Fig. 1.

Evolution of CAR construct design. (1) First generation CAR molecules contain a synthetic extracellular receptor for target antigen recognition, a transmembrane domain and one intracellular signaling domain. (2,3) Second and third generation CAR constructs are endowed with one or more intracellular co-stimulatory domains for augmented signaling. (4) Fourth generation constructs, dubbed armored CARs, contain molecular payloads which endow CAR-engineered cells with additional features and functionality. (5) Multi-specific targeting refers to genetic engineering strategies which aim to simultaneously target multiple tumor antigens. (6) Logic-gated CAR molecules are engineered to express both an activating (aCAR) and inhibitory (iCAR) CAR receptor on their surface. The former recognizes tumor-specific epitopes and is able to initiate immune cell activation and target engagement. The latter detects epitopes exclusively present on healthy tissues and inhibits immune cell activation. By integrating both signals, logic-gated CAR-modified NK cells can differentially kill tumor cells while sparing healthy tissues with shared antigens. (7) Bispecific Killer engagers (BiKes) are bispecific antibodies with binding sites for both FcRγIII (CD16A) and a tumor-specific epitope. Acting as a molecular glue, BiKes can bind and bring NK cells in close proximity of tumor cells for specific target elimination in a CAR-like manner. aCAR, activating chimeric antigen receptor; GzmB, granzyme B; HSC, hematopoietic stem cell; iCAR, inhibitory chimeric antigen receptor; IFNγ, interferon gamma; Il-15, interleukin 15; mAb, monoclonal antibody; PFN, perforin; T-NHL, T non-Hodgkin lymphoma; TNFα, tumor-necrosis factor alpha. Image created in BioRender