Several methods of indirect antigen binding have been devised to allow for a single CAR-To target different antigens. These “universal” CARs allow the same CAR design to be used across different tumor types. This can be achieved through the use of a bridging molecule that binds to a tumor-associated antigen and provides a complimentary binding domain for the CAR. For example, an scFv can be linked to a leucine zipper that binds to a cognate leucine zipper CAR (also known as a SUPRA CAR). When the leucine zippers interact, the CAR self-assembles, and the T cell is activated. A variation of this strategy is to use a tagged tumor antigen anti body with an anti-tag CAR, such as a biotinylated antibody and a CAR with an avidin binding domain or a fluorescein (FITC)-tagged antibody and a FITC-binding CAR. Similarly, CARs can be engineered with a CD16 binding domain that recognizes the Fc constant region of tumor-specific antibodies when bound to their target antigen. These methods are highly dependent on T cell persistence and the half-life of the adaptor.
Adaptor systems can also improve the safety of CAR-T cells by enabling dynamic control of cell activation. Among the safety issues posed by CD19-directed CAR-T cells is overactivation of the T cells leading to cytokine release syndrome (CRS) or immune cell-associated neurotoxicity syndrome (ICANS). The ability to rapidly turn off CAR-T cells when toxicity becomes apparent can circumvent these potentially fatal irAEs in patients. Low molecular weight adapters can be designed to bridge FITC to a molecule that will bind a receptor on the tumor cell, such as folate and the folate receptor. When coadministered with FITC-binding CAR-T cells, this adaptor molecule induced tumor killing in a mouse model. Importantly, cessation of adaptor infusion or administration of a competing ligand halted cytokine- mediated toxicity. A similar system uses a tumor-specific antibody fragment (Fab) containing a neo-epitope peptide that will bind to a CAR with an scFv specific to the neoantigen (termed a switch CAR). When this strategy was implemented using a Fab against CD19 in a xenograft model of B cell leukemia, it enabled dose-adjusted tuning of activity with comparable efficacy and lower systemic cytokine levels relative to standard CD19-targeted CAR-T cells.
AR-T cell activation can also be controlled through the addition of small molecules that trigger CAR assembly. One version of an “ON-switch” CAR uses a split CAR with an antigen-binding domain that assembles with the intracellular signaling domain only in the presence of a heterodimerizing small molecule. This CAR, nick named a remote-control CAR, provides a tunable system to control the CAR-T cell activation. Another “ON-switch” CAR, termed a SWIFF-CAR, uses a CAR construct linked by a protease site to a protease that is connected to a degron. In the absence of a protease inhibitor, the protease will cleave the site, disconnecting the CAR and allowing its expression on the cell membrane. Conversely, in the presence of a protease inhibitor, this activity is blocked and the CAR construct is targeted for proteolytic degradation by the degron. Another degron-based system directly targets the CAR for degradation by including a C-terminal zinc finger degron motif that recruits a ubiquitin ligase in the presence of lenalidomide, thus targeting it for degradation (Fig. 1c). Lenalidomide, which is FDA-approved for treating multiple myeloma and non-Hodgkin lymphoma, can be used in a similar system to activate CAR expression on the T cell surface. A split CAR can be engineered with one subunit containing the extracellular antigen- binding and CD28 intracellular domains connected to the zinc finger degron and the other subunit made up of CD28 and CD3ζ domains flanking a CRBN lenalidomide binding motif (normally found as part of the E3 ubiquitin ligase complex.) In this system, addition of lenalidomide induces dimerization of the CAR and activation in the presence of target antigen (Fig. 1d).
Fig1. Next generation CAR-T cell modifications. Chimeric antigen receptor (CAR)-T cells can be designed to evade tumor antigen escape and mitigate off tumor effects. (a) Antigen escape can be evaded by infusing a mixture of CAR-T cell products targeting different antigens or multi-targeted CAR-T cells designed to target multiple tumor antigens either via the same or different CAR constructs or (b) CAR-T cells engineered to secrete bi-specific T cell engagers which recruit endogenous T cells to engage in tumor killing. (c) To improve safety, one approach is to turn “OFF” the CAR by attaching a zinc finger degron motif to the C-terminus, which binds lenalidomide. When lenalidomide is added, a ubiquitin ligase is recruited, which targets the CAR for degradation and inactivates the CAR-T cell. (d) Triggered assembly CARs improve safety by enabling dynamic control of CAR-T cell activation via exogenously administered agents. In one example, lenalidomide-binding (CRBN) and zinc finger degron (IKZF3) domains can be engineered into SPLIT CARs to enable dimerization and thus activation only in the presence of lenalidomide. (e) Logic-gated CAR-T cells can mitigate on-target off-tumor effects by introducing another layer of specificity. Multi-targeted SPLIT CARs activate only when two tumor antigens are bound by separate scFvs. (f) Conditional expression of a CAR against one antigen is activated by binding of a separate synthetic Notch (synNotch) receptor against a different tumor antigen. (g) CAR-T cells can inactivate upon binding to an antigen on non-malignant cells. Thus, activation depends on binding to one tumor antigen but not the other in a “NOT” logic gate approach. (iCAR, inhibitory chimeric antigen receptor; ITIM, immunoreceptor tyrosine- based inhibitory motif; PD-1, programmed cell death 1)
