Design and properties of BiTE and CiTE molecules. (A) Schematic representation of the BiTE-like molecule. scFv domains, consisting of 1 variable heavy chain (V_H) joined to 1 variable light (V_L) domain, are connected into a single polypeptide chain. One of the 2 distal scFv domains (white) is specific for CD3ε, and the other (gray) is specific for CD33. Black connecting lines represent flexible Gly4Ser linker between the domains. (B) Schematic representation of the CiTE molecule. The CiTE molecule was constructed by fusing the PD-1_ex to the CD3 × CD33 BiTE. (C) Mechanism of BiTE action. To induce activation of and cytotoxic activity by a T cell, a BiTE protein must engage both a T cell and tumor cell (AML blast) simultaneously. Single-cell binding to a T cell or a target cell causes no activation. Simultaneous binding of multiple BiTE molecules to both T cell and tumor cell promotes the formation of an immunological synapse leading to T-cell activation, release of cytokines (IFN-γ), and cytotoxicity of the tumor cell. This T-cell activation leads to upregulation of checkpoint molecules like PD-1 on T cells and PD-L1 on AML blasts. PD-L1 interacts with PD-1 on T cells to suppress the T-cell–mediated tumor cytotoxicity. (D) Mechanism of CiTE action. Blockade of the interaction between PD-1 on T cells and PD-L1 of the tumor by the PD-1_ex domain of the CiTE prevents T-cell anergy and exhaustion, leading to enhanced T-cell proliferation and tumor cell killing.