Bioanalytical Methods

An immunoprecipitation liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been developed and validated to measure total soluble TL1A in human serum.¹⁶

An immunoprecipitation LC-MS/MS assay was developed and validated to measure free soluble TL1A in human colon tissue. Tissue samples were homogenized using a BioSpec BeadBeater, and free TL1A was isolated from human colon tissue using immuno-enrichment technology. The samples were incubated and shaken with a biotinylated DcR3 capture reagent at 4°C overnight. Dynabeads T1 streptavidin-coupled magnetic beads were added to each sample and incubated at room temperature for 1 hour to extract TL1A bound to biotinylated DcR3capture reagent. The beads were then washed 3 times, followed by elution of free soluble TL1A from the beads under acidic conditions. After the addition of the extended-sequence, stable, isotope-labeled peptide (internal standard) to each sample, trypsin digestion was performed at 37°C overnight. All sample processing was performed in a 96-well format on an automated liquid handling robot (Microlab STAR, Hamilton, Bonaduz, Switzerland). A 120-µL sample extract was injected onto a 3-dimensional Dionex UltiMate 3000 nano-LC system comprising a conventional flow immunoaffinity capture with a custom 2.1-mm inner diameter (ID) antipeptide antibody column, in addition to an elution to a 300-µm ID C18 trap column and a 75-µm ID nano-LC column. The signature peptide was eluted from the nano-LC column with a mobile phase gradient at a flow rate of 0.6 µL/min. A Thermo Scientific Vantage Triple Quadrupole mass spectrometer, with a Thermo Scientific EASY-Spray ionization source, was used for MS/MS analysis. One selected transition for the signature peptide was used to quantify TL1A in the positive ion mode, and all data were normalized to the internal standard response. The validated analytical range was 10 to 400 pg/mL. The tissue assay was qualified and was precise and accurate with an interbatch imprecision of <14.6% and interbatch inaccuracy of –0.8% to 6.0% at all concentrations investigated during assay validation. Samples with low but valid values were used in the final analyses. For the samples that were below the level of quantification (BLQ), sample values were rescued by testing whether the total protein levels of these samples were accurately measured; and if the total protein of these samples was measured with confidence, the imputed values of the BLQ/√2 were used in the association analyses. Otherwise, the samples were labeled as missing.

Transcriptomic profiling of gut biopsies from participants with UC was evaluated using RNA sequencing technology. All samples were extracted from blood and tissue, and the library was prepared by BGI Americas Corporation using a GLOBINclear+TrueSeq Stranded mRNA sample preparation kit. Next generation sequencing was performed using the Illumina HiSeq4000 with a read length of 100 PE, resulting in 40 million reads.

Transcriptomic analysis was performed by estimating the fold change (FC) for the comparisons of inflamed and noninflamed tissue at baseline and change from baseline under the general framework for linear models using limma and voom packages.¹⁷ The P values from the paired t test were adjusted for multiple hypotheses using the Benjamini-Hochberg procedure, which controls for the false discovery rate (FDR).¹⁸ Differences in baseline gene expression between inflamed and noninflamed biopsies were calculated. The change from baseline in responders and nonresponders was calculated using a linear mixed-effect model using time, tissue, and response (defined as responder and nonresponder) as factors. This analysis was then used to correlate transcriptomic changes with clinical response. The primary efficacy end point was the endoscopic index at week 14, defined by a Mayo endoscopic subscore of 0 or 1 without friability. To ensure objective and consistent assessment of the primary end point, the Mayo endoscopic subscore was determined through blinded, centrally read colonoscopy images with built-in adjudications.

Enrichment analyses using Ingenuity Pathway Analysis (IPA; QIAGEN, Silicon Valley, California) were performed using treatment-modulated genes that passed a significance cutoff to further explore the potential mechanisms underlying the findings. The P values were calculated using a right-tailed Fisher exact test to determine statistically significant over-representation of genes in enriched pathways. The z scores as reported by IPA predict the activation (positive z score) or inhibition (negative z score) of pathways based on the observed gene expression changes and the underlying knowledge in the database. A P value of < .05 and a z score >2 were established as significant cutoffs for pathway enrichment.