Precise Tumor Targeting with NOT Logic-Gated Chimeric Antigen Receptor Gene Circuits

Immune cells engineered with chimeric antigen receptors (CARs) have produced breakthrough efficacy in cancer immunotherapy. Still, the lack of uniquely defined cancer-specific antigens brings risk of life-threatening on-target, off-tumor toxicity. CAR suppression in response to a “safety antigen” selectively expressed on healthy cells could broaden the spectrum of addressable cancers and enable more aggressive on-target, on-tumor treatments. Here, we engineered NK and T cells with a synthetic NOT GATE circuit: an activating CAR (aCAR) killed target cells expressing activating antigen, while an inhibitory CAR (iCAR) suppressed cytotoxicity against target cells also expressing a safety antigen.

We have identified a panel of functional iCARs with different intracellular domains in NK cells and T cells. NOT GATE-equipped CAR-NK or -T cells pairing an iCAR with a second-generation aCAR significantly reduced cytotoxicity and secretion of inflammatory cytokines in response to safety antigen-expressing target cells, while aCAR-only expressing cells exhibited no such significant differences. The NOT GATE gene circuit did not interfere with aCAR performance in the absence of the safety antigen. We demonstrated the persistence of iCAR functionality with repetitive exposure of engineered immune cells to the target cells.

We have developed a NOT GATE gene circuit and validated its function in CAR-NK and -T cells. NK cells can be used in an allogeneic fashion, which may lead to a reduced cost of therapy and increased patient access relative to autologous products. Immune cell therapies equipped with our CAR NOT GATE gene circuits may improve safety for currently validated cancer antigens and enable new treatments for previously unaddressed indications.