4-1BB (CD137; TNFRSF9) binds 4-1BBL, which triggers subsequent proliferation and activation of immune cells, T and NK cells in particular. We designed and generated PVR-4-1BBL, a hexameric protein of 4-1BBL fused to PVR (CD155), the major ligand for TIGIT that marks exhausted T cells. This fusion protein efficiently bind to its targets TIGIT and 4-1BB on cells as well as in soluble form.
To evaluate the in vitro efficacy of PVR-4-1BBL, T cells were isolated from PBMCs of healthy individuals or AML patients and then stimulated with anti-CD3 alone or in combination with PVR-4-1BBL. The percentages and numbers of CD25+CD69+ T cells were measured by flow cytometry. Secreted INF-γ and TNF-α were measured by ELISA. In order to test the activity of 4-1BB signaling induced by the fusion protein, we performed 4-1BB-stimulated- NFκB reporter assay.
As a result, PVR-4-1BBL activated human T cell to induce cellular proliferation and activation as well as production of cytokines, such as INF-γ and TNF-α. The fusion protein increased T cell activation particularly under suboptimal conditions of TCR stimulation. Functional activity of this fusion protein on 4-1BB-mediated cellular NF-κB signaling was more potent than a 4-1BB agonist antibody in the presence of TIGIT-positive cells in close proximity. Our fusion protein showed target-specific potency in T cell activation in the presence of both TIGIT and 4-1BB. Therefore it is expected to have a lower side effect than 4-1BB agonist antibody which may affect peripheral T cells. According to a recent report, TIGIT was up-regulated on CD8+ T cells of AML patients. We confirmed that 4-1BB was also induced in T cells from AML patients’ PBMCs along with TIGIT when stimulated by anti-CD3. The fusion protein increased T cell activation and IFN-γ production much more than agonistic anti-4-1BB antibody in AML patients’ PBMCs.
In conclusion, PVR-4-1BBL confers a dual targeting function, effectively enhancing T cell proliferation, activation and cytokine production. Therefore, we propose that the dual-targeting fusion protein, PVR-4-1BBL is a promising drug candidate for AML treatment designed by a novel immunotherapeutic approach.
MOGAM Institute for Biomedical Research.
MOGAM Institute for Biomedical Research.
All authors have declared no conflicts of interest.