Poster Session II - Abstract # 17
Engineering CAR T Cells Targeting Osteosarcoma
Siddharth Subham1, John Jeppson2, Bryan Schatmeyer2, Jie Zhao2, David Akhavan2
1Bioengineering Program, University of Kansas, Lawrence, KS, USA; 2Department of Radiation Oncology, University of Kansas Medical Center, Kansas City, KS, USA
Background : Pediatric Osteosarcoma (OS) is the most common primary malignant bone tumor with poor overall survival. Treatment includes a highly toxic combination of chemotherapy followed by surgery and radiation. Given the poor prognosis, pediatric sarcoma has an unmet medical need. Chimeric Antigen Receptor (CAR) T cell immunotherapy combines the cytolytic potency of a T cell with the tumor specificity of an antibody. The clinical trial experience of CAR T cells in sarcomas has identified multiple obstacles, including immune evasion via tumor antigen loss. The current study addresses this shortcoming by designing CAR T constructs to overcome the Tumor Associated Antigen (TAA) heterogeneity in OS.
Methods : We designed 2nd generation CARs targeting EGFR and B7H3 antigens on tumor cells. We expressed these CARs in Jurkat cells which are immortalized human T cells and then performed a killing assay by treating these CAR expressing Jurkat cells with KHOS osteosarcoma cells and evaluated the live cells at end of the experiment. We also isolated human CD4 T cells from blood using magnetic beads and expressed these CARs on them to perform a similar killing assay on the OS KHOS cells.
Results : We found high expression of EGFR and B7H3 at RNA and protein levels in OS tumor samples as compared to normal tissue by evaluating the TCGA database. We confirmed this by performing immunofluorescence staining on patient tissue samples and found them co-expressing EGFR and B7H3 highly. We also evaluated their expression on established OS tumor cell lines by flow cytometry and found highest expression in the KHOS cell line. We found nearly 50% killing of tumor cells with the EGFR CARs and roughly 40% killing with the B7H3 CARs after 18 hours of co-culture. We also observed high expression levels of EGFR and B7H3 in orthotopic KHOS xenograft tumors which will serve as an appropriate murine model to evaluate these CARs.
Conclusion : This data suggests that the EGFR CARs and B7H3 CARs can effectively kill OS tumor cells. We are optimizing the conditions for maximum killing and engineering tandem / bispecific CARs to synergize their effect for maximum killing.