BAFF-based trifunctional T-cell engagers trigger robust tumor immunity against B-cell malignancies.

Advancements in protein engineering have driven the continuous optimization of T-cell engagers (TCEs), resulting in remarkable clinical outcomes in the treatment of B-cell malignancies. Moreover, developing tri- or multispecific TCEs has emerged as a promising strategy to address the challenges of tumor heterogeneity and antigen escape. However, considerable obstacles remain, primarily in format design. In this study, we engineered BAFF-based TCEs with various formats that incorporate anti-CD3 Fab or IgG domains fused with BAFF ligands to target BAFF receptors (BAFFR, BCMA, and TACI). These constructs varied in valency and the presence or absence of long-acting elements such as Fc domains or the albumin binding domain consensus sequence (ABDCon). Although the inclusion of an Fc domain did not enhance sustained tumor eradication, variations in valency and spatial configuration profoundly influenced cytotoxicity. We identified TriBAFF/CD3/ABDCon as the optimal trifunctional construct, featuring an anti-CD3 Fab backbone with BAFF and ABDCon fused to the C-termini of the heavy and light chains. This design facilitates optimal immune synapse formation between the target cells and T cells and effectively controls tumor burdens in various B-cell malignancy models with good tolerability. Notably, TriBAFF/CD3/ABDCon outperformed conventional therapies, including blinatumomab and BAFF-based CAR-T cells, in models of heterogeneous leukemia and aggressive lymphoma. These findings underscore the potential of using natural ligands as antibody-targeting modules and provide valuable insights into the design of the next generation of multispecific TCEs, which hold promise for improving treatment outcomes in a wide range of malignancies and beyond.