Table 2 Immunological screening assays used to test for neoantigen recognition

cDNA libraries

Interrogates all transcribed sequences

Interrogates mutated and non-mutated sequences; Biased toward highly transcribed genes; Influenced by the size, expression levels or GC-richness of transcripts encoding for T-cell epitopes; Labor intensive and time-consuming

Peptide-HLA multimers

Allows the isolation of antigen-specific T cells; Overcomes the need of autologous APCs

Multimers are available for a limited number of HLA molecules; Exclusively interrogates a selected list of mutated epitopes based on in silico prediction or validated by immunopeptidomics; Not optimal for CD4⁺ cells

Minimal epitopes

HLA-matched target cells (based on in silico prediction) can be used instead of autologous APCs; Cost-effective

Requires autologous or HLA-matched cells as target cells; Exclusively interrogates a selected list of mutated epitopes based on in silico prediction or validated by immunopeptidomics; Not optimal for CD4⁺ cells

Tandem minigenes or peptide pools

Allows potential processing and presentation of candidate neoantigens on HLA-I and HLA-II; Does not require prior knowledge of the minimal epitope or HLA restriction; Can be used to interrogate all or a large portion of mutated epitopes;

Peptide processing by immunoproteasome in APCs might differ from processing by the proteasome in tumor cells; Availability of APCs/effectors can limit this approach, especially when > 250 epitopes are tested; Cost increases in patients with high mutation burden; Requires autologous APCs as target cells;