P0002 Identification of Infliximab and Adalimumab -derived peptides using MHC class II antibody mediated immunopeptidomics and mass spectrometry: implications for drug re-design
Authors: Bedke N , Liao H , Nicastri A , Parker R , Dzhoneva A , Noble A , Al-Hillawi L , Brain O , Satsangi J , Ternette N 
Published: January 2025

Abstract

Background
Monoclonal antibodies against tumour necrosis factor (TNF) such as Infliximab (IFX) and Adalimumab (ADA) are used as first line therapy against inflammatory bowel disease (IBD) and have proven efficacy. However, in up to 40% of IBD patients these drugs become ineffective after the first year, principally due to the development of anti-drug immunogenicity. Distinct allelic variants at the HLA DQ1 locus predispose patients to develop immunogenicity to IFX or ADA (1, 2, 3). The aim of this study is to identify peptides derived from IFX and ADA that are presented on class II MHC of antigen presenting cells, in order to determine the epitopes that may play a role in T cell mediated immunogenicity against anti-TNF therapy. This may allow rational drug re-design.

Methods
Monocyte-derived dendritic cells (MDDCS) from 8 different donors, as well as EBV-transformed B cell lines from 3 different donors of known HLA-type were cultured and pulsed with IFX and ADA. Unpulsed cells were used as a negative control. Cells were processed for sequential immune precipitation with HLA-DQ, HLA-DP and HLA-DR antibodies. Peptides were then purified and analysed by mass spectrometry. These were run against a database of known proteins, including IFX and ADA, using the Peaks analytical software and then mapped to the original protein. Peptides were subjected to a binding prediction algorithm, to determine the likelihood of individual peptides binding to each HLA allele combinations. Peptides were then ranked from highest to lowest binder.

Results
Analysis of peptides from all three MHC class II antibody fractions showed that the highest number of peptides immunoprecipitated were between 14 to 16 amino acids in length, in line with the predicted length for class II MHC proteins. Additionally, there was an enrichment of peptides predicted to bind to specific class II MHC molecules, with an average of 77% binders versus 23% non-binders for MDDCs, and 84% binders versus 16 % non-binders for B-cells, after immune precipitation. Several overlapping peptides from the variable regions of both the heavy and light chain of IFX and ADA were identified, some of which are predicted to bind to risk alleles for anti-TNF immunogenicity, including DQA1*05:01, DRB1*11:01 and HLA-DQA1*05:05 (Figures 1 and 2).

Conclusion
We have characterised a series of IFX and ADA derived peptides from MDDCs and B cell lines, that were pulsed with anti-TNF antibody drugs. These peptides represent candidate T cell epitopes that may play a role in eliciting anti-drug antibody responses, and these data will therefore contribute to the understanding of the molecular mechanism of anti-TNF treatment failure.

References
1)Doherty R, Liao H, Satsangi J, Ternette N. Extended Analysis Identifies Drug-Specific Association of 2 Distinct HLA Class II Haplotypes for Development of Immunogenicity to Adalimumab and Infliximab. Gastroenterology. 2020, 159: 784-787.
2)Sazonovs A, Kennedy N, Moutsianas L, et al. HLA-DQA1*05 Carriage Associated With Development of Anti-Drug Antibodies to Infliximab and Adalimumab in Patients With Crohn’s Disease Gastroenterology. 2020, 158:189-199
3)Ternette N, Liao H., Satsangi J. Association of the HLA DQA1*05 allelic gene variants with immunogenicity to anti-TNF therapeutics - important differences between infliximab and adalimumab. Journal of Crohn's and Colitis. 2024