As more patients have been treated with CAR-T cells, a common mechanism of relapse is antigen escape—where the tumor downregulates or mutates the target antigen, resulting in outgrowth of antigen-negative tumor cells. In fact, up to 25% of ALL patients treated with CD19-targeted CAR-T cells relapse with CD19-negative disease. To overcome this limitation, CARs targeting more than one tumor antigen have been developed to increase tumor cell elimination and decrease the potential for antigen negative tumor outgrowth. In addition to promiscuous CARs discussed previously, multi-targeted CAR-T cells use two or more antigen binding domains to target multiple antigens simultaneously. This can be achieved through the transduction of multiple CAR constructs into the same T cell, either via the same or separate construct(s), or coadministration of separately transduced CAR-T cells (Fig. 1a). Alternatively, two scFvs specific to different target antigens can be expressed in tandem and attached to a single intracellular signaling domain (Fig. 1a). Each of these combinations has required optimization of the CAR design, including the use of different configurations of the linker and antigen binding variable regions.
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)
CAR-T cells can also target multiple tumor antigens by secretion of a bispecific T cell engagers (Fig. 1b). T cell engagers are composed of two scFvs in tandem: one specific to a tumor antigen and the other to the CD3 domain of the TCR. Upon binding the tumor antigen, T cell engagers can recruit endogenous T cells in the tumor microenvironment to kill tumor cells. T cell engagers have known efficacy when delivered as monotherapy, though significant cytokine-mediated toxicity can occur. Local secretion by CAR-T cells enables antigen targeting without systemic dissemination of the antibody or downstream immune-related adverse effects (irAEs) due to target antigen expression in normal tissue.
