Modular Design of T Cell Nanoengagers for Tumor Immunotherapy via Genetically Engineered Lipid-Tagged Antibody Fragments

T cell engagers, which bind tumor-associated antigens and T cell specific molecules, represent a promising class of immunotherapies for enhancing targeted immune responses. Here, we introduce a “plug-and-display” platform for engineering T cell nanoengagers by anchoring antibody fragments into lipid-based nanoparticles. This approach utilizes a genetically engineered lipoprotein fused with single-chain variable fragments (scFv) and nanobodies, which spontaneously integrated into lipid bilayer of the nanoparticles, achieving a high surface density of at least 0.102 scFv/nm2 (approximately 3200 scFv per particle). We designed modular bi-specific (Lipo-BiTE) and tri-specific (Lipo-TriTE) immunoliposomes to enhance anti-tumor T cell immune responses. The Lipo-BiTE, integrating anti-CD3 and anti-HER2 scFv at an optimized surface density of 1.28 × 10-3 scFv/nm2, exhibited enhanced CD8+ T cell-mediated cytotoxicity in HER2-positive tumor models by simultaneously engaging tumor cells and T cells. Incorporating anti-PD-L1 nanobodies to create Lipo-TriTE further addressed T cell exhaustion. This modular platform provides a robust foundation for designing immune cell engagers, with broad applications in targeted immunotherapy. Overall design: To elucidate the underlying mechanisms of treatment response, RNA-seq analysis was performed on tumor-infiltrating CD3+ T cells isolated from mice treated with PBS, Lipo-BiTE, or Lipo-TriTE. Tumors were harvested at the experimental endpoint, and CD3+ T cells were sorted for transcriptomic profiling.