High-Sensitivity Targeted Mass Spectrometry Enables Quantification of HLA-Presented Antigens for TCR-T Therapeutic Target Discovery

Human leukocyte antigens (HLA) are key mediators of adaptive immunity, presenting antigenic peptides derived from self and non-self proteins for immune surveillance. Tumor-specific neoantigens generated by somatic mutations represent key determinants of immune recognition and are central to the development of targeted immunotherapies. Recent advances in immunopeptidomics have enabled systematic identification of these peptides; however, accurate quantification of immunogenic epitopes remains essential to assess their therapeutic relevance. Precise measurement of (neo)antigen abundance can inform the design of T-cell receptor (TCR)–based therapeutics, as well as personalized vaccines and cell therapies. Highly sensitive, quantitative methods for profiling peptide presentation across cell or tissue materials are therefore critical to advancing precision immuno-oncology and immune-modulatory therapies.

To develop and optimize such a method, we employed a well-characterized model system: binimetinib treated SK-MEL5 melanoma cells. Binimetinib is a potent, selective, non-ATP-competitive allosteric inhibitor of MEK1/2, approved by the FDA in 2018 in combination with encorafenib for the treatment of metastatic BRAF-mutant melanoma. Beyond its direct anti-proliferative effects, MEK inhibition has emerged as a powerful modulator of tumor immunogenicity: inhibition of the overactivated MAPK pathway upregulates HLA class I surface expression and key components of the antigen processing machinery, including TAP transporters and β2-microglobulin. Critically, MEK inhibition selectively enriches the presentation of melanoma differentiation antigens such as DCT and PMEL in a dose-dependent manner1, with individual peptides reaching abundances exceeding 1,000 copies per cell. This dynamic and quantifiable response to treatment makes binimetinib treated SK-MEL5 cells an ideal benchmark system for absolute copy number estimation of HLA-presented antigens.