(P 124) UNLOCKING THE POTENTIAL OF MEASLES VIRUS ONCOLYTIC IMMUNOTHERAPY FOR SARCOMA: BIOMARKER DISCOVERY AND THERAPEUTIC VACCINE DEVELOPMENT

Objective: The objective of this preclinical study is to advance immunotherapy for sarcoma using oncolytic measles virus (MeV). MeV are promising therapeutic vectors due to their preferential replication in malignant cells, immunogenic tumor cell death leading to priming of anti-tumor immunity, a versatile reverse genetics system and an excellent safety record. In this study, we aim to identify predictive biomarkers for the sensitivity of sarcoma to measles virotherapy, assess potential sub-entity specific differences and demonstrate a proof-of-concept for using tumor-associated antigen (TAA)-encoding MeV as a therapeutic vaccine for the treatment of sarcoma.

Methods: A panel of 17 sarcoma cell lines representing 5 sarcoma sub-entities (leiomyosarcoma, liposarcoma, synovial sarcoma, osteosarcoma, and undifferentiated pleomorphic sarcoma) were used to assess the receptor expression, viral gene expression, production of viral progeny and virus-mediated cytotoxicity across different sarcoma subtypes. Based on the outcome, cell lines were stratified into high sensitive and low sensitive groups. This was followed by transcriptomic analyses to uncover underlying pathways and predictive gene signatures contributing to differences in sensitivity towards measles virotherapy.
Additionally, we designed and produced recombinant MeV encoding two variants of the NY-ESO-1 TAA: MeVac ld-NY-ESO-1 containing the complete coding sequence of NY-ESO-1, and MeVac ld-Ub-SLLMWITQCx6 featuring an epitope cassette harboring the known immunodominant HLA-A*02:01 restricted epitope NY-ESO-1-157–165. To evaluate whether this construct may act as an oncolytic vaccine to enhance antigen-specific T cell-mediated killing, NY-ESO-1-TCR+ T cells were generated from HLA-A*02:01-positive donors and co-cultured with MeVac ld-NY-ESO-1 or MeVac ld-Ub-SLLMWITQCx6 infected sarcoma cells. Tumor cell killing was monitored using xCELLigence technology.

Results: Flow cytometry showed high expression of the main MeV entry receptor CD46 on all 17 investigated cell lines. Cytotoxicity post-MeV infection was also observed in all sarcoma cell lines, underlining the applicability of oncolytic MeV approaches for a broad spectrum of sarcoma sub-entities. Interestingly, varying levels of viral protein and progeny production as well as virus-mediated cytotoxicity after MeV infection were observed across cell lines which were not sub-entity dependent, suggesting a requirement for a systematic investigation of the cellular response to MeV. Comparative transcriptomic analysis revealed enrichment of cell cycle, mitosis, angiogenesis, and DNA repair pathways in highly sensitive sarcoma cell lines, and inflammatory, interferon and cytokine response pathways in those with low sensitivity.
Additionally, we successfully cloned and produced the two recombinant MeVs, i.e, MeVac ld-NY-ESO-1 and MeVac ld-Ub-SLLMWITQCx6. These viruses were shown to markedly enhance antigen-specific T cell-mediated killing of sarcoma cells when co-cultured with NY-ESO-1-TCR+ T cells.

Conclusion: Our ongoing study highlights the potential of measles virotherapy to both mediate direct tumor cell lysis, as well as enhance anti-tumor immune response as oncolytic vaccine in multiple sarcoma sub-entities. Currently, we are refining MeV sensitivity signature by integrative transcriptomics and proteomics analysis that may act as a predictive biomarker for the response to measles virotherapy. In an in vitro setting, we demonstrate that an oncolytic MeV vaccine carrying NY-ESO-1 can enhance the T cell-mediated killing of sarcoma cells, thereby providing a successful proof-of-concept for the application of MeV-based oncolytic vaccine.