Through rational protein design and library screening, the investigators generated a protein-protein interaction of human origin that can be disrupted by venetoclax. Published in Nature Chemical Biology, this interaction was incorporated into a drug-regulated off-switch protein–protein interaction (DROP)-CAR configuration comprising a transmembrane signaling chain containing CD28 and CD3ζ intracellular domains and a receptor domain fused to BCL2. The signaling chain noncovalently engages the R-domain through the LD3:Bcl-2 interaction, which can be disrupted by venetoclax, resulting in receptor disassembly.
In Jurkat cells expressing a prostate-specific membrane antigen-targeted DROP-CAR, venetoclax induced near-total receptor disassembly after 24 hours of incubation at 1 µM, yielding a half-maximal inhibitory concentration of 59 nM. At 1.25 µM, receptor disassembly occurred with a half-life of approximately 2.8 hours. Venetoclax reduced DROP-CAR-mediated cell-cell interaction strength in tumor-binding assays, and no impact was observed on an equivalent second-generation CAR.
In primary human T cells, DROP-CAR constructs targeting prostate-specific membrane antigen or epithelial cell adhesion molecule were evaluated for cytotoxic function in vitro. Venetoclax reduced interferon-γ production and cytotoxicity in DROP-CAR T cells, whereas second-generation CAR T cells were unaffected. Live-cell imaging showed disruption of immune synapse formation, reduced calcium flux, and altered microtubule-organizing center polarization following venetoclax treatment.
In an in vivo adoptive T cell transfer study (n = 7 mice per group), tumor control curves were evaluated over 22 days. Treatment groups included mice receiving DROP-CAR or second-generation CAR T cells, with venetoclax administered as indicated.
“Our work introduces a simple and clinically realistic way to reversibly dial down CAR-T cell activation using as a remote control a cancer drug, venetoclax, that is already in clinical use as a cancer therapy,” said Melita Irving, who led the research, in a press release.
Comparative in vitro experiments with degron-based CAR systems regulated by lenalidomide indicated that regulation of DROP-CAR T cells occurred through receptor detachment. In serial coculture assays, venetoclax reduced interleukin-6 production in DROP-CAR T-cell cultures.
“Unlike previous controllable CAR designs, our system uses only human protein components and a clinically approved, non-immunosuppressive drug to directly disrupt tumor cell binding by the CAR-T cells,” said co-author Giordano Attianese.
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