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Review
. 2024 Jan 3;9(1):7.
doi: 10.1038/s41392-023-01680-5.

Programmable synthetic receptors: the next-generation of cell and gene therapies

Fei Teng #  1 , Tongtong Cui #  2   3 , Li Zhou  4   2   3 , Qingqin Gao  4   2   3 , Qi Zhou  5   6   7   8 , Wei Li  9   10   11   12
Affiliations
Review

Programmable synthetic receptors: the next-generation of cell and gene therapies

Fei Teng et al. Signal Transduct Target Ther. .

Abstract

Cell and gene therapies hold tremendous promise for treating a range of difficult-to-treat diseases. However, concerns over the safety and efficacy require to be further addressed in order to realize their full potential. Synthetic receptors, a synthetic biology tool that can precisely control the function of therapeutic cells and genetic modules, have been rapidly developed and applied as a powerful solution. Delicately designed and engineered, they can be applied to finetune the therapeutic activities, i.e., to regulate production of dosed, bioactive payloads by sensing and processing user-defined signals or biomarkers. This review provides an overview of diverse synthetic receptor systems being used to reprogram therapeutic cells and their wide applications in biomedical research. With a special focus on four synthetic receptor systems at the forefront, including chimeric antigen receptors (CARs) and synthetic Notch (synNotch) receptors, we address the generalized strategies to design, construct and improve synthetic receptors. Meanwhile, we also highlight the expanding landscape of therapeutic applications of the synthetic receptor systems as well as current challenges in their clinical translation.

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Conflict of interest statement

The authors declare no competing interests. As an editorial board member of the journal, Q.Z. did not participate in any reviewing process of this review article.

Figures

Fig. 1
Fig. 1
Landmark research achievements of the synthetic receptor over the past three decades. A timeline is shown with brief summaries of some of the key research milestones in the synthetic receptor field published in the past 30 years. CAR chimeric antigen receptor, RASSL receptor activated solely by a synthetic ligand, DREADD designer receptors exclusively activated by designer drug, TRUCK T-cells redirected towards universal cytokine killing, TEVp tobacco etch virus protease, synTF synthetic transcription factor, MESA modular extracellular sensor architecture, synNotch synthetic Notch, FDA the U.S. Food and Drug Administration, dCas9-synR dCas9 synthetic receptor, GPCR G protein-coupled receptor, iTango inducible Tango, Cal-Light calcium- and light-gated switch, FLARE fast light- and activity-regulated expression, SPARK specific protein associated tool giving transcriptional readout with rapid kinetics, SUPRA CAR split, universal and programmable CAR, RASER rewiring of aberrant signaling to effector release, LOCKR latching orthogonal cage-key protein, SPOC split-protease-cleavage orthogonal-coiled coil-based logic circuit, GEMS generalized extracellular molecule sensor, CHOMP circuits of hacked orthogonal modular protease, esNotch enhanced synNotch, TMD transmembrane domain, GEAR generalized engineered activation receptor, TCS two component system, POST phosphoregulated orthogonal signal transduction system, SNIPR synthetic intramembrane proteolysis receptor, AMBER advanced modular bispecific extracellular receptor, OCAR orthogonal chemically activated cell-surface receptor, DocTAR double-cut transcription activation receptor
Fig. 2
Fig. 2
Programming cell and gene therapies using synthetic receptors. a Mammalian cells can be designed and engineered to sense and respond to a variety of stimuli such as chemicals and disease biomarkers, and subsequently trigger downstream signaling pathways, which can finetune customized therapeutic effects (e.g., gene expression, protein activity and secretion, etc.)., b Engineering CAR T cell therapy., T cells are genetically engineered to express specific CAR proteins on their surface. When infused back into the body, CARs interact with the targeted antigens on cancer cells, causing the activation of CAR T cells for cancer-killing. PCMV cytomegalovirus promoter, scFv single-chain fragment variant, TMD transmembrane domain, CD costimulatory domain, CD3ζ CD3 zeta signaling domain, pA poly(A) signal. c Synthetic receptor applications in CAR T cell therapy., T cells can be engineered with a combination of CARs and synthetic receptors like synNotch for a more precise tumor recognition to reduce off-target toxicity. Synthetic receptors like MESA could also be used in combination with CAR to sense a soluble biomarker. In addition to driving CAR expression, synthetic receptors are also able to express additional beneficial payloads alongside the CAR, such as cytokines, chemokines, enzymes, single-chain fragment variants (scFvs), mono-antibodies (mAbs), ligands or receptors. TF transcription factor, SynP synthetic promoter, Prom promoter, synR synthetic receptor, pA poly(A) signal. d Therapeutic cell engineering., By incorporating synthetic receptors, therapeutic cells are designed to act as ‘smart drugs’ that can sense disease biomarkers or user-defined inputs, and trigger a therapeutic response, such as the release of a drug or a therapeutic protein. These engineered cells present clinical potentials as they were encapsulated and implanted in mouse to treat diseases in proof-of-concept studies., STAT3, signal transducer and activator of transcription 3; P, phosphorylation; shGLP-1, synthetic human glucagon-like peptide 1. e Rewiring of aberrant signaling to effector release (RASER)., In RASER, a hepatitis C virus protease (HCVp) and an effector protein (e.g., OFP-Bid) are fused to two different domains that can sense overactive ErbB signaling. When ErbB activity is detected, the two domains are co-recruited together, causing HCVp to cleave and activate OFP-Bid. This leads to the induction of apoptosis specifically in ErbB hyperactive cancer cells, sparing normal cells. The compact size of RASER construct makes it suitable for AAV-delivered gene therapy. PCMV cytomegalovirus promoter, TMD transmembrane domain, SH2 Src homology 2 domain, CS cleavage site, Bid BH3 interacting domain death agonist, OFP orange fluorescent protein, P2A 2A peptide derived from porcine teschovirus-1, PTB phosphotyrosine-binding domain; NS3, hepatitis C virus nonstructural protein 3, pA poly(A) signal. f Engineering multicellular behaviors with synthetic receptor systems., (Left) To construct a three-layer structure, two separate cell lines are constructed using synNotch systems., CD19 ligands on the A-type cells can activate anti-CD19 synNotch receptors on the B-type cells, which induces the expression of Ecadhi (E-cadherin, high expression) and GFPlig (surface-bound GFP) in the B-type cells. Subsequently, these cells will form a two-layer structure with a green core and blue outer layer. Then, GFPlig on the B-type cells can send reciprocal signals to the A-type cells via anti-GFP synNotch, leading to the activation of Ecadlo (E-cadherin, low expression) and mCherry, which will induce the stepwise formation of the three-layer structure. (Right, Upper) Synthetic diffusive morphogen systems can be engineered using synNotch. In these systems, soluble ligands can form an artificial morphogen gradient and activate synthetic receptors on receiver cells. The gradient patterns can be tuned by modulating the expression level of synthetic morphogens (e.g., soluble GFP). The synNotch-based synthetic morphogen systems require an extra anchor protein to be expressed on the hybrid anchor/receiver cells (as shown here) or solely on the anchor cells. (Right, Lower) Another possible synthetic diffusive morphogen system using the synthetic receptor, such as MESA. In this supposed system, soluble ligands induce the dimerization of synthetic receptors, activating downstream gene transcription. GFP green fluorescent protein, mCherry a red fluorescent protein, BFP blue fluorescent protein, CD19 cluster of differentiation 19
Fig. 3
Fig. 3
Design and engineering of the chimeric antigen receptor (CAR). a The architecture of CARs comprises an extracellular sensor domain, a hinge, a transmembrane domain (TMD) and an intracellular signaling domain (actuator domain). The extracellular sensor domain, also known as antigen-binding domain, is usually a single-chain variable fragment (scFv) derived from a monoclonal antibody by fusing its light (VL) and heavy (VH)-chain variable domain with a flexible linker peptide. Other proteins like nanobodies, designed ankyrin repeat proteins (DARPins), natural ligands and small peptides can also function as the antigen-targeting moiety., The hinge derived from T cell proteins or immunoglobins can function as a flexible linker, providing sufficient conformational freedom to overcome steric hinderance to facilitate the access to the target antigen. T cell protein-derived or de novo designed TMD not only anchors the CAR in the cell membrane but also affects the stability and function of CAR. The intracellular signaling domain generally contains a CD3ζ signaling domain and CD28/4-1BB costimulatory domains (CDs), which facilitates T cell persistence and activity. Several other costimulatory domains including ICOS, OX-40, CD27, MyD88/CD40 and NKG2D are already underway., CAR architectures can be further engineered to express an ‘armor’, which aims to enhance the in vivo persistence and efficacy of CAR T cells. sbL, surface-bound ligand; ITAM, immunoreceptor tyrosine-based activation motif. b First-generation (1 G) CARs only contain a CD3ζ signaling domain in the intracellular domain (ICD), which outperforms the less popular FcεR1γ signaling domain. Second-generation (2 G) CARs harbor one CD, and third-generation (3 G) CARs contain more than one CDs in their intracellular signaling domain. Fourth-generation (4 G) CARs are based on 2 G CARs with additional expression of transgenic products (armor), such as cytokines, antibodies, enzymes, ligands or receptors. Fifth-generation (5 G) CARs are also based on 2 G CARs with the addition of a cytoplasmic domain derived from cytokine receptors (e.g., IL-2Rβ chain fragment) or synapse formation proteins (e.g., PDZbm scaffolding anchor domain). NFAT nuclear factor of activated T cells, IL-12 interleukin 12, IL-2Rβ interleukin 2 receptor beta-chain, JAK Janus kinase, STAT signal transducer and activator of transcription. c Numerous approaches to improve the safety and efficacy of CAR T cell therapy. Tandem CARs using bispecific single-chain variable fragments (scFvs) can operate an OR gate and overcome obstacles caused by tumor heterogeneity and antigen loss. Dual CAR engaging split CARs can perform AND gate to provide and enhance the specificity through targeting multiple antigens. Switch CARs with ON/OFF switches utilizing small molecule-triggered dimerization or degradation mechanisms can timely control CAR activity and overcome systemic cytokine toxicities of CAR T cells. Switchable CARs are specific to bispecific adaptors, such as folate-FITC, biotinylated antibody, PNE-Fab and Co-LOCKR,, and can direct a universal CAR T cell to target distinct antigens. Split, universal and programmable (SUPRA) CARs consist of a set of leucine-zipper universal CARs (zipCARs) and leucine-zipper scFv (zipFv) domains, which specifically bridge the zipCARs to various antigens. The SUPRA CAR system can fine-tune T cell activation and perform combinatorial logic operations (AND, NOT, OR, AND-NOT)., Inhibitory CARs contain inhibitory domains derived from immune checkpoint proteins (PD-1 or CTLA-4), which are able to reduce off-tumor toxicities of CAR T cells by inhibiting T cell activation upon binding an antigen expressed on non-malignant cells. SynNotch CARs employ a co-expressed synNotch receptor to drive the expression of a CAR to achieve AND logic. The synNotch and CAR can target different antigens, resulting in improved specificity and sensitivity of CAR T cell therapy., deg degron, INH inhibitory domain, TF transcription factor
Fig. 4
Fig. 4
Design and engineering of the synthetic Notch (synNotch) receptor. a The architecture of synNotch receptors consists of an extracellular sensor domain, a transmembrane Notch core region and an intracellular actuator domain (transcription factors, TFs). In synNotch receptors, the extracellular and intracellular domains (ICDs) can be completely swapped with diverse recognition domains (scFv, nanobody, or peptide tags) and TFs (transcriptional activators or repressors). The core Notch regulatory region comprises the transmembrane domain (TMD) and multiple proteolytic cleavage sites of wild-type Notch. Ligand binding to synNotch leads to the intracellular proteolytic cleavage and release of the membrane-tethered TF to translocate into the nucleus and regulate gene expression. sbL surface-bound ligand, scFv single-chain fragment variant, JMD juxtamembrane domain. b Evolution of the development of synNotch receptors. (Right, Upper) The modular configuration of prototype synNotch. (Middle, Upper) Enhanced synNotch (esNotch) incorporates an intracellular hydrophobic sequence (QHGQLWF, name as RAM7) derived from native Notch which significantly decreases ligand-independent activation. (Left, Upper) Synthetic intramembrane proteolysis receptors (SNIPRs) are fully humanized transcriptional receptors through systematic modular engineering of the original synNotch. (Right, Lower) The diffusible synNotch system can detect diffusible ligands anchored by engineered anchor cells, which enables creating a synthetic morphogen signaling system. sL soluble ligand. (Middle, Lower) Orthogonal chemically activated cell-surface receptors (OCARs) are engineered by replacing the extracellular sensor domain of synNotch into a chemically induced dimerization (CID) domain, which can achieve small molecule-triggered activation in a cis fashion. (Left, Lower) In OCAR-synNotch system, one part of cis-acting OCAR is sequestered by synNotch through the incorporation of coiled-coil dimer-forming peptides into them, which prevents small molecule-induced activation of OCAR when synNotch is in an inactive state. Once synNotch is activated by surface-bound ligands, the sequestered OCAR part is liberated and OCARs can be activated by the addition of inducers, which subsequently enhance synNotch signaling
Fig. 5
Fig. 5
Design and engineering of modular extracellular sensor architecture (MESA) and generalized extracellular molecule sensor (GEMS). a, b The architecture of MESA (a) and GEMS (b) comprises the extracellular sensor domain, the transmembrane domain (TMD) and the intracellular actuator domain. The extracellular sensor domain potentially includes single-chain variable fragments (scFvs), nanobodies (Nbs), chemically induced dimerization (CID) proteins, or ligand-binding domains from native receptors. scFv-/Nb-based extracellular sensor domains must bind to non-overlapping epitopes on a single-ligand molecule. a The MESA receptor contains two different transmembrane chains, target chain (TC) and protease chain (PC). The TC intracellular domain (ICD) contains an engineered transcription factor (TF) and a protease cleavage sequence between the TMD and the TF. The PC ICD consists of a cognate protease (e.g., tobacco etch virus protease (TEVp) shown here). Ligand binding induces the heterodimerization of the MESA receptor, causing the TEVp to cleave its cognate cleavage sequence on the TC and releasing the TF to translocate into the nucleus and modulate gene expression. Depending on the types of TFs, synthetic promoter-driven transgene can be induced (e.g., tTA) or endogenous gene expression can be regulated (e.g., dCas9-VP64 activator)., sL soluble ligand. b The GEMS receptor also contains two transmembrane chains, of which the ICDs are derived from various receptor tyrosine kinases (RTKs) and cytokine receptors. Ligand binding induces the dimerization of the GEMS receptor, activating intracellular signaling cascade. By rewiring natural signaling cascades, transgene expression can also be induced. At the intracellular juxtamembrane region alanine residues are inserted to modulate the conformation of ICD, thus reducing ligand-independent signaling. EpoR erythropoietin receptor, D1 EpoR D1 domain, D2 EpoR D2 domain, F93A substitution of phenylalanine at position 93 with alanine, Ala alanine. c Evolution of the development of MESA receptors. (Right, Upper) The modular configuration of prototype MESA., (Left, Upper) Systematic evaluation of TMD reveals that the choice of TMD significantly affects MESA performance. The TMD-modified MESA utilizing two different TMDs in the TC and PC can achieve reduced background signals and/or increased ligand-induced signals. (Right, Lower) In split-TEVp MESA system, computationally optimized split TEVp can be reconstituted via ligand-induced dimerization and therefore restore TEVp function. The split-TEVp enables MESA to achieve low background and high fold induction. (Left, Lower) dCas9-synRTK (dCas9- and RTK-based chimeric receptor) as an example of dCas9-synRs (synthetic dCas9-based receptors), employs split-dCas9-VP64 and split-TEVp as the intracellular actuator domain, by fusing them to different RTKs. The difference between dCas9-synRTK and split-TEVp MESA is that dCas9-synRTK can only sense native ligands since the extracellular domain and TMD of a dCas9-synRTK are derived from an intact RTK. nTEVp N-terminal TEVp, cTEVp C-terminal TEVp, dCas9n N-terminal deactivated Cas9, dCas9c C-terminal deactivated Cas9, RTK receptor tyrosine kinase. d Engineering of chimeric cytokine receptors to mimic cytokine receptor signaling using scFv and EpoR scaffold. ScFv/c-Mpl (S-Mpl) chimera contains a scFv-based extracellular sensor domain, the extracellular EpoR D2 domain and transmembrane/cytoplasmic domains of cytokine receptors (e.g., c-Mpl).– Chimeric cytokine receptor constructs with different combination of the domains containing the extracellular scFv, EpoR scaffold and intracellular domain of cytokine receptors (e.g., gp130). Compared to Sg, SD1D2g-1A additionally contains the extracellular D1D2 domain and one alanine residue at the intracellular juxtamembrane region. But the extracellular D1/D2 domain is dispensable for signaling. e Evolution of the development of GEMS receptors. GEMS should be considered as an evolutionary version of prototype SD1D2g-1A. Through modular engineering, the GEMS platform is able to specifically target a range of soluble ligands and robust transgene expression with high signal-to-noise ratios. Based on GEMS, generalized engineered activation regulators (GEARs) capitalize on MS2 bacteriophage coat protein (MCP)-nuclear factor fusion proteins and the dCas9/sgRNA-MS2 system to rewire induced receptor signaling to endogenous gene expression. Advanced modular bispecific extracellular receptors (AMBERs) combine the GEMS system and designed ankyrin repeat proteins (DARPins). The high-throughput binder-screening technology, DARPin, can generate various new binders and endow AMBER with desired sensitivity and specificity towards new inputs. In addition to customizing target gene expression, GMES and its derivatives inevitably perturb the endogenous gene regulatory network. dCas9, deactivated Cas9; sgRNA, single guide RNA; MS2, MS2 hairpin
Fig. 6
Fig. 6
Metrics- and “design-build-test-learn” (DBTL) cycle-based framework for synthetic receptor engineering. a Performance metrics can be used for quantitative assessment of the performance of synthetic receptor systems. Signaling output can be quantified by reporter fluorescence measurement, luciferase assay or various other approaches. The performance of a synthetic receptor is determined not only by its background signal and signal-to-noise ratio, but also by the therapeutic thresholds in practical applications. An optimal receptor should exhibit a low background signal (OB) which is lower than the minimum therapeutic threshold (Tmin), and meanwhile exhibit a high induced output signal (Oi) (Right, Upper). A synthetic receptor exhibiting low OB and high Oi may still fail in practical applications, which might be due to OB > Tmax (maximum therapeutic threshold) (Right, Lower) or Oi < Tmin (Right, Lower). In another case when OB > Tmin (Left, Lower), leaky expression should be cautious of. b A modified DBTL-based framework can direct researchers to choose or engineer synthetic receptor systems for their application in cell therapy or gene therapy. A “goal” step is to define design objectives for engineered cell or gene therapy and the standards to quantify the performance of synthetic receptors using performance metrics. In the DBTL cycle, apart from conventional approaches, more advanced and powerful approaches like computation-guided design, high-throughput automation techniques, machine learning and computational modeling can further accelerate the engineering and improvement of synthetic receptors for better clinical applications. The figure is adapted from refs. ,
Fig. 7
Fig. 7
The expanding potential therapeutic applications of synthetic receptors. a A variety of immune cells, such as T cells, innate T cells, NK cells, macrophages, dendritic cells and myeloid cells, can be directly isolated from patients and genetically engineered with CARs to enhance their antitumor capacity., Alternatively, these immune cells can be differentiated from CAR-engineered pluripotent stem cells (PSCs) as ‘off-the-shelf’ products. After being infused back into patients, these engineered immune cells interact with antigens on the tumor cells, leading to the activation of CAR immune cells to achieve cancer-killing., b, c Synthetic receptors could be integrated into PSCs to enhance original or program novel functionalities of the differentiated derivates for developing next-generation cellular therapeutics. b PSCs can differentiate into various cell types, like hepatocytes, neurons, muscle cells, etc., which are suitable for transplantation. One could imagine after being transplanted, cells engineered with synthetic receptors could sense and respond to host microenvironmental cues to promote the survival, proliferation and enhance tissue repair or regeneration. c Differentiated derivates from synthetic receptor-engineered PSCs might also be encapsulated and implanted in patients to avoid immunogenicity. These implantable therapeutic cells can sense various serum biomarkers (e.g., glucose, uric acid or thyroid hormone) and then trigger the activation of the corresponding therapeutic functions. d Synthetic receptor systems can be further designed and engineered with compact size and lower immunogenicity easier for in vivo gene therapy., These suitable synthetic receptor constructs can be delivered by non-viral vectors (e.g., lipid nanoparticle (LNP)), or viral vectors (e.g., AAV)
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