Figure 11.

Schematic structure and putative mechanism of action of uPAR-directed immunotherapy approaches. (a) Wang et al. developed third-generation uPAR-targeted CAR T-cells using ATF as antigen recognition domain instead of the usual antibody-derived single-chain variable fragment (scFv) [251]. Besides the CD3 ζ-signaling domain, third-generation CARs incorporate endodomains from co-stimulatory molecules, in this case, CD28 and CD137 (also known as 4-1BB), to deliver the additional signals necessary to fully activate the T-cell, physiologically provided by the antigen-presenting cells (APC) [258]. (b) ARM-U2 is a uPAR-targeted antibody-recruiting small molecule comprising a derivative of the high-affinity uPAR inhibitor IPR-803 (bottom box), functioning as the target-binding molecule (TBM, shown in red), and a 2,4-dinitrophenol (DNP) moiety as the antibody-binding domain (ABM, depicted in blue). (c) Putative action mechanisms of ATF-CAR T cells (upper) and ARM-U2 (bottom). Upon uPAR-recognition and binding, mediated by the ATF domain, ATF CAR T-cells (upper) form a non-classical immune synapse (IS) with the target cancer cell, which mediates the T-cell cytolytic effector functions, including perforin and granzyme release, ultimately leading to selective tumor cell death [258]. The simultaneous binding of ARM-U2 to surface uPAR on the target cancer cell (by the TBM) and endogenous anti-DNP antibodies (recruited via the DNP moiety, ABM) results in the formation of a ternary complex, which can subsequently trigger selective immune-mediated cytotoxicity through different mechanisms. In the complement-mediated cytotoxicity, the C1q complement protein binds to the Fc part of the antibodies and activates the complement cascade, which culminates in the formation of the membrane attack complex (MAC) on the cancer cell surface and its lysis. Alternatively, the antibody’s Fc can also interact with the Fc-receptors expressed on the surface of various immune cells, such as macrophages or natural killer cells, followed by target cell phagocytosis (ADCP) or tumor-cell lysis via NK releasing potent oxidizing agents and protein toxins, such as granzyme and perforin (ADCC) [255]. Created with BioRender.com.