CARs: Synthetic Immunoreceptors for Cancer Therapy and Beyond

Abstract

Chimeric antigen receptors (CARs) are versatile synthetic receptors that provide T cells with engineered specificity. Clinical success in treating B-cell malignancies has demonstrated the therapeutic potential of CAR-T cells against cancer, and efforts are underway to expand the use of engineered T cells to the treatment of diverse medical conditions, including infections and autoimmune diseases. Here, we review current understanding of the molecular properties of CARs, how this knowledge informs the rational design and characterization of novel receptors, the successes and shortcomings of CAR-T cells in the clinic, and emerging solutions for the continued improvement of CAR-T cell therapy.

Keywords: adoptive T-cell therapy; chimeric antigen receptor (CAR); immunotherapy; protein engineering; synthetic biology.

Figure 1. Chimeric Antigen Receptor (CAR) Structure and Designs

(A) CARs are modularly constructed fusion receptors comprising the following protein domains (from N- to C-terminus): extracellular antigen-binding domain, extracellular spacer, transmembrane domain, costimulatory domain(s), and T-cell activation domain. (B) First-generation CARs contain a single intracellular signaling domain, most commonly CD3ζ, that is capable of triggering T-cell activation. Second- and third-generation CARs incorporate one or two costimulatory domains, respectively, and enhance productive T-cell stimulation compared to first-generation CARs. ScFv: single-chain variable fragment; Fc: crystallizable fragment of an antibody; V_L: light-chain variable fragment; V_H: heavy-chain variable fragment; ITAM, immunoreceptor tyrosine-based activation motif.

Figure 2. An Integrated Mechanistic Model of CAR Signaling Initiation

Research on T-cell receptor (TCR) triggering and the specific signaling domains utilized in CARs suggests the following potential mechanisms working in concert to initiate CAR signaling. (A) Ligand binding could generate mechanical forces that lead to the dissociation of CAR intracellular domains from the plasma membrane, thereby unmasking critical binding sites for downstream signaling molecules. (i) At rest, CAR intracellular domains (e.g. CD28 and CD3ζ) may interact with the plasma membrane, as they do in their native receptor contexts, through basic residue motifs that bind to the negatively charged inner leaflet of the plasma membrane [–16]. (ii) Upon antigen binding, CAR intracellular domains dissociate from the plasma membrane and adopt a signaling-competent conformation that allows interactions with downstream signaling molecules, including kinases such as ZAP-70 and Lck [15,16]. Phosphorylation of the intracellular domains is thought to lock the domains in the membrane-free state [15]. (B) Extending the receptor deformation model of TCR triggering to CARs suggests that the changes in CAR conformation from (A, i) to (A, ii) may arise from mechanical pulling or pushing between the T cell and the target cell. (i) A pulling force can be transmitted via tension in the CAR extracellular and transmembrane domains to dislodge the intracellular domains from the plasma membrane. (ii) A pushing force may alter the local membrane curvature, thereby reducing the stability of the membrane-associated state of the CAR intracellular domains. (C) In the kinetic segregation model of TCR triggering, bulky phosphatases must be physically segregated from TCRs for T-cell activation domains to transduce signal. Thus, in addition to having accessible (i.e. membrane-free) intracellular signaling domains, CARs may also need to be segregated from phosphatases to initiate signal transduction. (i) Segregation of phosphatases and CARs can occur when CAR/ligand interactions force the T cell and target cell into close apposition and exclude bulky phosphatases from the immunological synapse (IS). (ii) By this logic, CARs with excessively long extracellular spacers that allow phosphatases to comingle with CARs at the IS would not be able to robustly activate T-cell signaling. (D) CARs in resting T cells are localized diffusely together with other surface receptors such as CD45. As the events in (C) take place in response to target-cell engagement, ligated CARs can coalesce into microclusters, which have been confirmed to exclude CD45 and transduce T-cell activation signals [26]. With time, microclusters at the CAR-containing immunological synapse are hypothesized to coalesce and organize with other native surface receptors into the supramolecular activation cluster (SMAC), commonly observed at TCR synapses.

Figure 3. Comparison of Stimulatory Immunoreceptors

Standard CARs combine the antibody-like target-binding properties of B-cell receptors (BCRs) with the T-cell activation abilities of T-cell receptors (TCRs). T-cell costimulatory properties are further incorporated into CARs with the addition of costimulatory domains. K_D, dissociation equilibrium constant (with typical range shown for each receptor type); V_L: light-chain variable fragment; V_H: heavy-chain variable fragment; ITAM, immunoreceptor tyrosine-based activation motif.

Figure 4. Increasing Targeting Specificity of CARs by Boolean Logic Calculations

(A) NOT-gate CAR developed by the Sadelain group paired a conventional CAR or TCR with an inhibitory CAR (iCAR) that contains either PD-1 or CTLA-4. Antigen binding to the iCAR triggers an inhibitory signal that overrides the activation signal from the conventional CAR or TCR [79]. (B-D) Three AND-gate CAR designs. (B) A chimeric costimulatory receptor (CCR) that is equivalent to a third-generation CAR lacking the CD3ζ chain was developed. This CCR is paired with a first-generation CAR, and both receptors must be triggered by their respective cognate antigens to achieve full T-cell activation [78]. (C) The Wang group engineered a “masked CAR” whose antigen-binding domain is blocked by a masking peptide until the peptide is removed via cleavage by a tumor-associated protease [81]. (D) The Lim group developed a synNotch receptor that releases a synthetic transcription factor (TF) upon ligand binding (signal 1). The TF subsequently drives the expression of a CAR from a synthetic, cognate promoter, and the CAR can then responds to its cognate antigen (signal 2) [80]. V_L: light-chain variable fragment; V_H: heavy-chain variable fragment; scFv: single-chain variable fragment; Ag, antigen; DAP10: DNAX-activating protein 10; FKBP: FK506-binding protein; FRB: FKBP-rapamycin binding domain; tm: transmembrane; ecto: ectoplasmic; cyto: cytoplasmic

Figure 5. Strategies to Enhance T-cell Persistence and Effector Function

Several engineering approaches have been shown to increase T-cell proliferation, persistence, and anti-tumor effector functions such as cytotoxicity and cytokine production. These strategies include: (A) Using less differentiated cell types as starting material for CAR-T cell manufacturing. T_N: naïve T cell; T_SCM: stem-cell memory T cell; T_CM: central memory T cell; T_EM: effector memory T cell; T_E: effector T cell. (B) “Armoring” CAR-T cells with additional transgenic receptors or receptor ligands provide costimulation, enhance cytokine signaling, and/or promote migration [,–98]. (C) Equipping T cells with stimulatory cytokines expressed from constitutive or inducible promoters (pNFAT, pEF1alpha) [99,163]. (D) Blocking inhibitory signaling pathways through either the expression of transgenic peptides or the administration of pharmaceutical drugs. RAID: regulatory subunit I anchoring disruptor. PKA: protein kinase A. IDO: indoleamine 2,3-dioxygenase [102,103]. (E) Generating chimeric receptors that either abolish endogenous signaling pathways or convert inhibitory ligand inputs into stimulatory signal outputs [,,–109]. TGFBR: TGF-β receptor; DNR: dominant-negative TGF-β receptor. PD-1: programmed-death 1; IL-12: interleukin 12; IL-4Rα and IL-7Rα: interleukin receptors 4 alpha and 7 alpha; CCR4: C-C Motif Chemokine Receptor 4; CXCR2: C-X-C chemokine receptor type 2; tm: transmembrane; ecto: ectoplasmic; cyto: cytoplasmic