The activation fragment of PAR2 is elevated in serum from patients with rheumatoid arthritis and reduced in response to anti-IL6R treatment
Target selection: neo-epitope
PRO-PAR2 target sequence is a N-terminal neo-epitope formed after proteolytic cleavage of the full length PAR2 receptor (SKGR36↓37SLIGKV). A sequence of 10 amino acids adjacent to the cleavage site (↓27QGTSRSSKGR36↓) was chosen as the target (Fig. 5). The sequence was analyzed for homology to other human and animal proteins as well as for its uniqueness relative to the entire human proteome by Basic Local Alignment Search Tool (BLAST) using NPS@: Network Protein Sequence Analysis. Synthetic peptides (American Peptide, California, US and GenScript, Piscataway, NJ, US) were used for monoclonal antibody production as well as validation of the immunoassay that was developed. An overview of the peptides used is shown in Table 4.
Generation of monoclonal antibody
For the initiation of the development of this biomarker assay, antibodies were raised against the 10 amino acid sequence 27QGTSRSSKGR35 next to the cleavage site of PAR2. The method for the generation of monoclonal antibodies has been previously described by Shu et al.34. Briefly, 6–7-week-old Balb/C mice were immunized by subcutaneous injection of 200 μL emulsified antigen with 50 μg immunogenic peptide. Immunizations were repeated every 2nd week until stable serum antibody titer levels were obtained. Mice with the highest titer were selected for fusion and boosted intravenously with immunogen 3 days before isolation of the spleen for cell fusion. The mouse spleen cells were following fused with SP2/0 myeloma cells as described by Gefter et al.35, to produce hybridoma cells.
After fusion, the clones were plated into 96-well microtiter plates for further growth. Hybridoma cell lines with the highest specificity to the selection peptide were selected for sub-cloning according to the ‘limiting dilution’ method ensuring the growth of single clones. Two clones were used for further growth and for antibody generation. Supernatants from these clones were collected and antibodies purified using protein HiTrap Protein G HP columns according to the manufacturer’s instructions (GE Healthcare Life Sciences, Little Chalfont, UK). The clone with the highest specificity and selectivity was selected for assay development.
Development and optimization of the PAR2 immunoassay
A competitive chemiluminescence immunoassay (CLIA) was developed with a monoclonal antibody targeting PAR2 pro-fragment (PRO-PAR2). Briefly, the development process included optimization of the concentrations of biotinylated peptide (coater) and detection antibody to achieve the best signal to noise ratio, determination of the optimal buffer concentrations, incubation times and temperatures, generation of the standard curve, determination of the measuring range and technical validation36.
In the final assay procedure 96-well streptavidin-coated plates (cat. no. 655995, Greiner Bio-One, Austria) were coated with 100 μL/well of 2 ng/mL biotinylated peptide dissolved in assay buffer (25 mM PBS-BTB, 1% BSA, 0.1% Tween-20, 0.36% Bronidox, 8 g/L NaCl, pH 7.4) and incubated for 30 min at 20 °C in the dark with shaking at 300 rpm. Plates were then washed five times in washing buffer (20 mM TRIS, 50 mM NaCl, pH 7.2). 20 μL of selection peptide, control or sample were added to appropriate wells, followed by 100 μL of horseradish peroxidase (HRP) conjugated monoclonal antibody (final concentration 200 ng/mL). Plates were incubated for 20 h at 4 °C with shaking and followingly washed 5 times in washing buffer. One hundred μL per well of BM Chemiluminescence ELISA Substrate (POD) (cat. no. 11582950001, Roche, Switzerland) solution was added to the plate and incubated for 3 min at 20 °C with shaking. Lastly, the plate was analyzed by a SpectraMax M5 reader (Molecular Devices, CA, USA) with settings: All wavelengths. SoftMax Pro Software was used for data analysis. Sample concentration was determined based on a standard curve generated by serial dilution of the selection peptide and plotted using a 4-parametric mathematical model. Standard concentrations used for the curve were 250, 125, 62.50, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49 and 0 ng/mL.
Technical validation of the PAR2 immunoassay
Different technical tests were performed to assess the quality, robustness, and other parameters of the ELISA assay. The antibody specificity test was performed by assessing reactivity towards the selection peptide, an elongated peptide, and a truncated peptide. Robustness of the assay was evaluated with the intra- and inter-assay variations, with the intra-assay variation to be the mean coefficient of variation (CV%) within the plates and the inter-assay variation the mean CV% among the plates. These variations were calculated after ten independent runs including 7 kit controls, 3 of them were healthy human serum samples and 4 of them were selection peptide spiked in assay buffer. Each run consisted of two replicates of double determinations of the samples. Measurement range was defined as the range between LLMR (lower limit of measurement range) and ULMR (upper limit of measurement range) of the assay and was determined based on the 10 individual standard curves of the intra-inter variation. Apart from the measurement range, the lower limit of detection (LLOD) was determined as the concentration of three standard deviations above the zero standard (buffer).
To assess the linearity of the assay, four healthy human serum samples were used and diluted in assay buffer and the percentage recovery of several dilutions from the samples diluted with assay buffer was calculated. Accuracy was verified by spiking two-fold dilutions of the 36-amino-acid full length pro-peptide in three healthy human serum samples. The recovery was determined as the percentage recovery of the expected concentration, after combining the concentration of the analyte present in serum sample and the peptide concentration. The analyte stability was determined for three healthy human serum samples which were incubated at either 4 °C or 20 °C for 2, 4, 24 and 72 h, respectively. One more test was performed to assess the stability of the samples by subjecting them to four freeze and thaw cycles and calculating the percentage recovery using the zero freeze/thaw cycle as reference samples. Finally, interference was measured in healthy human serum spiked with two different concentration of biotin (low = 5 ng/mL, high = 100 ng/mL), hemoglobin (low = 2.5 mg/mL, high = 5.0 mg/mL), or lipids (low = 1.5 mg/mL, high = 5.0 mg/mL) and calculated as the percentage recovery of the analyte in non-spiked serum.
In vitro cleavage of PAR2
In vitro cleavage with recombinant human PAR2 as the substrate (cat.no. H00002150-P01, Novus Biologicals, Centennial, Colorado, USA) and matriptase as the enzyme (cat.no. 3946-SEB-010, R&D systems, Minneapolis, Minnesota, USA,) was performed. Cleavage buffer for the reaction contained 50 mM Tris-HCL, 50 mM NaCL and 0.01% Tween 20. Cleavage buffer only, PAR2 recombinant protein in cleavage buffer, and matriptase in cleavage buffer without PAR2 protein were used as controls. All ingredients were mixed thoroughly and incubated for 2 h and 24 h at 37 °C. Cleavage products were stored at − 20 °C until further analysis.
Human synovial explants (HEX) model
Explants were isolated from synovial membranes of OA patients who underwent total knee replacement. The use and the methodology of HEX experiments have been previously described37. Briefly, explants were cultured for 21 days with media being changed every 2–3 days and the following treatments were added accordingly: (1) medium without any treatment (w/o), (2) OSM [10 ng/mL] combined with TNF-α [20 ng/mL] (O + T), (3) recombinant human matriptase [30 nM] (matriptase), 4) O + T combined with matriptase (O + T + matriptase). The culture media were harvested every 2–3 days and stored until biochemical analysis. Metabolic activity was evaluated by the AlamarBlue assay (Thermo Fisher Scientific, Waltham, MA, USA) on day 0, 14, and day 21 when the experiment was terminated.
Serum samples from individuals with OA and RA were acquired from Discovery Life Science (AL, USA). Serum from healthy donors were used as controls and were obtained from BioIVT (West Sussex, UK). All samples were processed immediately after collection according to standard operating procedures and stored at − 80 °C for long-term storage. Samples were measured un-diluted in duplicates and CVs were below 15%.
A subset of serum samples from a phase III RA clinical trial was measured. The RADIATE study has been previously described by Karsdal et al.38 (clinicalTrials.gov identifier: NCT00106522). This study is a randomized, double-blind, placebo-controlled, parallel-group phase III trial with RA patients. Patients were randomly assigned to either tocilizumab (4 or 8 mg/kg) or placebo intravenously every 4 weeks, along with concomitant stable methotrexate (10 to 25 mg weekly) in all treatment groups. Clinical assessment and blood samples were obtained at baseline and after 16 weeks of follow-up. Samples were processed immediately and stored at − 80 °C until analysis. In the current study only samples from the placebo and the 8 mg/kg treatment groups were used. Furthermore, only patients with remaining baseline and follow up sample were included. Samples were measured in duplicates with CV below 15%. Due to unavailability of material a subset of patients was measured and any statistical differences in the demographic characteristics between the two groups (total and subset) at baseline were investigated. Differences in the demographic characteristics between placebo and treated patients were investigated as well.
Means, standard deviations, medians, and ranges were used to describe continuous variables, while counts/frequencies and percentages were used to describe categorical variables. Statistical differences between categorical variables were assessed using Chi-square test. For the cleavage experiment, one-way ANOVA with Dunnett’s multiple comparison test was performed to assess the difference in the mean levels of the PRO-PAR2 in the different experimental groups setting as control the protein-only (PAR2) group. Area under the curve (AUC) from treated synovial explants was compared to the untreated group using Kruskal–Wallis test with Dunn’s post-hoc test for multiple comparisons. For continuous variables, analysis of covariance (ANCOVA) was performed to examine the difference in mean levels of PRO-PAR2 among healthy controls, OA, and RA patients adjusting for age and gender. Receiver operating characteristics (ROC) analysis was performed to assess the diagnostic accuracy of the PRO-PAR2. Wilcoxon’s paired signed-rank test was used to determine if there is a difference in mean levels of PRO-PAR2 between baseline and week 16 for the 2 different groups (placebo and treated). Mann–Whitney test was performed to assess the difference in the percentage change from baseline between placebo and treated patients. For all statistical analysis performed, statistical significance was set to 0.05. For the analysis of the data GraphPad Prism, version 8.3.0 (GraphPad Software, Inc., CA, USA) and R version 4.0.3 were used.
The production of monoclonal antibodies performed in mice was approved by the Danish Animal Ethics Council under approval number 2013–15-2934–00,956. All animal experiments were carried according to relevant guidelines and regulations and all protocols were performed according to ARRIVE guidelines. The use of human tissues for the ex vivo experiments was approved by the Danish Scientific Ethics Committee for the Capital Region of Denmark (Den Videnskabsetiske Komité for Region Hovedstaden) under permit number H-D-2007–0084. All experimental protocols were performed according to relevant guidelines and regulations and informed consent was obtained from all participants. Healthy, OA and RA serum samples used for biological validation were commercially available and were ethically obtained, following all applicable HHS/OHRP, ISBER, and NCI/BBRB regulations, guidelines, and best practices. For the collection of samples informed consent was obtained from all the participants. The treated RA serum samples were subset of a clinical study (RADIATE study). The study was conducted throughout North America and Western Europe and was approved by institutional review boards, ethics committees and regulatory authorities. Informed consent was obtained from each patient in compliance with the Helsinki Declaration, and the study was registered at ClinicalTrials.gov (NCT00106522).