Overcoming on-target, off-tumour toxicity of CAR T cell therapy for solid tumours

Abstract

Therapies with genetically modified T cells that express chimeric antigen receptors (CARs) specific for CD19 or B cell maturation antigen (BCMA) are approved to treat certain B cell malignancies. However, translating these successes into treatments for patients with solid tumours presents various challenges, including the risk of clinically serious on-target, off-tumour toxicity (OTOT) owing to CAR T cell-mediated cytotoxicity against non-malignant tissues expressing the target antigen. Indeed, severe OTOT has been observed in various CAR T cell clinical trials involving patients with solid tumours, highlighting the importance of establishing strategies to predict, mitigate and control the onset of this effect. In this Review, we summarize current clinical evidence of OTOT with CAR T cells in the treatment of solid tumours and discuss the utility of preclinical mouse models in predicting clinical OTOT. We then describe novel strategies being developed to improve the specificity of CAR T cells in solid tumours, particularly the role of affinity tuning of target binders, logic circuits and synthetic biology. Furthermore, we highlight control strategies that can be used to mitigate clinical OTOT following cell infusion such as regulating or eliminating CAR T cell activity, exogenous control of CAR expression, and local administration of CAR T cells.

Figure 1.. CAR architecture.

Standard second-generation chimeric antigen receptor (CAR) with a single-chain variable fragment (scFv) derived from a monoclonal antibody linked via a transmembrane domain (TM) to a co-stimulatory signalling domain (for example, from CD28 or 4–1BB) and an intracellular CD3ζ signalling domain. CD28 allows rapid expansion but less durability, and 4–1BB promotes sustained effector function and persistence. ITAM, immunoreceptor tyrosine-based activation motif; V_H, heavy chain variable region; V_L, light chain variable region.

Figure 2.. CAR T cell cytolytic mechanisms and paracrine effects.

Upon recognition of a tumour-associated antigen (TAA) by a chimeric antigen receptor (CAR) T cell, an immune synapse is formed, followed by CAR T cell activation. Subsequently, cytotoxic granules containing perforin and granzymes are released, with the latter entering the target cell via perforin channels, triggering intrinsic apoptosis by damaging mitochondria and activating caspases^,. Additionally, CAR T cells upregulate FAS ligand, which engages the FAS receptor on target cells, triggering the extrinsic pathway of apoptosis and caspase-mediated targeted cell death. Further, CAR T cells release IFNγ and TNF that activate immune cells such as macrophages. CAR T cells might recognize TAAs on non-malignant cells, leading to undesired lysis of healthy tissues. Next-generation CAR T cells can also be equipped with additional effector functions (such as IL-12 secretion) that extend the immune response to include endogenous T cells^,. OTOT, on-target, off-tumour toxicity.

Figure 3.. Publicly available protein expression densities of selected solid tumour TAAs on non-malignant tissues.

Expression scores were established using immunohistochemistry and are based on staining intensity, the fraction of stained cells (<25%, 25–75% and >75%) and subcellular localization^,. TAA, tumour-associated antigen. *B4GALNT1 is analysed as a proxy for GD2 expression (B4GALNT1 is involved in the biosynthesis of GD2). Data are correct as of October 2022.

Figure 4.. Principles of Boolean logic-gating to circumvent OTOT.

a, AND-logic chimeric antigen receptor (CAR) T cells are activated by binding to two different tumour-associated antigens (TAAs) on target cells. b, IF/THEN tumour microenvironment-based logic-gated CAR T cells can only be activated when certain tumour microenvironment-specific characteristics are present. Examples include pH-restricted binding of TAAs, hypoxia-dependent expression of CARs or protease-dependent liberation of the antigen-binding site. c, NOT-logic CAR T cells co-express a stimulatory CAR and an inhibitory CAR targeting distinct antigens. The inhibitory CAR suppresses T cell activation when the target cell expresses selected ligands associated with non-malignant tissues. d, In IF/THEN TAA-based logic circuits, CAR T cells become activated only when encountering two different TAAs on target cells. The recognition of one TAA by a constitutively expressed synthetic Notch receptor subsequently induces the expression of a CAR targeting the other TAA. scFv, single-chain variable fragment; TM, transmembrane domain.

Figure 5.. Examples of logic-gating strategies tested in CAR T cells.

a, Conventional second-generation chimeric antigen receptor (CAR) T cells combine the two signals required for optimal CAR T cell function within a single construct. b, Dual AND-logic CAR T cells accomplish signalling and co-stimulation via two independent receptors that bind distinct tumour-associated antigens (TAAs). c, Synthetic Notch (synNotch) CAR T cells constitutively express a synNotch receptor. Antigen binding by this receptor leads to exposure of protein cleavage sites in the Notch regulatory region and transmembrane domains, allowing sequential cleavage via the enzymes ADAM and γ-secretase. This releases a tethered, intracellular transcription factor, which promotes CAR expression. The expressed CAR can recognize its own cognate antigen and trigger a cytotoxic response. d, Split, universal and programmable (SUPRA) CAR T cells express a leucine zipper as an extracellular binding domain. The leucine zipper can bind a leucine adaptor molecule carrying a single-chain variable fragment (scFv). This soluble adaptor–scFv endows the CAR T cells with antigen recognition capabilities toward a specific target. As the adaptor–scFv conjugate is administered independently to the CAR T cells, infusing a different scFv allows in vivo switching of the CAR target. A soluble zipper–scFv can competitively bind the adaptor–scFv, preventing CAR T cell signalling. This system can also be designed using the AND-logic split architecture as in b. e, AND-logic avidity-controlled CAR T cells contain low-affinity scFvs that rely on avidity for TAA encounter. CAR T cell activation is dependent on antigen recognition and dimerization of two low-affinity CARs in the presence of a soluble dimerizer. f, The Co-LOCKR system uses cage and key intermediary proteins to recognize and bind to target cells. The cage and key molecules carry a TAA binding domain, whereas only the cage carries a latch that contains a hidden Bim domain (binding site). Once the cage and key bind their cognate antigens, the latch is released, exposing the Bim domain and allowing recognition by CAR T cells. Co-stimulation is either via CD28 or 4–1BB. TF, transcription factor; TM, transmembrane domain; V_H, heavy chain variable region; V_L, light chain variable region.

Figure 6.. Control switches in CAR T cells.

a, Reversible inhibition of the tyrosine kinase LCK with dasatinib prevents phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) within CD3ζ, interrupting a necessary signal for chimeric antigen receptor (CAR) T cell effector functions^,. b, Administration and binding of AP1903 to FKBP12-F36V leads to dimerization and activation of an inducible caspase 9-based suicide construct (iCaspase 9), which triggers apoptosis of the CAR T cell. c, The CAR transgene can be engineered to also encode the selection marker RQR8, which can be targeted with rituximab to trigger complement-dependent cytotoxicity (CDC) and/or antibody-dependent cell-mediated cytotoxicity (ADCC) and thus depletion of the CAR T cells. d, A protease target site, a protease and a degron are fused to the intracellular C terminus of the CAR. The protease can cleave itself and the degron from the CAR, preventing CAR degradation and maintaining its expression on the cell surface. However, inhibition of the protease with asunaprevir prevents cleavage, leading to retention of the degron and thus degradation of the entire CAR construct. e, The inclusion or exclusion of exons during mRNA splicing can be altered using so-called splicing modifier drugs. By inserting the start codon (ATG) of the CAR into a site that is responsive to a splicing modifier, CAR translation and expression can be controlled. In the presence of this drug, the start codon of the CAR remains part of the mRNA. This allows translation of the mRNA and permits assembly and expression of the CAR. In the absence of the drug, the start codon is removed and CAR translation is not initiated. LTR, long terminal repeat; scFv, single-chain variable fragment; TM, transmembrane domain; V_H, heavy chain variable region; V_L, light chain variable region.

Figure 7.. Regional restriction of CAR T cell activity.

a, The chimeric antigen receptor (CAR) transgene can be placed under regulatory control of a heat shock protein (HSP), such that focused ultrasound can be used to generate a localized, transient increase in temperature. This activates the HSP and thus reversibly upregulates CAR transcription and expression. b, Blue light-induced CAR transcription can be achieved using the 3-component light-inducible nuclear translocation and dimerization (LINTAD) system. In addition to the CAR transgene, the T cells express a LexA-CIB1-biLINuS (LCB) construct. The biLINuS-responsive element is activated upon localized application of blue light, resulting in exposure of a nuclear localization sequence (NLS) and translocation of the LCB construct into the nucleus. LexA (as part of LCB) can bind to a LexA binding sequence (BS) engineered in the CAR transgene. The CIB1 component of LCB can then recruit a co-expressed CRY2–VPR fusion construct that can target the minimal promoter and trigger the expression of the CAR. c, Locoregional intraventricular administration of CAR T cells as an alternative to systemic intravenous infusion. CNS, central nervous system.