Abstract 6613: Identification of targetable HLA*A2:01 restricted peptides in osteosarcoma
Authors: Milewski David , Sindiri Sivasish , Wei Jun S. , Brohl Andrew , Song Young , Wen Xinyu , Qi Andrew , Guha Udayan , Khan Javed 
Published: September 2020

Abstract
Background: Osteosarcoma is the most common primary tumor of bone with 800-900 new cases diagnosed annually in the United States. The development of effective chemotherapeutic agents in the early 1970's led to a dramatic increase in osteosarcoma survival from less than 10% to over 60%. Unfortunately, little progress has been made in the past several decades despite intensified treatment and the application of targeted therapies with the 5-yr overall survival stalled at ~70%. Several phase I studies have reported occasional clinical responses to immune checkpoint inhibitors in osteosarcoma patients, although the role of immunotherapy in osteosarcoma treatment is largely unexplored. The goal of this work was to perform a deep immuno-genomic and proteomic profile 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 lentiviral construct targeting PRAME and co-cultured with U2OS cells to evaluate specific PRAME peptide targeting by T cells.
Results: Gene expression profiling of extracranial pediatric solid tumors identified high T cell infiltration in osteosarcoma patient tumors. High 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. We identified a set of tumor associated antigens, such as cancer germline antigens (CGA), which are highly expressed in osteosarcomas and display low/absent expression in normal tissues. PRAME, a CGA expressed in multiple solid tumors and hematological malignancies, was identified as one of the most frequently overexpressed CGAs in osteosarcoma. Immunoprecipitation of MHC Class I complexes followed by LC-MS/MS from osteosarcoma cells identified peptides derived from PRAME and other tumor associated antigens that are presented by HLA*A2:01 in osteosarcoma. In a proof-of-concept experiment, we demonstrate effective T cell cytotoxicity against osteosarcoma using a TCR which specifically recognizes an PRAME HLA*A2:01 peptide identified by mass spectrometry.
Conclusions: Osteosarcoma tumors have prognostic high CD8+ T cell infiltration, expression of cancer testis antigens, and presentation of tumor associated peptides on MHC class I complex. These data warrant a broad evaluation of the efficacy of immunotherapy in osteosarcoma.
Citation Format: David Milewski, Sivasish Sindiri, Jun S. Wei, Andrew Brohl, Young Song, Xinyu Wen, Andrew Qi, Udayan Guha, Javed Khan. Identification of targetable HLA*A2:01 restricted peptides in osteosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6613.