Background: Despite successes in haematological tumours, CAR T-cell therapy remain ineffective in solid tumours owing to the immunosuppressive tumour microenvironment (TME). The lack of T-cell costimulation remain a major challenge within the TME, preventing sufficient T-cell activation and effector function. 4-1BB presents a promising costimulatory molecule to overcome such deficiencies, being associated with CAR T-cell persistence and favourable responses to immune checkpoint blockades. Additionally, the immunosuppressive cytokine TGF-β also suppresses T-cell proliferation and effector functions within the TME, dampening the anti-tumour immune response.
Aims: To enhance CAR T-cell efficacy in solid tumours by targeting both 4-1BB and TGF-β.
Methods: We designed a bi-specific molecule capable of sequestering free TGF-β while activating 4-1BB on T-cells, named Bifunctional Activator Trap (BAT). BAT features a 4-1BB binding domain, IgG Fc region, and a TGF-β binding domain. Purified BAT were produced with the CHO expression system and evaluated both in vitro and in vivo.
Results: BAT reduced high concentrations of free TGF-β to undetectable levels even at low molar ratios in vitro; and completely prevented downstream activations of SMAD signalling in reporter cells. BAT also activated 4-1BB via the NF-κB pathway similar to existing anti-41BB monoclonal antibodies. Such 4-1BB activation were primarily driven by NF-κB rather than AP-1 or MAPK signalling pathways. When co-cultured with activated CAR T-cells, BAT further upregulated the activation markers CD25 and CD69, while simultaneously preventing TGF-β from downregulating CD25 and upregulating PD1. Intraperitoneal administration of BAT in tumour-bearing mice resulted in stable serum drug concentrations, significant BAT distribution in the tumour, and a complete reduction of tumour TGF-β levels. Lastly, systemic administration of BAT enhanced the anti-tumour effect of CAR T-cells without notable toxicities.
Conclusions: BAT effectively prevented TGF-β immunosuppression and provided 4-1BB costimulation in vitro, while showing sufficient stability and efficacy in vivo, allowing for further MoA studies.