Grant: Catalyst Grant: Systems Biology Approaches to Immunotherapy

The MHC class I peptide (MIP) repertoire comprises a collection of peptides that are presented at the cell surface in the context of MHC class I molecules for scrutiny by cells of the CD8+ lineage. Under conditions of microbial infection, CD8 T cell responses specific for pathogen-derived MIP peptides are elicited and these antigen-specific cells are primarily responsible for eradicating infection. However, under normal, steady-state conditions, peptides derived from "self" proteins are continually degraded as part of the constitutive activity of the proteasome and are presented on the cell surface as the "self" MIP repertoire. Likewise, tumor-associated antigens expressed by cancer cells are processed to become part of the cancer MIP repertoire and in doing so represent ideal target antigens for potential immunotherapeutic intervention. Despite the tremendous potential importance of the cancer MIP repertoire, relatively little is known about its composition or complexity. In the current proposal we intend to catalogue the MIP repertoire present on the surface of ovarian cancer cells. Specifically, we will utilize malignant ascites samples from patients with stage III-IV ovarian cancer as a source of cells for mass spectrometry-based identification of MHC-associated peptides. Malignant ascites removed from ovarian cancer patients via paracentesis, or as part of de-bulking surgery, frequently contains extraordinary numbers of tumor cells (>108) which can be readily purified from any infiltrating immune cells. Two complimentary approaches for peptide purification will be utilized, 1) peptides will be directly eluted from surface MHC using mild acid treatment and 2) peptides will be acid eluted from specific MHC molecules that have been immuno-precipitated from tumor cell lysates. Peptides isolated using these approaches will be identified by tandem mass spectrometric approaches and a database corresponding to the ovarian cancer MIP repertoire will be established.