GD2-specific car-T cells co-expressing the IL15 membrane form effectively lyse GD2-positive cell targets

1 - FSBI "NMITs DGOI named after Dmitry Rogachev" of the Ministry of Health of Russia, Moscow. 2 – FSBIS "Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikov" RAS, Moscow, Russia

Genetically modified T cells expressing chimeric receptors (CAR-T) have become an important tool for the treatment of the CD19+ hematological tumors. Due to a single-chain variable fragment of the antibody as a part of the CAR receptor, the cells acquire their specificity against selected tumor-associated antigens. The 3rd generation CAR also includes transmembrane, co-stimulatory domains, and tyrosine activation motifs derived from CD3z. This design allows the T-cells to recognize antigens in an MHC-independent manner and provides activation and release of cytotoxic cytokines upon recognition of the target. There are several limitations to the effective use of the CAR-T cells for the treatment of solid tumors, among them tumor stroma suppressive activity, poor persistence, and migration of effectors in a tissue.

The aim of our project is to improve the CAR-T cells targeting at GD2 and L1CAM neuroblastoma antigens by co-expression with membrane-anchored cytokines.

Results . At the present time, we have succeeded in achieving a stable 60-90% efficiency of lentiviral transduction of the primary T cells with an anti-GD2 CAR construction of the 3rd generation. At the same time, the CAR+ cells are equally represented by the CD4+ and CD8+ populations. To increase the persistence of the CAR-T cells, bicistronic co-expression of anti-GD2 CAR with the membrane form of IL15 (up to 40% CAR+; 30% IL15+; 30% IL15Ra+) was performed. Although the expected benefits of the 4th generation CAR receptor remain to be tested in in vivo models, its expression correlates with a better T cell survival in vitro . Both the IL15/IL15Ra positive and negative anti-GD2 CAR-T cells have been shown to specifically kill the GD2+ neuroblastoma and the glioma target cells (IMR-32, T98G) at an E:T ratio of 5:1 - 20:1. At the same time, degranulation, analyzed by the level of the surface CD107a, in contrast to anti-CD19 CAR-T, was practically not observed in short incubations.

Conclusion . The GD2-targeted CAR-T cells armored with the membrane form of IL15 specifically kill the GD2+ tumor cells in vitro and may serve as a promising tool for immunotherapy of neuroblastoma, ganglioneuroblastoma and glioma.