P06.04.b new approaches for expanding glioblastoma infiltrating lymphocytes

Abstract BACKGROUND Adoptive cell therapy using tumor infiltrating lymphocytes (TILs) has shown promise in melanoma and Non-Small Cell Lung Cancer. TILs culture and expansion from GBM remains challenging due to paucity of infiltrated T-cells that are also exhausted. This hinders creation of potent TILs. We have developed a new efficient method to manufacture potent TILs from GBM samples. METHODS We have developed efficient TILs manufacturing methods using both solid and aspirated liquid samples collected during vacuum assisted excision containing tumor fragments that may stem predominantly from the tumor margins and provide a source for TILs. We followed the traditional two steps: initial expansion (preREP) where T-cells are expanded with tumor cells to selectively expand tumor antigen-reactive TILs, followed by a large-scale expansion (REP) after further activation. For both steps we optimized media and reagents. RESULTS TILs cultures from solid tumors were established from enzymatically digested tissue and by fragment plating. Successful TILs yield from liquid samples required enzymatic digestion, debris removal and RBC lysis prior to culture initiation. PreREP TILs yields ranged from a few million to 2 billion viable cells for solid and liquid samples. REP of TILs from liquid samples expanded similarly from solid samples. REP of TILs reached up to 556-fold expansion (mean: 346, range: 95-556) providing promising numbers of activated TILs. The potency of the TILs was demonstrated with tumor specific Interferon-gamma production when cultured with matched tumor digest or neurospheres. CONCLUSIONS We successfully expanded TILs that are rich in cytotoxic T-cells, demonstrated their tumor reactivity and further expanded the preREP TILs to reach therapeutic doses. We have characterized the TILs by scRNA and TCRseq and analyze paired TCR reads using scRNA sequencing. Overrepresented TCR alpha and beta chains will be linked by a P2A site and will be cloned into a lentiviral vector to establish a TCR library which will be transduced into novel Jurkat Triple Parameter Reporter Cell Lines under the control of different T cell activation programs. TCR Jurkat Libraries will be cocultured with original tumor material and sorted to identify tumor reactive TCRs; enabling us to define the percentage of TCRs that are enriched by TIL isolation that are specifically reactive to tumor vs bystander expanded TCR cells. Ultimately, we aim to bring this therapy to patients in a clinical trial.