Engineering strategies to overcome the current roadblocks in CAR T cell therapy

Abstract

T cells genetically engineered to express chimeric antigen receptors (CARs) have proven - and impressive - therapeutic activity in patients with certain subtypes of B cell leukaemia or lymphoma, with promising efficacy also demonstrated in patients with multiple myeloma. Nevertheless, various barriers restrict the efficacy and/or prevent the widespread use of CAR T cell therapies in these patients as well as in those with other cancers, particularly solid tumours. Key challenges relating to CAR T cells include severe toxicities, restricted trafficking to, infiltration into and activation within tumours, suboptimal persistence in vivo, antigen escape and heterogeneity, and manufacturing issues. The evolution of CAR designs beyond the conventional structures will be necessary to address these limitations and to expand the use of CAR T cells to a wider range of malignancies. Investigators are addressing the current obstacles with a wide range of engineering strategies in order to improve the safety, efficacy and applicability of this therapeutic modality. In this Review, we discuss the innovative designs of novel CAR T cell products that are being developed to increase and expand the clinical benefits of these treatments in patients with diverse cancers.

Fig. 1. Blueprint of CAR design.

Chimeric antigen receptors (CARs) have a modular design consisting of an antigen-binding domain, a hinge, a transmembrane domain and an intracellular signalling domain. In preclinical and clinical studies of CAR T cells, investigators have used reference sequences from a myriad of molecules within each of these domains. The antigen-binding domain is usually a single-chain variable fragment (scFv) molecule derived from a monoclonal antibody (mAb; from mouse anti-human CD19 antibodies, for example, in the currently FDA-approved CAR T cell products). The intracellular signalling domain generally contains a T cell activation domain derived from the CD3ζ chain of the T cell receptor as well as co-stimulatory domains that often comprise immunoreceptor tyrosine-based activation motif-containing regions of CD28 or 4-1BB (also known as CD137 and TNFRSF9). CAR gene constructs can be further modified to engineer CAR T cells with expression of an ‘armour’ protein, which is typically a cell-surface or secreted immunomodulatory molecule that enhances T cell function or favourably modifies the tumour microenvironment. Variation of each of these component parts of CAR constructs enables fine tuning of the functionality and antitumour activity of the resultant CAR T cell product, and CARs with various designs are being developed to improve the safety and efficacy of these therapies across various cancers. In addition, gene editing of the engineered T cells to further enhance CAR T cell function is a promising avenue of research in this area. BiTEs, bi-specific T cell engagers; DARPins, designed ankyrin repeat proteins; ICOS, inducible T cell co-stimulator; IL-1Ra, IL-1 receptor antagonist; KIR2DS2, killer cell immunoglobulin-like receptor 2DS2; V_HH, variable domain of a heavy chain antibody.

Fig. 2. Overcoming systemic cytokine toxicities of CAR T cells.

The activation and rapid expansion of chimeric antigen receptor (CAR) T cells in patients treated with these agents is associated with high systemic levels of cytokines. To counter this effect in the event of systemic cytokine-related toxicities, researchers are engineering methods to control CAR expression or activity. A | CAR T cells with on/off switches predicated on small-molecule adapter ligands (a), CAR subunit-dimerizing agents (b), inhibitors of signalling downstream of the CAR (c), or protease inhibitors used to control CAR protein expression (d). B | Suicide gene systems that enable elimination of CAR T cells via induction of apoptosis (a) or antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) (b). C | CAR T cells engendered with the intrinsic ability to secrete factors that neutralize relevant cytokines. FcR, fragment crystallizable region receptor; GM-CSF, granulocyte–macrophage colony-stimulating factor; iCasp9, inducible caspase 9; IL-1R, IL-1 receptor; IL-1Ra, IL-1 receptor antagonist; mAb, monoclonal antibody; NK, natural killer; scFv, single-chain Fv; SMASh-CAR, small molecule-assisted shutoff chimeric antigen receptor.

Fig. 3. Overcoming on-target, off-tumour toxicities of CAR T cells.

Chimeric antigen receptor (CAR) T cells are typically designed to target tumour-associated antigens (TAAs); however, expression of these antigens on healthy tissues can result in ‘on-target, off-tumour’ CAR T cell-mediated toxicities. A | Engineering strategies aiming to overcome this include mechanisms whereby the tumour specificity of CAR T cells is enhanced by ensuring dependency of functional activation on the recognition of multiple TAAs (a), the absence of an antigen selectively expressed on non-malignant cells (b), or the presence of factors that are typically enriched on tumour cells, such as the phosphoantigens that can be recognized via γδT cell receptors (TCRs) (c), or in the tumour microenvironment (TME) such as the immunosuppressive cytokine IL-4 (d). B | Alternative strategies are based on logic gating and/or conditional expression systems, whereby expression of a CAR targeting a particular TAA is dependent on activation of another engineered transgenic receptor, such as synthetic Notch (synNotch) receptors, by a different TAA (a) or is driven by a factor associated with the TME such as hypoxia (b). C-VHL, HIF1α C-terminal von Hippel–Lindau tumour suppressor protein recognition site; HIF; hypoxia-inducible factor; iCAR, inhibitory chimeric antigen receptor; IL-2Rγ, IL-2 receptor γ chain; IL-4R, IL-4 receptor; IL-7Rα, IL-7 receptor α chain; ITIM, immunoreceptor tyrosine-based inhibitory motif; NTAD, HIF1α N-terminal transactivation domain; N-VHL, HIF1α N-terminal von Hippel–Lindau tumour suppressor protein recognition site; PD-1, programmed cell death 1; STAT5, signal transducer and activator of transcription 5; T_H1, T helper 1.

Fig. 4. Improving the efficacy of CAR T cell therapy.

Several innovative engineering strategies have been used to enhance the efficacy of chimeric antigen receptor (CAR) T cells. A | CAR T cell products designed to target multiple different tumour-associated antigens (TAAs) (a) can overcome antigen escape or heterogeneity; variations on this approach are predicated on the use of CAR T cells engineered to co-express and secrete bi-specific T cell engagers (BiTEs) (b) or the use of CARs targeting adapter molecules that can be linked to a range of soluble antigen-recognition moieties to enable simultaneous recognition of multiple antigens with a single CAR (c). B | The in vivo persistence of CAR T cells can be enhanced by using less-differentiated T cell subsets (a) or by engineering CAR T cells to express factors that foster a supportive microenvironment such as 4-1BB ligand (4-1BBL) (b). C | The trafficking and/or penetration of CAR T cells into solid tumours can be improved by engendering these cells with the ability to respond to tumour-associated chemokines (a) or to target physical barriers present in the tumour microenvironment (TME) (b). D | Finally, CAR T cells can be engineered to overcome the immunosuppressive factors present in the TME, for example, by circumventing the activity of inhibitory immune checkpoints, including programmed cell death 1 (PD-1) (a), or by promoting an inflammatory milieu via the expression of cytokines (b) or other immunostimulatory factors, such as CD40 ligand (CD40L) (c). APC, antigen-presenting cell; CAF, cancer-associated fibroblast; CCR2b, CC-chemokine receptor 2b; CCR4, CC-chemokine receptor 4; CSF-1R, macrophage colony-stimulating factor 1 receptor; CSR, chimeric switch receptor; DC, dendritic cell; DNR, dominant negative receptor; FAP, fibroblast activation protein; scFv, single-chain variable fragment; shRNA, short hairpin RNA; T_CM, central memory T cells; TCR, T cell receptor; T_eff cell, effector T cell; T_EM cell, effector memory T cells; T_H cell, T helper cell; T_SCM cell, stem cell-like memory T cell.

Fig. 5. Life cycle of a CAR T cell and associated challenges to safe and effective therapy.

Many obstacles must be overcome over the life cycle of chimeric antigen receptor (CAR) T cells, including production issues, physical barriers to tumour infiltration, tumour heterogeneity in antigen expression, immunosuppressive factors in the tumour microenvironment and challenging toxicities. Researchers have devised several novel engineering solutions to address each of these issues, as outlined in this figure. BiTEs, bi-specific T cell engagers; BTLA-4, B and T lymphocyte attenuator 4; CRS, cytokine release syndrome; CTLA-4, cytotoxic T lymphocyte protein 4; DC, dendritic cell; GM-CSF, granulocyte–macrophage colony-stimulating factor; ICANS, immune effector cell-associated neurotoxicity syndrome; MDSCs, myeloid-derived suppressor cells; PD-1, programmed cell death 1; PD-L1, programmed cell death 1 ligand 1; scFv, single-chain Ig variable fragment; TGFβ, transforming growth factor-β; T_reg, regulatory T cells.