Chemogenetics for sensing antigens

Antigen-based systems offer highly specific binding and customizability, broadening their application to various fields of cell biology. In a recent issue of Nature, Kalogriopoulos et al. design antigen-sensing G-protein-coupled receptors that exhibit programmable responses spanning exogenous gene expression, G-protein signaling, and receptor activation.

The desire to control cellular responses has long been an active area of research. In 1979, Francis Crick first proposed the potential of optogenetics, a light-dependent spatiotemporal control of cells.¹ Over 20 years later, researchers would manifest this idea by leveraging the light-sensitive ion-channel, channelrhodopsin.² Prior to the successful implementation of optogenetics, however, the first chemogenetic system was created by mutating the ligand-binding pocket of the beta-adrenergic receptor (β-AR), rendering it selectively responsive to rationally designed ligands.³

The β-AR belongs to a large family of transmembrane metabotropic receptors, commonly known as G-protein-coupled receptors (GPCRs). GPCRs exhibit innate affinity towards particular G-protein subunits (G_q, G_i/o, G_s, G_z, G_12/13, G₁₄, G_15/16, G_olf, G_t), dependent on receptor subtype. Differential G-protein coupling enables GPCRs to initiate a wide range of intracellular responses that heavily influence many physiological functions.

Further chemogenetic innovations led to the generation of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) which were developed through directed evolution.⁴ DREADDs harness the power of G-protein-coupled signaling cascades to control genetically manipulated cell populations using physiologically inert ligands. Additional GPCR-based technologies, such as GPCR activation-based (GRAB) sensors,⁵ bioluminescence resonance energy transfer (BRET) assays,⁶ and various second messenger reporting assays have bolstered the toolkit available to the GPCR research community.

In cancer biology and therapeutics, synthetic receptors with antibody/antigen targeting systems, such as chimeric antigen receptors (CARs), have been used for decades to stimulate therapeutic T cell effector responses.⁷ These synthetic receptors generally consist of three key components: (1) a protein-specific antibody tagged to the extracellular cell surface; (2) a transmembrane domain; and (3) an intracellular module that initiates a measurable response correlated to binding events at the extracellular surface. Modification of the extra- and intracellular components enable customizable responses, expanding the potential for therapeutic applications.

Recently, Kalogriopoulos et al.⁸ developed programmable antigen-gated G-protein-coupled engineered receptors (PAGERs), providing programmable control of GPCR signaling through an elegant combination of extracellular antigen-binding domains with existing intracellular GPCR-based technologies (Fig. 1). Using a nanobody conjugated to a peptide antagonist, the group creates a displaceable autoinhibitory fragment that is released by the addition of the nanobodies’ respective antigens. Removal of the peptide antagonist reveals the orthosteric binding pocket, endowing any added agonist with binding ability to the engineered receptor.

Fig. 1. Modular design of PAGERs.

Left, family of DREADDs used as building scaffolds for the different PAGERs. Top, example of nanobody library available for programming PAGER design of interest. Top right, peptide antagonist library compatible with chosen DREADD scaffold. Bottom, graphic display of different intracellular outputs engineered to PAGER_TF, PAGER_G, and PAGER_FL, respectively.

To prototype this concept, the group selected the kappa-opioid receptor DREADD (ΚORD)⁹ as the mainstay transmembrane domain segment and fused it to their Specific Protein Association tool giving transcriptional Readout with rapid Kinetics (SPARK) technology¹⁰ to create PAGER_TF. By genetically adhering the peptide antagonist Arodyn to the anti-GFP nanobody LaG17, the KORD-based PAGER resides in an autoinhibited state with transcription factor elements below the plasma membrane, awaiting activation. GFP binding to the LaG17 nanobody sterically prohibits Arodyn binding to KORD, rendering the receptor available for agonist binding. The SPARK technology in PAGER_TF design additionally requires light or administration of furimazine to uncage the LOV domain on the intracellular face, making the TEV cleavage site accessible to its TEV protease. Multiple nanobodies and their companion antigens, such as alternative fluorescence proteins (mCherry), growth factors, cytokines, chemokines, receptor tyrosine kinases, viral proteins, and other cancer-related surface proteins, show compatibility in the PAGER_TF design. Interestingly, the PAGER technology overcomes previous limitations by showing successful binding to soluble antigens, an area where previous synthetic receptors have struggled.

The group then pivoted to the original family of DREADDs, evolved from muscarinic acetylcholine receptors, to generate a suite of G-protein subtype-specific PAGERs (PAGER_G): PAGER_Gq, PAGER_Gs, PAGER_Gi and PAGER_G12. The authors validate PAGER_G in neuronal cells with GCaMP6s and a fluorescent DAG probe confirming intact downstream signaling mechanisms. Furthermore, an adeno-associated virus (AAV) encoding anti-mCherry PAGER_Gi (LaM6-PAGER-G_i-P2A-mEGFP) induced an appreciable decrease in electrical activity of CA1 neurons, indicative of G_i protein activation. In T cell cultures, exposure to deschloroclozapine (DCZ) alone drove chemotaxis specifically in the presence of the mCherry antigen, but not in the presence of a non-specific control (GFP).

To combine the strengths of PAGERs and GRAB sensors, the authors inserted a circularly permutated GFP (cpGFP) between transmembrane domain helices 5 and 6 on the intracellular side of the group’s adopted hM4Di DREADD scaffold (DCZ1.0) to generate PAGER_FL. This approach allows PAGER_FL to exhibit fluorescence-based sensitivity to conformational shifts that occur in the transmembrane domain upon receptor activation. Displacement of muscarinic toxin 3 (MT3) by mCherry antigen binding enables docking of DCZ ligand, instigating conformational shifts that change cpGFP fluorescence. The group confirmed the modularity of PAGER_FL by swapping out LaM6 for anti-TNF and anti-VEGF at the autoinhibitory domain and replicated their success in dose-response assays conducted in cell culture.

The authors acknowledged several avenues for PAGER evolution: removing the requirement for LOV domain uncaging, creating transducer-specific PAGER mutants (i.e., G-protein vs β-arrestin), further extending the modular design to other non-GPCR receptors, and appending the intracellular domain to additional existing technologies. In the future, research will benefit from packaging PAGER constructs into AAVs driven by cell type-specific promoters. The PAGER toolkit can also be employed for trafficking studies, leveraging its ability to bind soluble fluorescence protein-based antigens. Furthermore, as the available pool of nanobodies grows, so does the PAGER antigen library, enhancing PAGER versatility.

The authors highlight PAGER’s potential utility by demonstrating IFNγ-dependent M1 macrophage differentiation and tumor cell death. This approach reinforces the modular three-piece design, reminiscent of CARs, with significant therapeutic value to the field of cancer research. Notably, PAGER constructs can be generated with relatively rapid turnaround times compared to earlier methods that heavily relied on directed evolution⁴ or site-directed mutagenesis⁹ specific to each protein of interest. The only requirement is an established antigen–nanobody pair. PAGER technology shows promise for a wide range of interesting applications, garnering excitement for how the field will stretch therapeutic use of PAGERs.

Competing interests

The authors declare no competing interests.