Method for differentially quantifying naturally processed HLA-restricted peptides for cancer, autoimmune and infectious diseases immunotherapy development

A method for the identification and label-free quantification of at least one MHC ligand peptide suitable for use in an immunotherapeutic composition, said method comprising

• a) isolating at least one MHC ligand peptide from at least one primary healthy tissue sample and at least one primary diseased tissue sample by immune-precipitating the at least one MHC ligand peptide using an antibody that specifically binds to a human leukocyte antigen (HLA)-A, HLA-B, and/or HLA-C,
• b) performing an HPLC-MS analysis on said MHC ligand peptides in order to generate peptide signals therefor, wherein a plurality of replicate runs of HPLC-MS analysis is performed for each sample,
• c) extracting the precursor ion signal area for each of said MHC ligand peptide signals,
• d) identifying the sequences for each of said MHC ligand peptides,
• e) normalizing the areas within replicate runs by calculating a mean presentation value for each peptide in each replicate run;
• and computing a normalization factor for each peptide and each replicate run using the mean presentation value;
• and averaging over all peptides results in run-wise normalization factors;
• and applying the run-wise normalization factors to all peptides of a particular replicate run,
• f) determining relative quantities of the MHC ligand peptides that are comparable between samples,
• g) calculating a presentation profile and/or a presentation score for the MHC ligand peptide of said diseased sample and said healthy sample based on said average and relative quantities of the MHC ligand peptide, and
• i) label-free quantification of the MHC ligand peptides,
• j) formulating the at least one MHC ligand peptide in an immunotherapeutic composition if the at least one MHC ligand peptide is over-presented in the diseased sample.