Next generation LMP2A-targeting TCR-recombinant T-Cells with inducible expression of Il-18 as promising tool to treat various EBV-associated malignancies

Background

Epstein-Barr virus (EBV) infects >90% of the population and remains in B-cell compartments life-long, passing through several latency stages. In latency stages II and III, latent membrane protein 2A (LMP2A) is expressed and therefore associated with post-transplant lymphoproliferative disorder (PTLD) as well as with various lymphomas and carcinomas. Recently, a clinically protective TCR recognizing an LMP2A-derived peptide (CLG) in context of HLA-A*02 (A*02/CLG) was identified (LMP2A_TCR).

Methods

We developed TCR-engineered T cells based on the LMP2A_TCR (LMP2A_TCR-Ts) and further equipped these with an inducible cassette for locally restricted IL-18 release (LMP2A_iIL18_TCR-Ts) to enhance T-cell functionality. Engineered TCR-Ts were generated by lentiviral transduction and their memory phenotype, replicative capacity, activation and exhaustion state during generation and after target cell encounter were analyzed. Their cytotoxicity towards HLA-A*02⁺ SPI-801 cells loaded with the CLG peptide and HLA-A*02⁺ EBV-transformed B-lymphoblastoid cell lines (B-LCLs) were analyzed using flow cytometry-, microscopy- and impedance-based assays. Furthermore, cell avidity (z-MOVI) as well intracellular Ca²⁺ signaling were evaluated.

Results

LMP2A_TCR-Ts and LMP2A_iIL18_TCR-Ts recognized HLA-A*02+ SPI-801 cells loaded with different concentrations of CLG in a dose-dependent manner. No signs of HLA cross-reactivity or recognition of an irrelevant HLA-A*02-restricted peptide were observed. Specificity and activation capacity of LMP2A_iIL18_TCR-Ts was confirmed by Ca2+ signaling analysis. Release of IL-18, which was shown to convert T cells into pro-inflammatory effector cells and reshape the immunosuppressive tumor milieu, resulted in significantly increased cytotoxicity when compared to LMP2A_TCR-Ts. Of note, LMP2A_iIL18_TCR-Ts but not LMP2A_TCR-Ts recognized and efficiently eliminated HLA-A*02⁺ B-LCLs serving as PTLD model and described to produce an anti-inflammatory milieu.

Conclusion

Our data indicate that LMP2A_iIL18_TCR-Ts specifically recognize A*02/CLG, leading to effective elimination of EBV⁺ HLA-A*02⁺ target cells. The release of IL-18 improved the functionality of the engineered T cells. In conclusion, ex vivo isolated, protective TCRs could be redirected into T cells from third-party donors with the potential to attract innate immune cells and alter the tumor environment, thereby widening the applicability of T-cell therapy to refractory viral infections.

Offenlegung Interessenkonflikt:

The authors declare no conflict of interest.