Clinical-scale manufacturing of CD137+ enriched tumor-reactive T cells for glioblastoma in the CliniMACS Prodigy

Background Most tumor-infiltrating lymphocyte (TIL) studies and therapies have focused on tumors with high T-cell infiltration and mutational burden. Glioblastoma (GBM) represents a challenging indication for TIL therapy because of its limited T-cell infiltration and low mutational burden. We investigated whether targeted enrichment of tumor-reactive T cells using CD137-based selection could enable robust, clinical-scale production of TIL products from GBM. Methods Ultrasonic aspirates (UAs), routinely collected during GBM resections, were evaluated as a source of TILs for clinical manufacturing. TILs were generated from GBM UAs under GMP-like conditions at clinical scale using the closed, automated TRT Process on the CliniMACS Prodigy. Matched TRT products that were enriched for CD137+ cells, depleted of CD137+ cells, or left unselected were generated in parallel at full manufacturing scale. Products were characterized by cellular and transcriptomic analyses and evaluated in functional assays for cytotoxicity against autologous and non-autologous patient-derived GBM cell lines. Results Cellular and transcriptomic analyses revealed that UA-derived CD137+ (4-1BB/TNFRSF9) T cells were clonally expanded, exhibited activated and memory phenotypes, and displayed transcriptional profiles associated with tumor reactivity. The closed manufacturing process preserved phenotypic features previously associated with clinical benefit in other TIL indications. Functional assays demonstrated potent, tumor-reactive cytotoxicity of CD137-enriched TRT products against autologous GBM cell lines compared with TILs generated in parallel without selection. Discussion/Conclusions These findings establish the feasibility of a clinical-scale TIL manufacturing approach using the CliniMACS Prodigy that enables production of clinically relevant quantities of TILs from GBM while enriching for cells with tumor-reactive characteristics. This approach may broaden the application of TIL therapy to immunologically cold tumors with limited T-cell infiltration or low mutational burden that have historically been challenging to manufacture using conventional TIL production methods.