P211 Identification of linear and conformational epitopes on the infliximab molecule

Abstract

<h3>Introduction</h3> Antidrug antibodies (ADA) to infliximab occur commonly, may limit its therapeutic use, and are associated with a range of clinical outcomes. The aim of this study was to identify linear and conformational infliximab epitopes. <h3>Methods</h3> Pooled sera from 8 ADA positive inflammatory bowel disease patients treated with biosimilar infliximab, CT -P13 (Remsima ®, Celltrion, South Korea) with different clinical outcomes were used for selection of two phage display libraries: 1) Whole gene fragment library (GFL) &gt;10⁶ clones to select linear epitopes was synthesised using ~400 infliximab in–frame gene fragments of 150 base pairs with overlapping amino acids (Twist Bioscience, US). After three rounds of selection, phagemid clones were picked and the diversity of sequences compared to pre–selection. 2) Random peptide library (RPL) (Ph.D.™–12, New England BioLabs, US) 10⁹ clones of random 12–mer peptides was used to identify ‘mimitopes’– sequences that mimic conformational epitopes. After four rounds of selection, 70 plaques from pre–selection, rounds 3 and 4 were sequenced from ADA+ and control selections. Bioinformatic epitope prediction tool Pepitope, (http://pepitope.tau.ac.il) predicted the conformational epitopes using the infliximab Fab 3D protein structure. <h3>Results</h3> There was enrichment of three sequences from the GFL: all mapped to complementarity determining regions (CDRs) (Figure 1). From the RPL, three random peptide sequences showed selective enrichment. Conformational mimotopes were predicted using these sequences: RPL light chain peptide 1: SVALNVKRAPVT (predicted mimotope S14 V13 S12 L11 N103 A84 R39 R45 G57 P59 I58 S60), RPL heavy chain peptide 2: YNKETPQTGFRA (predicted mimotope Y201 N162 N204 N206 T212 P126 K124 T123 S122 F153 A121) and RPL heavy chain peptide 3: FHGPSKISSGES (predicted mimotope L45 E46 G44 P41 S40 Q39 V95 T115 T114 G113 E6 S21). There was overlap of selected amino acids (G57 P59 I58 S60) in GFL light chain peptide 3 and RPL light chain peptide 1. <h3>Conclusions</h3> All three linear epitopes identified from the GFL mapped to infliximab CDRs – sites where ADA could affect function and have a neutralising role. The possible position of conformational epitopes has been predicted. Further work is needed to confirm specificity of ADA+ sera for these peptides.