T cell engagers (TCEs) have emerged as an important therapeutic approach for hematologic malignancies, yet their effectiveness against solid tumors has been limited. Elevated expression of programmed cell death 1 (PD-1) is closely linked to T cell exhaustion and contributes to immunosuppression driven by the tumor. The creation of a novel nanobody-based trispecific T cell engager (Nb-TriTE) may offer an effective means to enhance treatment outcomes. Leveraging the favorable properties of nanobodies (Nbs), we first identified a nanobody specific for fibroblast activation protein (FAP). We constructed a Nb-based bispecific T cell engager (Nb-BiTE) directed at FAP. We then engineered a Nb-TriTE by linking an anti-PD-1 Nb to this Nb-BiTE. The functional properties and therapeutic potential of the Nb-TriTE were examined using in vitro assays and in vivo studies with both cell line- and patient-derived xenograft models in mice. We successfully isolated a FAP-specific Nb and used it to develop new Nb-BiTE and Nb-TriTE constructs that exhibited strong and specific binding to their intended targets. In vitro, the Nb-TriTE triggered efficient antigen-specific tumor cell killing, strong T cell activation, and improved T cell effector functions. In mouse models of multiple solid tumors, the Nb-TriTE also markedly reduced tumor progression, extended survival, and increased T cell infiltration into tumors compared with the Nb-BiTE, while demonstrating a favorable safety profile without detectable toxicity. The newly developed Nb-TriTE represents a versatile and promising platform capable of counteracting tumor-induced immunosuppression and potentially leading to better clinical results for patients in the future.
