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. 2023 Oct 19;42(7):1107–1117. doi: 10.1038/s41587-023-01945-y

Fig. 2. Generation of HLA class I monoallelic cell lines that stably express a polyantigen cassette containing 47 shared cancer neoantigens.

Fig. 2

a, Process overview for the cell engineering and MS analysis of peptides presented by polyantigen-expressing HLA monoallelic cell lines. KO, knockout. b, Vector map of the piggyBac polyantigen expression constructs used in this study. A single transcript containing 47 tandem neoantigens followed by seven control peptides and an IRES-linked mTagBFP2 (BFP) reporter is driven by an EF1a promoter. Neoantigens were either directly concatenated (no-linker) or interspersed by short flexible linker sequences (linker). c, Flow cytometric detection of HLA expression (W6/32 antibody-APC) and polyantigen cassette reporter (BFP) in selected cell lines or HLA knockout parental line. Here, ‘−’ indicates the absence of linkers and ‘+’ indicates presence of linkers in the polyantigen construct. d, Targeted immunopeptidomic detection of a previously described A*02:01-presented TP53 neoepitope (HMTEVVRHC, position 39) as well as two neoantigen control peptides from pp65 (NLVPMVATV, control 1) and IE-1 (VLEETSVML, control 4) known to be presented by the A*02:01 allele. a and b were created with BioRender.com.