a high-throughput screening-amenable assay for human poly(ADP-ribose) polymerase inhibitors

INTRODUCTION: Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. Inhibitors of PARP have been shown to reduce infarct size in animal models of myocardial ischemia. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, head trauma, and neurodegenerative disease, and as an adjunct therapy with chemotherapeutic agents/radiation in cancer therapy. METHODS: Assays reported in the literature and commercially available PARP assay kits are labor-intensive, use radioactive reagents, use antibodies, and are not readily amenable to high throughput screening (HTS) [corrected]. Here we report the development and the validation of a nonradioactive PARP assay suitable for HTS. This is a biotinylated NAD-based colorimetric assay in a 96-well plate format. RESULTS: The assay is sensitive, reproducible, and easy to use. The IC(50) values generated for the known PARP inhibitors are in agreement with those generated using the commercial radioactive kit and those reported in the literature. DISCUSSION: The present study demonstrates a sensitive and reproducible methodology capable of screening human PARP inhibitors in high-throughput format.     

Small-molecule modulators of the OX40–OX40 ligand co-stimulatory protein–protein interaction

Background and Purpose

The OX40–OX40L protein–protein interaction (PPI) is an important cell-surface signalling co-stimulatory regulator within the TNFR superfamily (TNFRSF) and a promising therapeutic target for immunomodulation. PPIs are difficult to modulate using small-molecules. Here, we describe the identification of a small-molecule OX40 modulator and confirm its partial agonist character.

Experimental Approach

Cell-free screening assays were developed and used to identify OX40–OX40L inhibitors. Modified versions of this assay were used to elucidate the binding partner and the binding nature of active compounds. OX40-transfected sensor cells with NF-κB reporters were constructed and used to confirm and characterize activity and specificity. Immunomodulatory activity and partial agonist nature were further confirmed by ex vivo T-cell polarization assays.

Key Results

Several compounds that concentration-dependently affected OX40-OX40L were identified. Cell assays indicated that they were partial agonists with low micromolar potency and adequate selectivity. Under polarizing conditions based on TGF-β, the most promising compound mimicked the effect of an agonistic anti-OX40 antibody in suppressing regulatory T-cell generation and diverting CD4⁺CD62L⁺Foxp3⁻ cells to T_H9 phenotype in vitro.

Conclusions and Implications

We identified, to our knowledge, the first small-molecule compounds able to interfere with OX40–OX40L binding and, more importantly, to act as partial agonists of OX40. This is particularly interesting, as small-molecule agonism or activation of PPIs is considered unusually challenging and there are only few known examples. These results provide proof-of-principle evidence for the feasibility of small-molecule modulation of the OX40–OX40L interaction and for the existence of partial agonists for TNFRSF-PPIs.