Generation and functional evaluation of bispecific T cell engaging antibodies.

T cell engagers (TCE) are a class of bispecific antibodies that simultaneously target a tumor antigen and CD3, acting as a bridge between T cells and tumor cells. They promote the formation of T cell receptor (TCR)-independent canonical immune synapses and potent tumor-specific cytotoxic responses at extremely low concentrations. TCE-based immunotherapeutic have revolutionized the treatment landscape for hematological cancers. Structurally, TCE can be divided into non-IgG-like formats consisting of antibody fragments fused to an Fc-free polypeptide, and IgG-like formats containing an Fc region for construct dimerization. For the former, tandem single-chain variable fragment (scFv) are the most common design. They are potent and small molecules with high tissue penetration and improved tumor penetration, but also have a short serum half-life. To maintain effective serum antibody levels, they require continuous infusion or other strategies such as engineering T cells to secrete the TCE in situ. On the other hand, to enable monovalent CD3 interaction, the binding domains of IgG-like TCEs are usually heterodimerized by incorporating complementary docking mutations in the CH3 domains. In addition, most IgG-like TCEs contain engineered silent Fc to eliminate unwanted Fc interactions and mitigate potential toxicity, while retaining the potential for half-life extension. Here we present detailed protocols for the design, generation and validation of both non-IgG-like and IgG-like TCE, as well as their recombinant production and characterization. The methodology covers construct generation, antibody expression in mammalian systems and purification by chromatography. Finally, structural and functional characterization includes biophysical studies including in vitro and in vivo studies.