P058 The identification of three linear epitopes on infliximab all map to complementarity-determining regions

Abstract

Abstract Background Infliximab (IFX) is a mainstay of treatment for inflammatory bowel disease (IBD). Its use is limited by the development of anti-drug antibodies (ADA), which can cause loss of response and adverse drug reactions. The antigenic epitopes have not been clearly defined, and large antigenic areas suggested. The aim of this study was to identify clinically relevant distinct epitopes in IBD patients treated with IFX with ADA and different clinical outcomes. Methods Sera were pooled from 8 ADA positive IBD patients (ADA >10 AU/mL; IDKmonitor® Infliximab total ADA) treated with CT -P13 (Remsima® Celltrion Inc., Korea) exhibiting: loss of response, response with therapeutic drug levels, response with subtherapeutic drug levels and adverse drug reactions. A novel whole gene fragment library (GFL) was synthesised using pools of overlapping synthetic oligonucleotides covering the IFX heavy chain and light chain (Twist Bioscience, California, United States). This was cloned into a phage display vector to generate a library >106 clones. Three rounds of selection of the GFL were performed using pooled sera. DNA from the selection outputs were analysed by next generation sequencing (NGS) (Illumina, San Diego, United States). Outputs from one round of selection using individual sera were also analysed by NGS. Results ADA ranged 17->600 AU/mL. 23,383 gene sequences analysed from the GFL showed complete coverage of the IFX gene. After three rounds of selection, DNA sequencing showed the enrichment of three peptides – all overlapping the IFX complementary-determining regions (CDR)- Figure 1. The three peptides enriched: heavy chain peptide 1 included heavy chain CDR 1, light chain peptide 2 overlap light chain CDR 1 and light chain peptide 3 overlap light chain CDR 2. NGS assessment of the outputs from round 1-3 using pooled ADA positive sera confirmed enrichment of the three peptides. NGS analysis of the selections on individual sera showed differences in enrichment profile of the three peptides. Conclusion Using ADA positive sera from IBD patients treated with IFX with differing clinical outcomes, three linear epitopes were identified from a novel whole IFX-GFL. All epitopes overlapped with CDRs which is consistent with being potential sites for neutralising ADA. NGS has suggested that individual ADA positive sera have differing recognition profiles for the selected epitopes. Identifying distinct epitopes may lead to the identification of a biomarker for loss of response to infliximab.