Steering CAR T cells to distinguish friend from foe

Abstract

CD19-specific chimeric antigen receptor (CAR)⁺ T cells have demonstrated clinical efficacy and long-lasting remissions, concomitant with tolerable normal B-cell aplasia. However, many tumor-associated antigens (TAAs) are expressed on normal tissues, the destruction of which would lead to intolerable toxicity. Thus, there is a need to engineer CAR⁺ T cells with improved safety profiles to restrict toxicity against TAA-expressing normal tissues. Bioengineering approaches include: (i) targeting CAR⁺ T cells to the tumor site, (ii) limiting CAR⁺ T-cell persistence, and (iii) restricting CAR activation. We review and evaluate strategies to engineer CAR⁺ T cells to reduce the potential of on-target, off-tissue toxicity.

Keywords: Bioengineering; T cell; chimeric antigen receptor; on-target toxicity.

Figure 1.

Potential mechanisms of toxicity mediated by infused chimeric antigen receptor (CAR)⁺ T cells. Cytokine-induced toxicity describes damage mediated by T-cell activation and release of inflammatory cytokines, as exhibited in clinical trials with CD19-specific CAR⁺ T cells. Anaphylactic response (anaphylaxis) describes development of an IgE-mediated immune response to foreign CAR moieties that degranulates mast cells. On-target, off-tissue toxicity describes CAR⁺ T-cell recognition of tumor-associated antigens (TAAs) expressed on normal tissue(s).

Figure 2.

Strategies to limit on-target, off-tissue toxicity by targeting CAR⁺ T cells to the tumor site. (A) Homing via introduced chemokine receptors. Genetically introduced chemokine receptors enable CAR-modified T cells to home to chemokine-secreting malignancies, enriching biodistribution in the tumor microenvironment. (B) Conditional expression of CAR in hypoxia. CAR fused to an oxygen-dependent degradation domain results in degradation of CAR in normoxic, normal tissue, and selective expression of CAR in hypoxia, a common environmental condition in many malignancies.

Figure 3.

Strategies to limit on-target, off-tissue toxicity by limiting CAR T-cell persistence. (A) Suicide genes. Conditional, selective ablation of CAR-modified T cells can be achieved through enforced expression of suicide genes. Drug-induced suicide relies on administration of a drug, here a chemical inducer of dimerization, which bioactivates in the T cell by inducing dimerization of inducible caspase 9 to result in initiation of a suicide program. Antibody dependent cell cytotoxicity (ADCC)-mediated suicide occurs via introduction of a biologically inert, truncated protein on the surface of CAR-modified T cells. Administration of a clinically available monoclonal antibody activates PBMC to mediate ADCC. (B) CAR expression by mRNA modification results in transient, self-limiting expression of CAR to reduce long-term antigen recognition and temporally limit toxicity. (C) Inducible CAR expression can be achieved by dual expression of two chimeric molecules in which antigen A activates a syn-NOTCH receptor, which in turn activates NOTCH responsive elements in the CAR promoter to selectively express a CAR specific for antigen B in the presence of antigen A. (D) Inducible CAR expression may potentially be achieved by expression under a drug-inducible promoter, such as the Rheo-Switch Therapeutic System (RTS)® to allow for selective CAR expression upon administration of an oral activating ligand.

Figure 4.

Strategies to limit on-target, off-tissue toxicity by restricting CAR T cell activation. (A) Dissociated signaling domains. Decoupling expression of CD3ζ and CD28 co-stimulation by expressing these intracellular endodomains in two separate chimeric proteins requires expression of two antigens to result in fully competent CAR activation. Selection of two antigens mutually co-expressed only on tumor cells limits recognition of normal tissue expressing only one antigen. (B) Inhibitory CAR to normal tissue. Expression of a CAR with an inhibitory intracellular signaling domain derived from PD-1 that is antigen-restricted to normal tissue can result in reversible inhibition of T-cell activation mediated by a second CAR specific for tumor antigen. (C) Pharmacological control of CAR activation can be achieved by inclusion of domains in the CAR construct that come together and render the CAR functionally active only in the presence of an administered drug. Thus, in the absence of drug, CAR is functionally inactive. (D) CARs recognizing adaptor molecules. Generation of tumor-specific scFvs tagged with unique molecules can be used as adaptor molecules to mediate recognition of tumor by CARs specific to the unique tag. (E) CARs sensitive to antigen density. Because tumor cells often express antigens at a higher density than that of normal tissue, CARs can be generated to be sensitive only to high-density antigen by reducing the affinity of the scFv, thereby sparing normal tissue with low antigen density from CAR recognition.