With the increasing expectation of GD 2 as a broad target for CAR T cell therapy and the expected benefit in applying TRUCKs with transgenic IL-18 release, there is a need to manufacture such cellular medicinal products in a safe, validated and reproducible fashion. Initial GD 2-CAR T cell clinical studies targeting neuroblastoma by first to third generation CAR T cells showed moderate or transient anti-tumor responses but failed to produce sustained remissions, emphasizing the need to modulate the T cell response ( 17– 19). GD 2 therefore is a promising target for redirected immunotherapy. GD 2 is highly and consistently expressed in childhood cancer neuroblastoma and can be found on the cell surface of other solid cancer entities including breast cancer ( 8), osteosarcoma ( 9), melanoma ( 10), glioblastoma ( 11), small cell lung cancer ( 12), retinoblastoma ( 13), soft tissue sarcoma ( 14) and Ewing sarcoma ( 15, 16). Physiological expression of GD 2 is restricted to low densities on neurons, skin melanocytes and peripheral pain fibers ( 7). In this study we focused on the manufacturing of TRUCKs targeting disialoganglioside GD 2. CAR T cells engineered with inducible IL-18 release improve T cell effector functions towards superior activity against pancreatic and lung tumors in mice that were refractory to CAR T cells without cytokines ( 5, 6). Innate immune cells are attracted and activated by IL-12 or IL-18 ( 4) to eliminate antigen-low expressing or antigen-negative cancer cells within the tumor ( 2). These factors include cytokines, such as IL (interleukin)-12 and IL-18, but also enzymes and costimulatory ligands augmenting T cell activation. TRUCKs are thereby used as “living factories” to produce and deposit substances with anti-tumor activity in the targeted tissue. TRUCKs are CAR T cells that release a transgenic protein upon CAR engagement of cognate antigen and signaling. A promising strategy to target solid tumors with their phenotypic heterogeneity has led to the fourth generation of CARs also known as TRUCKs or armored CARs ( 2, 3). To enable and improve CAR T cell proliferation, anti-tumor activity, and in vivo persistence, advanced generations of CARs have been developed ( 1). One of the most significant recent developments in cancer therapy is the CAR T cell technology. In conclusion, GMP-compliant manufacturing of TRUCKs is feasible and delivers high quality T cell products. Following antigen-specific CAR activation of TRUCKs, CAR-triggered release IL-18 was induced, and the cytokine was biologically active, as demonstrated in migration assays revealing specific attraction of monocytes and NK cells by supernatants of TRUCKs co-cultured with GD 2-expressing target cells. Manufactured TRUCKs showed a specific cytotoxicity towards GD 2-expressing target cells indicated by lactate dehydrogenase (LDH) release, a decrease of target cell numbers, microscopic detection of cytotoxic clusters and detachment of target cells in real-time impedance measurements (xCELLigence). IL-2, granzyme B, IFN-γ, perforin, TNF-α). CD25, CD69) on both CD4 + and CD8 + T cells and an enhanced release of pro-inflammatory cytokines and cytolytic mediators (e.g. IL-18 TRUCKs were activated in an antigen-specific manner by co-cultivation with GD 2-expressing target cells indicated by an increased expression of activation markers (e.g. The functional capacities of the clinical-scale manufactured TRUCKs were similar to TRUCKs generated in laboratory-scale and were not impeded by cryopreservation. Preclinical characterization demonstrated antigen-specific GD 2-CAR mediated activation after co-cultivation with GD 2-expressing target cells. Transduction efficiencies of 77.7% and 55.1% was obtained, and yields of 4.5 x 10 9 and 3.6 x 10 9 engineered T cells from the two donors, respectively, within 12 days. Starting with 0.84 x 10 8 and 0.91 x 10 8 T cells after enrichment of CD4 + and CD8 + we reached 68.3-fold and 71.4-fold T cell expansion rates, respectively, in two independent runs. We generated IL-18-secreting TRUCKs targeting the tumor antigen GD 2 using the CliniMACS Prodigy ® system using a recently described “all-in-one” lentiviral vector combining constitutive anti-GD 2 CAR expression and inducible IL-18. Based on our previous development and validation of automated and closed processing for GMP-compliant manufacturing of CAR T cells, we here present the proof of feasibility for translation of this method to TRUCKs. Thus, approaches using 4 th advanced CAR T cells secreting immunomodulatory cytokines upon CAR signaling, known as TRUCKs (“ T cells redirected for universal cytokine-mediated killing”), are currently under investigation. Chimeric antigen receptor (CAR)-engineered T cells can be highly effective in the treatment of hematological malignancies, but mostly fail in the treatment of solid tumors.
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