HLAII peptide presentation of infliximab increases when complexed with TNF

Abstract

CD4+ T-cell activation through recognition of Human Leukocyte Antigen II (HLAII)-presented peptides is a key step in the development of unwanted immune response against biotherapeutics, such as the generation of anti-drug antibodies (ADA). Therefore, the identification of HLAII-presented peptides derived from biotherapeutics is a crucial part of immunogenicity risk assessment and mitigation strategies during drug development. To date, numerous CD4+ T-cell epitopes have been identified by HLAII immunopeptidomics in antibody-based biotherapeutics using either their native or aggregated form. Antibody-target immune complexes have been detected in patients with ADA and are thought to play a role in ADA development by enhancing the presentation of CD4+ T-cell epitopes at the surface of antigen presenting cells (APCs). The aim of this study was to investigate the effect of biotherapeutic antibody-target immune complexes on the HLAII peptide presentation of biotherapeutics in human primary monocyte-derived dendritic cells (DCs). The trimeric tumor necrosis factor (TNF) and its biotherapeutic antagonists infliximab (INFL), adalimumab (ADAL), and a single armed Fab' were used as a model system. The HLAII immunopeptidome of DCs loaded with antagonists or their immune complexes with TNF was analyzed by trapped ion mobility time-of-flight mass spectrometry (timsTOF MS) leading to the identification of ~ 12,000 unique HLAII-associated peptides per preparation. Anti-TNF sequences were detected at a median of 0.3% of the total immunopeptidome, against a majority background of peptides from endogenous and media-derived proteins. TNF antagonist presentation spanned the variable and constant regions in a widespread manner in both light and heavy chains, consistent with previously discovered HLAII peptides. This investigation extends the collection of observed HLAII peptides from anti-TNF biotherapeutics to include sequences that at least partially span the complementary determining regions (CDRs), such as the LCDR1 for both INFL and ADAL. Although antagonist presentation varied significantly across donors, peptides from both bivalent antagonists INFL and ADAL were more highly presented relative to the Fab'. While TNF immune complexes did not alter overall HLAII presentation, a moderate increase in presentation of a subset of peptide clusters was observed in the case of INFL-TNF, which included HCDR2, HCDR3 and LCDR2 sequences.

Keywords: CD4+ T-cells; HLAII; TNF antagonist; dendritic cells; immunogenicity; immunopeptidomics; presented peptides; timsTOF mass spectrometry.

Figure 1

HLAII immunopeptidomics workflow for the identification of peptides presented from TNF antagonists by DCs. Freshly isolated PBMCs collected from fourteen healthy donors were isolated by density separation for subsequent CD14+ monocyte-purification. CD14+ monocytes were cultured and differentiated into DCs for six days. DCs were loaded with TNF antagonists or their preformed TNF immune complexes at day 5, followed by LPS stimulation for 22 h. DCs were harvested, washed and lysed to obtain whole protein extracts for HLAII immunoprecipitation using a pan-HLAII antibody. The HLAII immunopeptidome was purified by filtration and by C18 prior to LC-MS/MS analysis using trapped ion mobility time-of-flight mass spectrometry (timsTOF MS).

Figure 2

Size characterization of TNF antagonists and immune complexes of TNF with antagonists by dynamic light scattering (DLS). (A) Representative hydrodynamic diameter profile of TNF, INFL and INFL-TNF as measured by DLS. Each line denotes one of three technical replicates for each condition, which were then ran in fourteen independent experiments, i.e., one for each donor. (B) Size comparison of TNF (black), TNF antagonists and their complexes with TNF; INFL and INFL-TNF (blue), ADAL and ADAL-TNF (green) and Fab’ and Fab'-TNF (orange), where each bar correspond to the mean and the standard error of mean (SEM) from fourteen independent experiments (n=14, ****p < 0.0001, *p < 0.05).

Figure 3

Dendritic cell yields after monocytic purification from PBMCs, LPS stimulation and treatment with TNF, antagonists or immune complexes. (A) Total yield of DCs derived from fourteen independent sets of purified CD14+ monocytes. Bars represent the mean and SEM per donor (n=9), where each circle corresponds to one of the nine treatment conditions: UNSTIM, LPS, TNF, INF, ADAL, Fab’, INFL-TNF, ADAL-TNF and Fab’-TNF. (B) Yield of DCs plotted by treatment condition: TNF antagonist-free controls (black), INFL and INFL-TNF (blue), ADAL and ADAL-TNF (green) and Fab’ and Fab’-TNF (orange). Each bar represents the mean and SEM per fourteen independently stimulated DCs, where each circle corresponds to the number of cells per donor.

Figure 4

Total HLAII-associated peptides in monocytic-derived DCs. (A) Total number of unique peptides identified in the fourteen donors by HLAII immunopeptidomics, each box and whisker plot represent the mean and the SEM of independent data sets per donor. (B) Total number of unique peptides plotted against the number of DCs used as input, each point represents an experimental sample (n=124), where samples with >1x10⁷ DCs were not included (2 sets). (C) Total number of unique peptides identified in the fourteen donors upon HLAII immunopeptidomics, where each set has the mean and data distribution from fourteen donors per treatment condition corresponding to INFL (blue) ADAL (green) and Fab’ (orange).

Figure 5

Length distribution of HLAII-associated peptides in monocytic DCs. (A) Length of total HLAII immunopeptidome in 126 data sets where each dot represents a detected peptide. (B) Frequency distribution of peptide length from a representative donor (D1237), where each bar corresponds to the number of peptides identified at a given residue-length. (C) HLAII peptide length distribution from different conditions was analyzed by frequency (D1237). Each bar corresponds to the number of peptides identified at a given residue-length in samples treated with INFL (blue), ADAL (green) and Fab’ (orange) antibodies either alone (light color) or TNF-bound (bold color).

Figure 6

HLAII-presented sequences derived from anti-TNF compounds identified by HLAII immunopeptidomics. (A, B) Sequence coverage of identification within both light chain (LC) and heavy chain (HC) from antagonists detected in HLAII immunopeptidomes from antagonist-treated sets corresponding to INFL (blue) ADAL (green) and Fab’ (orange) (C) Length distribution of identified TNF antagonist-derived peptides from all donors.

Figure 7

HLAII-presented sequences derived from anti-TNF biotherapeutics. (A) Number of unique anti-TNF peptides derived from DCs loaded with test article alone (empty circles) or in complex with TNF (full circles) per donor, corresponding to INFL (blue), ADAL (green) and Fab’ (orange); (*p < 0.013 by Wilcoxon marched-pairs signed rank test) (B) Median fold-change anti-TNF vs. anti-TNF complexed with TNF based on the number of unique anti-TNF peptides detected per sample. ns, non-significant; *p > 0.036 by Mann-Whitney analysis.