Background Chimeric Antigen Receptor (CAR)-T cell therapy is very effective in the treatment of B cell leukemia but still inefficient in solid cancer treatment. Immunosuppression in tumor microenvironment (TME), tumor heterogeneity and immune escape dampen the efficacy of CAR-T cells in these tumor types. To overcome these issues, here we propose murine 4^th generation CAR-T cells secreting a modified Toll-Like Receptor (TLR) ligand. We assume that this mediator functions as a ‘danger signal’ that can activate immune cells in the TME and promotes the generation of an inflammatory milieu. Thus, we aim to combine direct CAR-dependent antitumor activity and TLR-mediated immunostimulation in one tool.

Materials and Methods MC38 murine cancer cells were engineered to express a truncated form of the human Epidermal Growth Factor Receptor (trEGFR) and used as a target. A second generation CAR was synthetized (cetuximab scFv - CD8 hinge - 4.1BB - CD3z) and cloned in the MSCV retroviral vector. Murine 2^nd generation CAR-T cells were engineered and killing and cytokines secretion assessed by luminescence-based assay and ELISA upon co-culture with target cells. Different constructs were tested combining the TLR ligand to different export signals. Colorimetric assays and western blot analyses were performed to evaluate its activity and its production and secretion, respectively. A repetition of the Nuclear Factor of Activated T cells (NFAT) with a synthetic TATA box (synTATA)(1) was tested for the inducible production of the TLR ligand only upon CAR-T cells activation.

Results EGFR-targetedmurine CAR-T cells recognized and killed target cells after 48 hours of co-culture. Meanwhile, TLR ligand constructs were cloned and expressed in HEK293T cells. Analysis of supernatants and cell lysates revealed high production and secretion of the glycosylated ligand when coupled with the IgK leader sequence, indicating Golgi transport. Whereas, when coupled with the cell penetrating peptides transportan or a repetition of eight arginines, the ligand was produced and released in the supernatant in its non-glycosylated form, bypassing Golgi. All the secreted ligands stimulated TLR-sensor cells, with different intensities and kinetics. TLR activation appeared to be dependent on ligand production and its glycosylation status as well. Finally, murine T cells transduced with the NFAT synTATA promoter expressed GFP upon CD3 activation, indicating inducible protein production.

Conclusions EGFR-targetedCAR-T cells activity, ligand-dependent TLR stimulation and NFAT synTATA inducible protein production represent valuable building blocks for the production of 4^th generation CAR-T cells. Next steps contemplate the construction of a vector encoding for both CAR and inducible TLR ligand, and to test its functionality in terms of improved CAR activity and reshaping of immune cell landscape in solid tumors both in vitro and in an immunocompetent mouse tumor model.

References

1. Zimmermann K, Kuehle J, Dragon AC, et al. Design and Characterization of an ‘All-in-One’ Lentiviral Vector System Combining Constitutive Anti-GD2 CAR Expression and Inducible Cytokines. Cancers (Basel) 2020;12(2):375.

Disclosure Information J. Magri: None. M. Menotti: None. A. Abbati: None. T.L. Haas: None.