Abstract B008: GPR64-targeting single-domain CAR-T cells for immunotherapy in Ewing sarcoma

Abstract

Abstract Ewing sarcoma (EwS) is the second most frequent bone and soft tissue cancer of childhood and adolescence. EwS is characterized by balanced chromosome translocation resulting in an aberrant tumor-specific transcriptional factor, EWSR1-FLI1, in 85% of cases. EwS is a highly aggressive cancer with a 5-years overall survival of only 30% for patients with metastases. These metastatic diseases are often resistant to intensive therapy and associated with acute and chronic adverse effects. Thus, there is a need for new treatment options to improve survival and to limit the long-term side effect. Immune-based approaches can offer alternative options to conventional treatments modalities. In this context, the aim of this study is to identify therapeutic target for immunotherapy. We carried-out an integrative mass spectrometry analysis of whole proteome and phosphoproteome of 42 cells lines including EwS and non-EwS cancer cell lines as well as mesenchymal stem cells (MSCs), the putative cell-of-origin for EwS. This multi-omics study revealed expected EWSR1-FLI1-associated signatures. Additional surfaceome data permitted to highlight 70 cell surface proteins up-regulated by EWSR1-FLI1. Among these selected proteins, potential candidates for immunotherapy were selected according to their healthy tissue expression profiles based on integrated transcriptomic analysis using public resources and RNA-seq data of EwS tumors. A particular interest was dedicated to the expression of proteins that may derive from the recently described neogenes (Vibert et al, Mol Cell 2022). We identified the G protein-coupled receptor 64 (GPR64), as a potential specific EwS target for immunotherapy. Immunochemistry assay highlighted a high expression of GPR64 specifically in EwS PDXs and patients-derived tumors though the level of expression varies from one case to the other. GPR64 expression in normal tissues is only restricted to human epididymis. Alternative splicing occurs intensively in the N-terminal region, generating nine splice isoforms of the protein with slight differences in the extracellular domain. All splice isoforms are expressed in Ewing's sarcoma, with a majority over-representation of protein isoforms 2 and 4, compared to healthy tissues. GPR64-specific single-domain antibodies (sdAb) were selected by phage display libraries binding to the recombinant protein corresponding to an extracellular part of the receptor. The specificity of sdAb was verified by flow cytometry on GPR64-wild type and GPR64-knock down cells as well as MSCs. The activating ability of sdAb to activate T cells and their cytotoxic potential against EwS cells are currently in progress. Thus, our multi-omics study identified GPR64 as a potential target for immunotherapy due to its EwS-specificity and its limited expression in normal tissues. This work could highlight new therapeutic opportunities for EwS patients with refractory disease. Citation Format: Floriane Petit, Florent Dingli, Jacob Torrejon-Diaz, Bérangère Lombard, Stéphane Liva, Sakina Zaidi, Didier Surdez, Renaud Leclere, Thomas Grünewald, Sandrine Grossetete, Berengère Ouine, Véronique Marsaud, André Nicolas, Franck Perez, Ahmed El Marjou, Damarys Loew, Sandrine Moutel, Amaury Leruste, Olivier Ayrault, Olivier Delattre. GPR64-targeting single-domain CAR-T cells for immunotherapy in Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B008.