Analysis of the human tumor HLA peptidome based on patient derived xenograft in mice

Introduction:

The direct biochemical HLA peptidome analysis provides very reliable information about the presentation levels and patterns of neoepitope HLA peptides, by the tumor cells. Using this information, one can select the best-presented neoepitope peptides for designing and prioritizing a vaccine suitable for the patients.
Here we describe the use of patient derived xenografts (PDX) tumor tissue models as a source for HLA peptidome analysis. PDX tumors are already used for testing different anti-cancer drugs, and as a source for large amounts of tumor tissues that maintain closer similarities to the original tumors and their microenvironments, relative to the in-vitro cultured cells. In this research, our goal was to establish PDX mice as an effective model for cancer specific HLA peptides antigen detection in order to implement them in the future into the clinical pipeline of personalized cancer immunotherapy.

Methods:
We used immunoaffinity purification to recover the HLA molecules from the PDX tumors, followed by capillary chromatography and tandem mass spectrometry (LC-MS/MS) analysis of extracted HLA peptides. We compared the original patients’ tumor tissues to the same tumors grown in PDX mice. The HLA peptides were identified using databanks of all human protein sequences, supplemented with sequences of the mutated proteins, as elucidated from exome data of the tumors.

Results and discussion:
We demonstrate here that the PDX tissues are a reliable source for discovery of large HLA peptidomes, larger than obtainable from the limiting amounts of original tumors. The HLA peptidomes of multiple PDX tumors grown in separate mice and originating from the same human tumors, were very similar, even after three generations of re-grafting. Furthermore, the HLA peptidomes of the PDX were very similar to those of the original tumors. Between 50% to 95% of the original tumors’ HLA peptides were also detected in their derived PDX tumors. The majority of the peptides fitted the patient`s HLA sequence motifs and peptides length distributions, supporting the conclusion that these are likely true human HLA peptidomes. Importantly, three new neoantigens HLA peptides were detected in the PDX tumors, which were not detected in the original tumors. These neoantigens fit the patients HLA sequence motif, and therefore can be used as a proof of concept for the implementation of the PDX mice models into the clinical pipeline of personalized cancer immunotherapy.