David Milewski, Jun S. Wei, Sivasish Sindiri, Andrew Brohl, Young Song, Xinyu Wen, Andy Qi, Udayan Guha, and Javed Khan.

• Submitter: David Milewski (Postdoc or fellow)
• email: david.milewski@nih.gov

Background: Osteosarcoma is an aggressive bone tumor that is one of the leading causes of cancer-related death in the pediatric population. Several phase I clinical trials have reported occasional clinical responses to immune checkpoint inhibitors in osteosarcoma patients. The key feature of immunotherapy involves T cell recognition of peptides from abnormal proteins (tumor-associated antigens; TAAs) presented by MHC class I of tumor cells which stimulates specific anti-tumor cytotoxicity. Currently, the role of immunotherapy for osteosarcoma remains to be determined. Furthermore, little is known about the TAAs presented by osteosarcoma tumors which can be targeted by T cells. The goal of this work is to perform a deep immunogenomic and proteomic profiling of osteosarcoma tumors as the basis for developing effective immune-based therapy for osteosarcoma.

Methods: Osteosarcoma tumors and cell lines were subjected to whole transcriptomes analysis. Intratumoral T cell receptor sequences were identified using MiXCR and VDJtools from the RNA-seq data. Immune signatures were scored in each tumor sample using single-sample GSEA. MHC class I bound peptides were isolated by immunoprecipitation of MHC class I complex, acid elution of bound peptides, and identification using LC-MS/MS. Healthy donor T cells were transduced with a reported TCR targeting a PRAME peptide and co-cultured with osteosarcoma cells to evaluate cytotoxicity.

Results: Gene expression profiling of extracranial pediatric solid tumors identified high T cell infiltration in osteosarcoma patient tumors. CD8+ T cell infiltration were associated with favorable outcome among osteosarcoma patients, suggesting the presence of an underlying endogenous T cell response against osteosarcoma tumors. In addition, we identified TAAs which are frequently overexpressed in osteosarcoma but display low/absent expression in normal tissues. To determine if any of these TAAs are presented on MHC class I in osteosarcoma, we performed large scale immunoprecipitation of MHC class I complexes, peptide isolation and identification by LC-MS/MS. We found that the immunopeptidome of osteosarcoma is rich in TAAs, including cancer germline antigens (CGAs), which may be immunogenic for T cells. Furthermore, we identified multiple peptides from PRAME, one of the most commonly overexpressed TAAs across pediatric solid tumors, highlighting it as a prime candidate target for T cell therapy. In a proof-of-concept experiment, we demonstrate effective T cell cytotoxicity against osteosarcoma using a TCR specific for the PRAME-MHC class I peptide identified by mass spectrometry.

Conclusions: Osteosarcoma tumors have prognostic high CD8+ T cell infiltration, expression of targetable TAAs, and presentation of tumor associated peptides on MHC class I complex which can be effectively targeted with engineered T cells. These data warrant a broad evaluation of the treatment of osteosarcoma using immunotherapy.