Alterations in the HLA-B*57:01 Immunopeptidome by Flucloxacillin and Immunogenicity of Drug-Haptenated Peptides

2021 FDA Science Forum

• Authors: Puig, Montserrat, FDA/CDER; Ananthula, Suryatheja, FDA/CDER; Venna, Ramesh, FDA/CDER; Polumuri, Swamy Kumar, FDA/CDER; Mattson, Elliot, FDA/CDER; Walker, Lacey M, FDA/CDER; Cardone Marco, FDA/CDER; Takahashi, Mayumi, FDA/CDER; Su, Shan, FDA/CDER; Boyd, Lisa F, NIH/NIAID; Natarajan, Kannan, FDA/CDER; Abdoulaeva, Galina, FDA/CBER; Wu, Wells W, FDA/CBER, Roderiquez, Gregory, FDA/CDER, Hildebrand, William H, University of Oklahoma Health Sciences Center; Beaucage, Serge L, FDA/CDER; Li, Zhihua, FDA/CDER; Margulies, David H, NIH/NIAID, Norcross, Michael A, FDA/CDER

• Center: Center for Drug Evaluation and Research

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Abstract

Neoantigen formation due to the interaction of drug molecules with human leukocyte antigen (HLA)-peptide complexes can lead to severe hypersensitivity reactions. Flucloxacillin (FLX), a β-lactam antibiotic for narrow-spectrum gram-positive bacterial infections, has been associated with severe immune-mediated drug-induced liver injury caused by an influx of T-lymphocytes targeting liver cells potentially recognizing drug-haptenated peptides in the context of HLA-B*57:01. To identify immunopeptidome changes that could lead to drug-driven immunogenicity, we used mass spectrometry to characterize the proteome and immunopeptidome of B-lymphoblastoid cells solely expressing HLA-B*57:01 as MHC-I molecules. Selected drug-conjugated peptides identified in these cells were synthesized and tested for their immunogenicity in HLA-B*57:01-transgenic mice. T cell responses were evaluated in vitro by immune assays. The immunopeptidome of FLX-treated cells was more diverse than that of untreated cells, enriched with peptides containing carboxy-terminal tryptophan and FLX-haptenated lysine residues on peptides. Selected FLX-modified peptides with drug on P4 and P6 induced drug-specific CD8+ T cells in vivo. FLX was also found directly linked to the HLA K146 that could interfere with KIR-3DL or peptide interactions. These studies identify a novel effect of antibiotics to alter anchor residue frequencies in HLA-presented peptides which may impact drug-induced inflammation. Covalent FLX-modified lysines on peptides mapped drug-specific immunogenicity primarily at P4 and P6 suggesting these peptide sites as drivers of off-target adverse reactions mediated by FLX. FLX modifications on HLA-B*57:01-exposed lysines may also impact interactions with KIR or TCR and subsequent NK and T cell function. Our results expand the knowledge on the mechanisms by which severe drug hypersensitivity reactions might occur which is of relevance to better understanding product safety.

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