Methods and compositions in programmable receptors for antigen-detection and customized cell responses

Abstract

The present disclosure provides programmable synthetic receptor fusion proteins comprising an input module, a G-protein coupled receptor (GPCR), and an optional output module. The input module comprises a peptide inhibitor of a GPCR, an antigen binding molecule, and / or a linker which can contain a protease cleavage site. The optional output module can comprise a polypeptide having a) a light-sensitive voltage sensitive (LOV) domain, b) a tobacco etch virus protease recognition sequence (TEVcs), and c) a transcription factor (TF), where the polypeptide is covalently linked to the carboxy terminus of the GPCR. Antigen binding or protease cleavage activates the receptor, leading to either G protein mediated cell signaling, or cleavage of the TEVcs, which releases the TF and activates transcription of a target gene. Thus, the disclosure allows user programmed input signals to activate gene expression or intracellular signaling pathways.

Description

PATENT Attorney Docket No.110221-1449371-009910WO Client Ref. No. CZB-280S-PC METHODS AND COMPOSITIONS IN PROGRAMMABLE RECEPTORS FOR ANTIGEN-DETECTION AND CUSTOMIZED CELL RESPONSES CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims benefit of priority to U.S. Provisional Patent Application No. 63 / 516,900, filed August 1, 2023, which is incorporated by reference for all purposes. STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under contract CA257159 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND OF THE INVENTION

[0003] Existing technologies for programmable synthetic receptors capable of antigen-induced cell signaling and cell behaviors include Chimeric Antigen Receptors (CARs), synthetic Notch receptor (synnotch), synthetic intramembrane proteolysis receptors (SNIPRs), Modular Extracellular Sensor Architecture (MESA) receptors, and generalized extracellular molecule sensor (GEMS) receptors. Limitations of these existing synthetic receptors include not being able to respond to soluble antigens (CARs, synnotch, and SNIPRs); not being able to be activated by specific protease cleavage (CARs, synnotch, SNIPRs, MESA, and GEMs); requiring two binding domains against a single antigen to function (MESA and GEMS); and limited signaling outputs (CARs are limited to immune receptor signaling cascades, synnotch is limited to notch signaling and transgene expression, MESA is limited to transgene expression and PPIs, and GEMS are limited to receptor tyrosine kinase signaling and PPIs). Importantly, none of these programmable synthetic receptors are capable of activating G protein signaling.

[0004] The present disclosure provides synthetic receptors that can overcome the limitations of existing technologies. BRIEF SUMMARY

[0005] In some embodiments, a synthetic receptor fusion protein is provided (e.g., expressed in a cell). In some embodiments, the synthetic receptor fusion protein comprises, from amino terminus to carboxy terminus, formula (I): [input module] – [G-protein coupled receptor (GPCR)] (I), wherein the input module comprises: (i) a polypeptide inhibitor of the GPCR; and optionally (ii) an antigen binding molecule (ABM), and optionally (iii) an amino acid linker between the input module and the GPCR.

[0006] In some embodiments, the synthetic receptor fusion protein comprises, in an amino terminus to carboxy terminus direction, a compound of formula (Ia): [polypeptide inhibitor of a GPCR] – [ABM] – [amino acid linker] – [GPCR] (Ia); a compound of formula (Ib): [ABM] – [polypeptide inhibitor of a GPCR] – [amino acid linker] – [GPCR] (Ib); a compound of formula (Ic): [polypeptide inhibitor of a GPCR] – [ABM] – [GPCR] (Ic); or a compound of formula (Id): [ABM] – [polypeptide inhibitor of a GPCR] – [GPCR] (Id).

[0007] In some embodiments, the synthetic receptor fusion protein further comprises an output module, wherein the synthetic receptor fusion protein comprises formula (II): [input module] – [G-protein coupled receptor (GPCR)] – [output module] (II).

[0008] In some embodiments, the synthetic receptor comprises, in an amino terminus to carboxy terminus direction, a compound of formula (IIa): [polypeptide inhibitor of a GPCR] – [ABM] – [amino acid linker] – [GPCR] – [output module] (IIa); a compound of formula (IIb): [ABM] – [polypeptide inhibitor of a GPCR] – [amino acid linker] – [GPCR] – [output module] (IIb); a compound of formula (IIc): [polypeptide inhibitor of a GPCR] – [ABM] – [GPCR] – [output module] (IIc); or a compound of formula (Id): [ABM] – [polypeptide inhibitor of a GPCR] – [GPCR] – [output module] (IId).

[0009] In some embodiments, the polypeptide inhibitor binds to the GPCR in the absence of the antigen and does not bind the GPCR in the presence of the antigen.

[0010] In some embodiments, the polypeptide inhibitor is an orthosteric binding peptide. In some embodiments, the orthosteric binding peptide is an antagonist or inverse agonist.

[0011] In some embodiments, the ABM is selected from the group consisting of a nanobody, scFv, Fab, VH, monobody, a designed ankyrin repeat protein (DARPin), and a miniprotein. In some embodiments, the ABM binds an antigen expressed on the surface of a cell, an antigen bound to a solid support, or a soluble antigen.

[0012] In some embodiments, the amino acid linker comprises a protease cleavage site.

[0013] In some embodiments, the output module comprises a polypeptide comprising a) a light-oxygen-voltage-sensing (LOV) domain, b) a tobacco etch virus protease recognition sequence (TEVcs), and c) a transcription factor (TF), and the polypeptide is covalently linked to the carboxy terminus of the GPCR. In some embodiments, antigen binding to the ABM results in recruitment of an arrestin-TEV protease fusion protein to the GPCR and proteolytic cleavage of the TEVcs and release of the TF.

[0014] In some embodiments, the GPCR is selected from a GPCR in Table 7.

[0015] Also provided is a nucleic acid encoding a synthetic receptor fusion protein as described above or elsewhere herein.

[0016] Also provided is a vector comprising the nucleic acid as described above or elsewhere herein.

[0017] Also provided is a cell comprising (i) the synthetic receptor fusion protein as described above or elsewhere herein, (ii) the nucleic acid as described above or elsewhere herein and / or (iii) the vector as described above or elsewhere herein. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a primary cell.

[0018] Also provided is a cell comprising (i) the synthetic receptor fusion protein as described above or elsewhere herein, or a nucleic acid encoding (i), wherein the GPCR binds to a G protein endogenous to the cell in the presence of antigen. In some embodiments, the endogenous G protein is selected from the group consisting of Gi, Gs, Gq / 11, and G12 / 13.

[0019] Also provided is a method of activating transcription of a target gene in a cell. In some embodiments, the method comprises: (i) providing a cell expressing a synthetic receptor fusion protein as described above or elsewhere herein on the cell surface, wherein the cell comprises an arrestin-TEV protease (TEVp) fusion protein; (ii) contacting the cell with an antigen that binds the ABM; (iii) contacting the cell with blue light, resulting in proteolytic cleavage of the TEVcs and release of the TF, thereby activating transcription of the target gene.

[0020] In some embodiments, the blue light is exogenous to the cell. In some embodiments, the blue light is produced intracellularly by a heterologous bioluminescent protein. In some embodiments, the arrestin-TEVp fusion protein further comprises a bioluminescent protein that emits blue light. In some embodiments, the bioluminescent protein comprises a luciferase or nanoluciferase. In some embodiments, the target gene is a reporter gene present in the cell.

[0021] In some embodiments, the cell is transfected or transduced with a nucleic acid encoding the synthetic receptor fusion protein, a nucleic acid encoding the arrestin-TEVp fusion protein, and a nucleic acid comprising the target gene operably linked to a promoter that binds the TF.

[0022] In some embodiments, the cell is transfected or transduced with one or more of the nucleic acids in vitro, ex vivo, or in vivo.

[0023] Also provided is a method for activating a G protein in a target cell. In some embodiments, the method comprising: (i) providing a target cell expressing a synthetic receptor fusion protein as described above of elsewhere herein on the cell surface; (ii) contacting the target cell with an antigen that binds the ABM; wherein antigen binding to the ABM results in binding of the GPCR to the G protein, thereby activating the G protein.

[0024] In some embodiments, the G protein is selected from the group consisting of Gi, Gs, Gq / 11, and G12 / 13.

[0025] In some embodiments, before step (i), the target cell is transfected or transduced with a nucleic acid encoding the synthetic receptor fusion protein. In some embodiments, before step (i), the target cell is transfected or transduced with the nucleic acid in vitro, ex vivo, or in vivo.

[0026] Also provided is a method of using the synthetic receptor fusion protein as described above or elsewhere herein, the method comprising providing a cell expressing the synthetic receptor fusion protein, wherein the synthetic receptor fusion protein is activated by either (i) binding of antigen to the ABM, or (ii) proteolytic cleavage of an amino acid linker sequence in the input module.

[0027] In some embodiments, binding of antigen displaces the peptide inhibitor and activates the GPCR or in the case of (ii), proteolytic cleavage releases a peptide inhibitor bound to the GPCR and activates the synthetic receptor.

[0028] In some embodiments, the method further comprises contacting the synthetic receptor fusion protein with blue light, wherein binding of antigen in the presence of blue light results in proteolytic cleavage of the TEVcs and release of the TF.

[0029] In some embodiments, binding of antigen results in activation of an endogenous G protein. In some embodiments, the G protein is selected from the group consisting of Gi, Gs, Gq / 11, and G12 / 13. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG.1A-E. Design and proof-of-concept for a GPCR-based antigen-detection system. A) A 7-transmembrane G protein-coupled receptor (GPCR) can be bound by a small molecule agonist drug. The agonist binds the orthosteric binding pocket and activates the GPCR. B) A peptide antagonist is fused to the N-terminus of the GPCR such that it binds and occupies the orthosteric site of the GPCR it is fused to. The bound antagonist prevents the agonist drug from binding and activating the GPCR, thus the GPCR is inactive. C) An antigen binding module, in this case a nanobody, that specifically binds an antigen of interest is included in the linker between the GPCR and the peptide antagonist. In the absence of the specific antigen, the peptide antagonist remains bound and the GPCR remains inactive. D) In the presence of a specific surface antigen on a neighboring cell (left), the nanobody binds the antigen and physical opposing forces between the two cells removes the peptide antagonist from the orthosteric site, thereby allowing the drug to bind and the GPCR to be activated. In this way, the GPCR is activated only in the presence of the specific antigen and the small molecule agonist drug. In the presence of a specific soluble antigen (right), the nanobody binds the antigen and steric clashes prevent simultaneous binding of the antigen and antagonist, again facilitating the removal of the peptide antagonist from the orthosteric site, thereby allowing the drug to bind and the GPCR to be activated. In this way, the GPCR is also activated only in the presence of the specific antigen and the small molecule agonist drug. This antigen-gated GPCR activation system is also referred to as PAGER (which stands for Programmable Antigen-gated G protein-coupled Engineered Receptor). E) Domain structure diagram for the general design of PAGERs.

[0031] FIG.2A-D. Characterization of KORD-SPARK for use in PAGER. A) Kappa opioid receptor DREADD (KORD) was previously engineered (Vardy et al., Neuron, Volume 86, Issue 4, P936-946, 2015, DOI: 10.1016 / j.neuron.2015.03.065) to not bind any of its native ligands, butstill be selectively activated by the exogenous small molecule agonist drug salvinorin B (Sal B). KORD activity can be converted to transgene expression by using the previously published SPARK readout (Kim et al., eLife, 2017, DOI: 10.7554 / eLife.30233; Kim et al., eLife, 2019, DOI: 10.7554 / eLife.43826). Briefly, GPCR-SPARK allows for proteolytic release of a GPCR- fused, membrane-tethered transcription factor (TF) only when there is both a protein-protein interaction to deliver a protease proximal to its cleavage peptide and blue light (supplied as exogenous light or provided via BRET by a proximal NanoLuc luciferase) to uncage the cleavage site. If functional, KORD-SPARK could be used to build PAGERs. A domain structure diagram for KORD-SPARK is shown. B) Schematic showing the mechanism of KORD-SPARK. Even in the presence of furimazine (NanoLuc substrate), if Sal B is absent, KORD is not activated, NanoLuc-arrestin-TEV (NL-Arr-TEV) is not recruited to the receptor, the TEVcs stays caged and uncut, and the transcription factor stays sequestered at the membrane. In the presence of furimazine and Sal B, KORD is activated, NL-Arr-TEV is recruited to the receptor, the TEVcs is uncaged by NanoLuc BRET, the TEV protease cleaves the TEVcs, and the transcription factor is released and goes on to induce transgene expression. Exogenous white light can be used to uncage the TEVcs instead of NanoLuc BRET. C) HEK293T cells expressing KORD-SPARK (with no antagonist and no nanobody) are stimulated to produce firefly luciferase transgene expression only in the presence of the small molecule agonist Salvinorin B (1^M Sal B) and exogenous white light (supplied simultaneously for 15 min). Luminescence displayed as bar graph. D) HEK293T cells expressing KORD-SPARK (with no antagonist and no nanobody) are stimulated to produce firefly luciferase transgene expression only in the presence of 1^M Sal B and 1x of the NanoLuc substrate furimazine (supplied simultaneously for 15 min). Luminescence displayed as bar graph.

[0032] FIG.3A-C. Screening for peptide antagonists to use in a KORD-based PAGER-TF. A) Adding the antigen-gating GPCR activation strategy (FIG.1A-D) to KORD-SPARK would create a PAGER receptor that could couple antigen presence to transgene expression; we refer to this SPARK-linked PAGER receptor as PAGER-TF. A domain structure of PAGER-TF is shown. B) Schematic showing the mechanism of PAGER-TF. Even in the presence of furimazine and Sal B, if antigen is absent, the peptide antagonist stays bound to KORD, Sal B cannot bind and activate KORD, NL-Arr-TEV is not recruited to the receptor, the TEVcs stays caged and uncut, and the transcription factor sequestered at the membrane. In the presenceof furimazine, Sal B, and antigen, the nanobody binds the target antigen thereby removing the peptide antagonist from KORD, Sal B binds and activates KORD, NL-Arr-TEV is recruited to the receptor, the TEVcs is uncaged by NanoLuc BRET, the TEV protease cleaves the TEVcs, and the transcription factor is released and goes on to induce transgene expression. In this way, antigen detection by PAGER results in transgene expression. Exogenous white light can be used to uncage the TEVcs instead of NanoLuc BRET. C) To screen for peptide antagonists that would work for antigen-gating in a KORD-based PAGER-TF, we fused various dynorphin peptide analogs to the N-terminus of KORD-SPARK along with the GFP nanobody LaG17 (un-used for now) and screened the fused peptides for antagonism. HEK293T cells expressing candidate PAGER-TF constructs were stimulated with 1^M Sal B and exogenous white light provided simultaneously for 15 min. Luminescence from the firefly luciferase reporter is displayed as a bar graph. Several candidate PAGER-TF constructs were not activated due to efficient peptide antagonism.

[0033] FIG.4A-C. Screening candidate PAGER-TF constructs for surface-antigen dependent activation. A) In order to test whether surface-expressed antigen could be detected by candidate PAGER-TF constructs and result in relieved antagonism and KORD activation, we developed a co-culture assay using surface-expressed GFP as the target antigen. HEK293T cells expressing GFP-specific (LaG17 Nb) PAGER-TF (GFP(LaG17)-PAGER-TF) constructs were co-cultured with non-expressing or surface-GFP expressing HEK293T cells and assessed for activation and transgene expression as before. Schematic showing the design of this co-culture assay is shown. B-C) HEK293T cells expressing candidate GFP(LaG17)-PAGER-TF constructs were co- cultured with surface-GFP expressing HEK293T cells and stimulated with of 1^M Sal B and exogenous white light (B) or 1x furimazine (C) (supplied simultaneously for 15 min). Luminescence from the firefly luciferase reporter is displayed as bar graphs. Several candidate PAGER-TF constructs were activated only in the presence of surface-GFP and Sal B.

[0034] FIG.5A-B. Screening candidate PAGER-TF constructs for soluble-antigen dependent activation. A) HEK293T cells expressing candidate GFP(LaG17)-PAGER-TF constructs were stimulated with of 1^M soluble GFP, 1^M Sal B, and exogenous white light (supplied simultaneously for 15 min). Luminescence from the firefly luciferase reporter is displayed as bar graphs. Several candidate PAGER-TF constructs were activated only in the presence of solubleGFP and Sal B. B) HEK293T cells expressing candidate GFP(LaG17)-PAGER-TF constructs were stimulated with of 1^M or 10^M soluble GFP, various concentrations of Sal B, and 1x furimazine (all supplied simultaneously for 15 min). Luminescence from the firefly luciferase reporter is presented as percent max luminescence for each construct and is displayed as scatter plots with variable slope (four parameter) non-linear regression lines. Candidate PAGER-TF constructs showed left shifts in Sal B dose response curves in the presence of soluble GFP, indicating they can detect and are responsive to soluble GFP. “Ant” in the figure legend refers to antagonist. Peptide antagonist for each PAGER-TF tested is indicated at the top of each graph.

[0035] FIG.6. PAGERs are made against a variety of antigens simply by swapping for other antigen-specific nanobodies. HEK293T cells expressing various antigen-specific PAGER-TF constructs made using nanobodies against different antigens are stimulated with soluble target antigen (100nM-1^M), Sal B (100nM-1^M), and 1x furimazine (supplied simultaneously for 15 min). See Methods section for specific nanobody and concentrations of antigen and Sal B used for each different PAGER. Soluble recombinant ectodomains were used for receptor target antigens. Luminescence from the firefly luciferase reporters are displayed as bar graphs. PAGER-TF constructs are activated in response to their cognate antigen.

[0036] FIG.7A-E. Building protease responsive PAGER receptors. A) Including a protease cleavage site between the GPCR and antagonist would make PAGER receptors responsive to protease activity; we refer to this receptor as Protease-PAGER. A domain structure of Protease- PAGER-TF is shown. B) Schematic showing the mechanism of Protease-PAGER-TF. Even in the presence of furimazine and Sal B, if the protease is absent, the peptide antagonist stays bound to KORD, Sal B cannot bind and activate KORD, NL-Arr-TEV is not recruited to the receptor, the TEVcs stays caged and uncut, and the transcription factor stays sequestered at the membrane. In the presence of furimazine, Sal B, and protease, the protease cuts the protease cleavage site thereby removing the peptide antagonist from KORD, Sal B binds and activates KORD, NL-Arr- TEV is recruited to the receptor, the TEVcs is uncaged by NanoLuc BRET, the TEV protease cleaves the TEVcs, and the transcription factor is released and goes on to induce transgene expression. In this way, detection of protease activity by PAGER results in transgene expression. Exogenous white light can be used to uncage the TEVcs instead of NanoLuc BRET. C-D) HEK293T cells expressing GFP(LaG2)-PAGER-TF (no TEVcs) (C) or GFP(LaG2) / TEV-PAGER-TF (with TEVcs) (D) constructs were treated with or without 1^M TEV protease for 90 min followed by stimulation with or without 1^M soluble GFP, various concentrations of Sal B, and 1x furimazine (all supplied simultaneously for 15 min). Luminescence from the firefly luciferase reporter is displayed as scatter plots with variable slope (four parameter) non-linear regression lines. The GFP(LaG2)-PAGER-TF (no TEVcs) construct was only responsive to GFP, while the GFP(LaG2) / TEV-PAGER-TF (with TEVcs) construct was responsive to GFP or TEV protease. E) HEK293T cells expressing a GFP(LaG17) / Thrombin-PAGER-TF (with Thrombin cleavage site; referred to as Thrombin-PAGER-TF) construct were treated with or without 12.5 units / mL Thrombin protease for 90 min followed by stimulation with 1^M Sal B and 1x furimazine (supplied simultaneously for 15 min). Luminescence from the firefly luciferase reporter is displayed as a bar graph. The Thrombin-PAGER-TF construct was activated only when treated with thrombin and stimulated with Sal B.

[0037] FIG.8A-B. Building PAGERs that activate endogenous heterotrimeric G proteins as an output. A) Instead of using SPARK as an output of PAGER to drive transgene expression in an antigen-dependent manner, we wanted to use the native function of GPCRs to drive antigen- dependent activation of heterotrimeric G protein; we refer to these as G protein-PAGERs. A schematic showing the mechanism of G protein-PAGER antigen-dependent G protein activation is shown. G protein-PAGERs have none of the SPARK components fused to it or included in the system. Instead, when the antigen is present, the activated PAGER receptor recruits and activates endogenous G proteins. B) In order to develop G protein-PAGERs that could couple to the 4 major classes of heterotrimeric G proteins (GĮi, GĮs, GĮq, and GĮ12), we built PAGERs using the muscarinic DREADD receptors (Armbruster et al., PNAS, vol.104, no.12, 5163–5168, 2007, DOI: 10.1073 / pnas.0700293104; Roth, Neuron, Volume 89, Issue 4, P683-694, 2016, doi: 10.1016 / j.neuron.2016.01.040) (or chimeric DREADDs with intracellular loops from other GPCRs) and muscarinic toxins as the fused peptide antagonists. TEV cleavage sites were also included between the nanobody and the GPCR to make these PAGERs responsive to TEV protease cleavage. Just like the muscarinic DREADDs, these PAGERs can be activated by several orthogonal small molecule agonists including clozapine-N-oxide (CNO), deschloroclozapine (DCZ), DREADD agonist 21, JHU 37152, JHU 37160, olanzapine, and perlapine. Domain structures for the developed G protein-PAGERs are shown.

[0038] FIG.9A-B. TEV and antigen specific G protein-PAGERs activate all 4 major classes of heterotrimeric G proteins. A) The TRUPATH G protein activation BRET assay (Olsen et al., Nature Chemical Biology, 16, pages 841–849, 2020, DOI: 10.1007 / 978-1-0716-2473-9_13) was used to readout G protein-PAGER activity. G protein activation results in a loss of BRET in the TRUPATH assay. HEK293T cells expressing GFP(LaG2) / TEV-G protein-PAGER constructs were treated with 1^M TEV protease for 90 min followed by stimulation with various concentrations of CNO and 20^M CTZ400a (substrate for TRUPATH assay) for 5 min before reading out BRET. Data is presented as NET BRET and displayed as scatter plots with variable slope (four parameter) non-linear regression lines. All 4 GFP(LaG2) / TEV-G protein-PAGERs activated their respective G protein in response to TEV activity. D) HEK293T cells expressing mCherry(LaM6)-G protein-PAGER constructs were treated with 1^M mCherry for 15 min followed by stimulation with various concentrations of CNO and 20^M CTZ400a for 5 min before reading out BRET. Data is presented as NET BRET and displayed as scatter plots with variable slope (four parameter) non-linear regression lines. All 4 mCherry(LaM6)-G protein- PAGERs activated their respective G protein in response to mCherry.

[0039] FIG.10A-D. G-protein-PAGERs drive changes in G protein second messengers. A) Schematic the canonical signaling pathways and second messengers for each G protein class. GĮi inhibits cAMP levels, GĮs stimulates cAMP levels, GĮq stimulates DAG production and increases in intracellular calcium, and GĮ12 activates RhoA. B) GloSensor assay (Promega) was used to measure relative cAMP levels in response to Gi- and Gs-PAGER activation. HEK293T cells expressing GFP(LaG2)-Gi-PAGER (left) were stimulated with 1^M GFP and various concentrations of CNO for 15 min followed by 100nM isopreteronol for 30 min before reading out luminescence. HEK293T cells expressing GFP(LaG2)-Gs-PAGER (right) were treated with 1^M GFP for 15 min followed by various concentrations of CNO for 2 min before reading out luminescence. Luminescence from the firefly luciferase reporter is displayed as scatter plots with variable slope (four parameter) non-linear regression lines. GFP(LaG2)-Gi-PAGER show reduced cAMP levels in response to GFP. GFP(LaG2)-Gs-PAGER show elevated cAMP levels in response to GFP. C) A fluorescent DAG sensor (C1(PKCȖ)-mCherry) is used to readout DAG production after GĮq activation. The fluorescent DAG sensor is cytosolic at low levels of DAG and translocates to the plasma membrane when DAG is produced in response to GĮq activation. HEK293T cells stably expressing the DAG sensor and GFP(LaG16)-Gq-PAGER were treatedwith 1^M GFP for 3 min followed by stimulation with a 133nM CNO for 1 min. Fluorescent microscopy images were taken for and used for quantification (right). Representative fluorescent microscopy images are shown (left). HEK293T cells expressing GFP(LaG16)-Gq-PAGER show increased DAG production only in the presence of GFP. D) A fluorescent calcium sensor (GCaMP6s; Chen et al., Nature, 499, pages 295–300, 2013, doi: 10.1038 / nature12354) is used to readout increases in intracellular calcium in response to GĮq activation. Fluorescence of the GCaMP6s increases when intracellular calcium levels increase. HEK293T cells stably expressing GCaMP6s and mCherry(LaM6)-Gq-PAGER were treated with 2^M mCherry for 3 min followed by stimulation with 133nM CNO for 1 min. Fluorescent microscopy images were taken for and used for quantification (right). Representative fluorescent microscopy images are shown (left). HEK293T cells expressing mCherry(LaM6)-Gq-PAGER show increased intracellular calcium levels only in the presence of mCherry. DEFINITIONS

[0040] The term “about” when referring to a numerical value or range includes normal variation expected by a person of ordinary skill in the art, and includes a range of plus or minus 0.1%, 0.5%, 1.0%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the recited value or range. Any numerical value or range disclosed herein can be modified by the term about, regardless of whether the term about is expressly recited.

[0041] The term “antigen” refers to a molecule that binds to an antigen binding molecule (ABM) of the disclosure.

[0042] The term “binding” refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. “Specific binding” refers to binding with an affinity of at least about 10-7M or greater, e.g., 5 x 10-7M, 10-8M, 5 x 10-8M, and greater.

[0043] The term “fusion protein” refers to a protein consisting of at least two domains that are encoded by separate open reading frames that have been joined so that they are transcribed and translated as a single unit, producing a single polypeptide.

[0044] The term “heterologous” refers to biological material that is introduced, inserted, or incorporated into a recipient (e.g., host) organism that originates from another organism. Typically, the heterologous material that is introduced into the recipient organism (e.g., a host cell) is not normally found in that organism. Heterologous material can include, but is not limited to, nucleic acids, amino acids, peptides, proteins, and structural elements such as genes, promoters, and cassettes. A host cell can be, but is not limited to, a bacterium, a yeast cell, a mammalian cell, or a plant cell. The term “heterologous” also refers to a nucleotide or polypeptide sequence that is not found in the native (e.g., naturally-occurring) or wild-type nucleic acid or polypeptide, respectively.

[0045] The terms “identical,” “substantially identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using the BLAST and PSI-BLAST algorithms, which are described in Altschul et al. (J. Mol. Biol.215:403-10, 1990), and Altschul et al. (Nucleic Acids Res., 25:3389-3402, 1997), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (see the internet at ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992).

[0046] All nucleic acid and amino acid sequences disclosed herein can include sequences that have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence or sequence identifier recited herein. All nucleic acid and amino acid sequences disclosed herein can include sequences that have about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a sequence or sequence identifier recited herein.

[0047] The term “pharmaceutically acceptable carrier” refers to a substance that aids the administration of an active agent to a cell, an organism, or a subject. “Pharmaceutically acceptable carrier” refers to a carrier or excipient that can be included in the compositions of the invention and that causes no significant adverse toxicological effect on the patient. Non-limiting examples of pharmaceutically acceptable carrier include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, cell culture media, and the like. One of skill in the art will recognize that other pharmaceutical carriers are useful in the present invention.

[0048] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0049] The terms “polypeptide” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include genetically coded and non- genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins a heterologous amino acid sequence, fusionswith heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

[0050] The term “peptide” refers to a polypeptide of less than or equal to 50 amino acids.

[0051] The term “modified polypeptide” refers to a polypeptide that differs from a natural, wild-type, or unmodified reference or parental polypeptide in amino acid sequence, and includes a nucleic acid molecule encoding a modified amino acid sequence. The term “modified” encompasses polypeptides having one or more amino acid mutations / substitutions, insertions or deletions that are not found in natural, wild-type or reference polypeptides. The terms “modified polypeptide” and “variant polypeptide” can also refer to a polypeptide comprising an amino acid sequence having one or more amino acid substitutions, insertions or deletions relative to a reference sequence.

[0052] The term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding region of a nucleic acid if the promoter affects transcription or expression of the coding region of a nucleic acid.

[0053] The term “orthosteric binding peptide” refers to a peptide that binds at the active site of a receptor, such as a GPCR of the disclosure, competing with the natural substrate or ligand.

[0054] A “vector” or “expression vector” is a nucleic acid replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e., an “insert”, may be attached so as to bring about the replication of the attached segment in a cell. Expression vectors typically contain nucleic acid sequences (e.g., a coding region or open reading frame) that encode a protein of interest, and can also include sequences that regulate transcription of the coding region (e.g., promoters, enhancers, transcription terminators) and translation of the protein (e.g., translation initiation codons or Kozak sequences) and well as sequences that control location of the translated protein in the cell (e.g., nuclear localization signals). DETAILED DESCRIPTION

[0055] The present disclosure provides compositions comprising a synthetic receptor that can be programmed to sense an antigen of interest (input) and produce a cellular response of interest(output), and methods of using the compositions. The disclosure provides the following advantages: (i) unprecedented user control of cell signaling and cell behavior via modular engineered receptors by expanding the type and scope of inputs and outputs that can be programmed; (ii) new capabilities for the programmable detection of and cellular response to extracellular antigens; (iii) antigen detection can lead to a variety of cellular outcomes, including but not limited to transgene expression, cell activation, cell inhibition, altered signal transduction, and changes in levels of second messengers; (iv) components regulating receptor function are modular and programmable; they can be easily swapped to alter the specificity of the conditional receptor quickly and easily; and (v) the synthetic receptors can respond to and be activated by surface and soluble antigens as well as protease cleavage, only require a single binding domain against an antigen of interest, and have expanded output capabilities that include transgene expression, G protein signaling, and induced protein-protein interactions. Synthetic Receptor Fusion Proteins

[0056] The present disclosure provides synthetic receptor fusion proteins (also referred to herein interchangeably as “synthetic receptors”) that can respond to one or more different input signals, such as antigen binding or protease cleavage, and generate multiple different output signals. The synthetic receptors of the disclosure comprise a modular structure, such that the synthetic receptor comprises one or more of an input module and a receptor module, and optionally an output module (e.g., when an output module is not naturally available in the cell). In some embodiments, the receptor module comprises a G-protein coupled receptor (GPCR). In some embodiments the receptor is referred to herein as a Programmable Antigen-gated G protein-coupled Engineered Receptor (PAGER).

[0057] In some embodiments, the synthetic receptor comprises, from amino terminus to carboxy terminus, a compound of formula (I): [input module] – [G-protein coupled receptor (GPCR)] (I).

[0058] The modules of formula (I) can be linked by peptide bonds, either directly to each other or indirectly via an amino acid linker. Thus, in some embodiments, the carboxy terminus of the input module is linked, directly or indirectly, via peptide bonds to the amino terminus of the GPCR. In addition, an optional output module (detailed below) can be fused to the carboxy terminus of the GPCR in a synthetic receptor of formula I. The various components of formula (I) are described below. Input Module

[0059] In some embodiments, the input module comprises a polypeptide inhibitor of a GPCR. In some embodiments, the input module further comprises an antigen binding molecule (ABM). As shown in the embodiment illustrated in Fig.1E, in the absence of antigen, the polypeptide inhibitor is bound to the GPCR and the GPCR is inactive. Upon binding of an antigen of interest to the ABM, the polypeptide inhibitor is no longer bound to the GPCR, and the GPCR becomes active. Therefore, in embodiments comprising an ABM, GPCR activity (output) is dependent upon binding to the antigen of interest.

[0060] In some embodiments, the polypeptide inhibitor of a GPCR is linked to the ABM by an amino acid linker. In some embodiments, the input module comprises a polypeptide inhibitor of a GPCR, an ABM, and an amino acid linker.

[0061] In some embodiments, the input module comprises, in an amino terminus to carboxy terminus direction: (i) polypeptide inhibitor of a GPCR; (ii) an ABM; and (iii) an amino acid linker. Thus, in some embodiments, the synthetic receptor comprises, in an amino terminus to carboxy terminus direction, a compound of formula (Ia): [polypeptide inhibitor of a GPCR] – [ABM] – [amino acid linker] – [GPCR] (Ia).

[0062] In some embodiments, the input module comprises, in an amino terminus to carboxy terminus direction: (i) an ABM; (ii) a polypeptide inhibitor of a GPCR; and (iii) an amino acidlinker. Thus, in some embodiments, the synthetic receptor comprises, in an amino terminus to carboxy terminus direction, a compound of formula (Ib): [ABM] – [polypeptide inhibitor of a GPCR] – [amino acid linker] – [GPCR] (Ib).

[0063] In some embodiments, the input module does not comprise an amino acid linker, such that the polypeptide inhibitor of a GPCR or the ABM is directly linked via a peptide bond to the amino terminus of the GPCR. Thus, in some embodiments, the synthetic receptor comprises, in an amino terminus to carboxy terminus direction, a compound of formula (Ic): [polypeptide inhibitor of a GPCR] – [ABM] – [GPCR] (Ic).

[0064] In some embodiments, the synthetic receptor comprises, in an amino terminus to carboxy terminus direction, a compound of formula (Id): [ABM] – [polypeptide inhibitor of a GPCR] – [GPCR] (Id).

[0065] Without being bound by theory, in the absence of antigen the polypeptide inhibitor is bound to the GPCR and the synthetic receptor is in an inactive state or conformation. In the presence of an antigen of interest, the antigen binds to the ABM, which results in loss of binding of the polypeptide inhibitor to the GPCR, and activation of the synthetic receptor (e.g., a synthetic receptor of formulas I, Ia, Ib, Ic, and / or Id).

[0066] In any of the embodiments of the disclosure, the amino acid linker can further comprise a protease cleavage site. As shown in the embodiment illustrated in Fig. 6A, a protease can digest the linker at the protease cleavage site, thereby activating the GPCR. Without being bound by theory, it is believed that protease cleavage of the amino acid linker results in loss of binding of the polypeptide inhibitor to the GPCR, thereby activating the synthetic receptor. Thus, in some embodiments, binding of antigen to the ABM is not necessary or required for activation of the GPCR. Polypeptide Inhibitors

[0067] The polypeptide inhibitors of the disclosure can inhibit the activity of a GPCR. In some embodiments, the polypeptide inhibitor is a peptide comprising less than or equal to 100,or 75 or 50 amino acids, e.g., less than or equal to 50, 45, 40, 35, 30, 25, or 20 amino acids. In some embodiments, the polypeptide inhibitor is a peptide between 5-100 amino acids long. In some embodiments, the polypeptide inhibitor is an orthosteric binding peptide. Examples of suitable polypeptide inhibitors include antagonists or inverse agonists. Exemplary polypeptide inhibitors include those shown in Table 1. In some embodiments, the polypeptide inhibitor comprises an amino acid sequence having at least, or greater than or equal to, 90% sequence identity (e.g., at least, or greater than or equal to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity) to a sequence selected from a row of Table 1. T l ^1^^E l ^ i ^ I hi i ^ ^ i ^ i ^ E ^ID^ p G D D D D D [ [ [ [ [ [ [ [ [ [ [ J A A A A A A AArodyn(1^5)^ KORD^ FFFRL^ 27^Muscarinic^Toxin^1^ hM1D,^hM2D,^ LTCVTSKSIFGITTENCPDGQNLCF 28^ A A A A A A A A A A A A A A A A A A A A A A AArodyn^like^pepƟde^24^(1^11)^ KORD^ FWWFWRRARPK^ 57^Arodyn^like^pepƟde^25^(1^17)^ KORD^ FLLFLRRARPKLKWDNQ^ 58^ A A A A A A A A A A A A A A A A A A AAntigen Binding Molecule (ABM)

[0068] The ABM of the disclosure can bind any antigen of interest. In some embodiments, the ABM is selected from the group consisting of a nanobody (e.g., a single variable domain located on a heavy chain, also known as VHH antibodies), a single chain variable fragment (scFv), an F(ab) fragment, a variable heavy chain (VH), a monobody (i.e., a synthetic protein derived from the 10th domain of human fibronectin type III), a designed ankyrin repeat protein (DARPin), and a miniprotein binder (see, e.g., Cao et al., Nature volume 605, pages 551–560 (2022))}. In some embodiments, an ABM of the disclosure is selected from a row in Table 2 below. In some embodiments, the ABM comprises an amino acid sequence having at least, or greater than or equal to, 90% sequence identity (e.g., at least, or greater than or equal to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity) to a sequence selected from a row of Table 2. Nanobodies have been found to be particularly useful as ABMs. Without intending to limit the scope of the invention, it is believed spatial proximity between the CDRsof the nanobody and the N-terminus provides a steric clash between N-terminal fused antagonist and bound antigen, improving function of the constructs described herein. Table 2. ABM sequences. AnƟgen^Binding^ Target^ ABM^sequence^(amino^acid^sequence)^ SEQ^ID^ N N N N N N N N C NRQAPGKEREFVAGISWSGSSSYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADPVGRADLYEYD N N N C N C N N N N N a N iDYWGQGTQVTVSS^Nanobody^ MBP^ VQLVESGGGSVQAGGSLRLSCVASGDIKYISYLGWFR ^97^ L N L N L s N N N s NFRQAPGKEREGVAGITSLVSGVAYYKYYTDSVKGRFTIFRDDDKNTVDLQMNSLKPEDTAIYYCAASRSGLRARL N N s B s N N N N NRBD^ RQAPGKEREFVAAISWSSTPTYYGESVKGRFTISRDNAKNTVYLQMNRLKPEDTAVYFCAADRGESYYYTRPT N N R N R N N N N N E N NGQGTRVTVSS^Nanobody^ CD38^ VQLQESGGGLVQAGHSLRLSCVGSGSRFDNYAMG ^126^ N N N N N s E E E E N 2TQVTVSS^scFv^Gancotamab^ HER2^ VQLVESGGGLVQPGGSLRLSCAASGFTFRSYAMSWV ^138^ D A N N s B D D DHRAAWGHLELVEKLLKNGADVNAADIDGYTPLHLAAFSGHLEIVEVLLKYGADVNADDQAGFTPLHLAAIFGH T T s N N N N N N N NREYIFRGQGTQVTVS^Nanobody^LaG27^ GFP^ VQLVESGGGLVQAGGSLRLSCTASGLTISTYNIGWFR ^158^ N N N N N N N N N NRQAPGKEREWVSVILISSGVAYYKWYADSVKGRFTISQDNAKNTVYLQMNSLKPEDAAIYYCAASMGRPNSA N V N S N N O N s

[0069] In some embodiments, the antigen of interest comprises a molecule that does not bind a GPCR. In some embodiments, the antigen of interest is selected from a growth factor, a cytokine, a hormone and a chemokine. In some embodiments, the antigen of interest is soluble or in solution. In some embodiments, the antigen of interest is bound to a cell surface or cell membrane, or bound to the extracellular matrix. Exemplary antigens of interest are provided in Table 3 below. Table 3. Exemplary antigens of interest. Target n gen UnprotGreen^Fluorescent^Protein^(GFP)^ P42212^ E m V H P T I I I C E R A P S U [ [ 4 5 A a A a a a a agou reatedproten O00 53agrin^ O00468^ a a a a a a a a a a a a a a a a a a a a a a a a a a Aartemin^ Q5T4W7^ a a B B B b b b b b b b b b B b B B B B B B B B B B BQbovine^adrenal^medulla^pepƟde^8^22^ P01210^ b b C C C C c c c c c C C C C C C C C C C C C C C C CCCL22^ O00626^ C C C C C C C C C C C C C C c c c c c c c C C C C C conac n QcontacƟn^3^ Q9P232^ c c c c C C C C C C C C C C C C C C C C C C C C C C CCXCL9^ Q07325^ c c d d d d d d d d D d d d d e e E E E E e e e e e ep rnephrin^A4^ P52798^ e e e e e e e F F F F F F F F F F F F F F F F Į Į Į Įronec nFLICE^like^inhibitory^protein^subunit^p12^ O15519^ F F f F g g g g g g g g g g g g g g g G G g G g g g gucagon epep e ( )am eglucagon^like^pepƟde^1^(7^37)^ P01275^ g g g G G G G g g g g g g g g g g g G g g g H h h h hepc nhepcidin^25^ P81172^ H h h h h h I I I I I I I I I I I I I I I I I I I I IFomegaIL^1^alpha^ P01583^ I I I I I I I I I I I I I I I I I I I I I I I I I I ILap a QIL^36^beta^ Q9NZH7^ I I I I I I I I i i i i i I I I I I I i i i i i k k ksspep n QkisspepƟn^54^ Q15726^ k k l l L L L li L l L l l m m m m m m m m m m nern gan Qneuregulin^1^ Q02297^ n n n n n n n n n n n n n n n n n n n n n n n n n n neur urn QNGF^ P01138^ n N n o o o o o o O o P P P p P P P p p P P p p p p pro ne cn QprokineƟcin^2^ Q9HC23^ p p p p p p P P P P P P P P Q R R R R r r r r r r r RQ QRFRP^2^ Q9HCQ7^ R R r r R R R R s s s s s s s s s S s S S s s S T T TeaTGF^beta^3^ P10600^ t t t t t T T T T T Ɵ T T T T T T t t T u u u u u u vascuarce a esonproenvasopressin^ P01185^ V V V V V V v v v v Wn QWnt^9a^ O14904^ X X x 5 A A A A A A B B B C C C C C C C C C D E G F F G G G G G G G HHydroxycarboxylic^acid^receptors^^ KisspepƟn^receptor^ ^ L L L N N N N N N O O O P P P P P P P Q R S S T T T U V V 5 A E G GIonotropic^glutamate^receptors^^ IP3^receptors^ ^ N P Z C C P R T V V V A C C P S S T R P R R L V H R T T C E N F G D E 3 R T TType^III^RTKs:^PDGFR,^CSFR,^Kit,^FLT3^receptor^family^^ Type^IV^RTKs:^VEGF^(vascular^endothelial^growth^factor)^receptor^family^ ^ T T T T T T T T T T T T T T T T C I I I I P I I I I F G I I P T N R R T N R TATP^binding^casseƩe^transporter^family^^^ F^type^and^V^type^ATPases^^ ^ P S A A A C I N A C O NLinkers

[0070] The input module of the disclosure can also include an optional amino acid linker. As noted above, the amino acid linker can be located between the ABM and the GPCR, or between the polypeptide inhibitor of a GPCR and the GPCR, or one linker can link the ABM and the GPCR and a second linker can link between the polypeptide inhibitor of a GPCR and the GPCR. Suitable linkers include flexible Glycine-Serine (GS) linkers (e.g., a linker in which a majority or all of the amino acids are glycine or serine) and XTEN linkers.

[0071] In some embodiments, an epitope tag is included in the linker for antibody detection of the synthetic receptor. Exemplary epitope tags can include but are not limited to an ALFA tag (SRLEEELRRRLTE) (NanoTag Biotechnologies GmbH, Germany) (see Götzke, H. et al. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat Commun10, 4403 (2019).

[0072] In some embodiments, the linker is resistant to endogenous proteases. In some embodiments, a protease cleavage site is included in the linker to render the synthetic receptor cleavable by protease activity.

[0073] In some embodiments, the linker comprises between 1 and 25 amino acids in length, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. Exemplary linkers are provided in Table 4 below. In some embodiments, thelinker comprises an amino acid sequence having at least, or greater than or equal to, 90% sequence identity (e.g., at least, or greater than or equal to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity) to a sequence selected from a row of Table 4. Table 4. Exemplary Linkers. Linker^Name^ Linker^sequence^(amino^acid^sequence)^ SEQ^ID^NO:^ G G G G A X XProteolytic Cleavage

[0074] As noted above, the amino acid linker of the input module can comprise a protease cleavage site to render the synthetic receptor responsive to protease activity. In some embodiments, cleavage of the linker by an extracellular protease can activate the GPCR module of the synthetic receptor.

[0075] Exemplary proteases and their cognate cleavage sites are shown in Table 5.

[0076] In some embodiments, the protease cleavage site comprises an amino acid sequence selected from a row of Table 5. Table 5. Exemplary Protease Cleavage Sites. P s E L T LPLG / LAG^ MMP9^ P14780^IEPD / VSQV^ Granzyme^B^ P10144^In some embodiments, the linker comprising a protease cleavage site comprises an amino acid sequence selected from a row of Table 6. In some embodiments, the linker comprises an amino acid sequence having at least, or greater than or equal to, 90% sequence identity (e.g., at least, or greater than or equal to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity) to a sequence selected from a row of Table 6. Table 6. Exemplary Linkers comprising Protease Cleavage Sites. Li ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ N G T G T G G G u G GEPGGGSGGGSGT^GPCR

[0077] In some embodiments, the synthetic receptor comprises a G-protein coupled receptor (GPCR) covalently linked to an input module described herein. Different GPCRs can bind different signaling molecules (agonists or ligands). After a GPCR binds its cognate agonist or ligand, the GPCR changes its conformation, which results in intracellular signaling through a heterotrimeric G protein comprising GĮ bound to a GȕȖ dimer. Specifically, the activated GPCR undergoes a conformational change which catalyzes the release of GDP and subsequent binding of GTP on the alpha subunit, which leads to dissociation of the GȕȖ dimer from GĮ and thus activates multiple intracellular signaling responses.

[0078] In some embodiments, the GPCR is a wild-type or unmodified GPCR. In some embodiments, the GPCR is a variant or modified GPCR. In some embodiments, the GPCR is modified so it no longer binds any of its native ligands. In some embodiments, the GPCR is modified so it binds an inhibitor described herein.

[0079] Exemplary GPCRs and cognate polypeptide inhibitors useful in the present disclosure are provided in Table 7 below. In some embodiments, the GPCR comprises an amino acid sequence having at least, or greater than or equal to, 90% sequence identity (e.g., at least, or greater than or equal to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity) to a GPCR in Table 7. Table 7. Exemplary GPCRs and cognate inhibitors G K ^ r ( 1RLLSGSREKDRNLRRITRLVLVVVAVFVVCWTPIHIFILVEALGSTSH K ^ r ( K ^ r ( K ^ r (HPVKALDFRTPLKAKIINICIWLL K ^ r ( V aNSTINPMCYALCNKAFRDTFRLL a a a C aM1^DREADD^x^G^protein^coupled^receptor^183^ DLIIGTFSMNLYTTYLLMGHWAL C ( a a C a c C (NSTINPMCYALCNKAFRDTFRLLLLCRWDKRRWRKIPKRPGSVHR a a a a a D ^ r K ^ r N ^ ^ r 5 r 5 r 5 r 5 r 5 r 5 r 5receptor^2B^^ ^ 5^hydroxytryptamine^ P28335^ ^^ r 5 r 5 r 5 r 5 r A A A A A c A c A c A c A c A c C s r C s r C s r A c A c A c Acoupled^receptor^E2^^^^^ ^ Adhesion^G^protein^ Q9BY15^ ^^ c P p r A c A c A c A c A c A c A c A c A c A c A c A c A c A c A c A c A c A c Areceptor^^^^^^ ^ Alpha^1B^adrenergic^ P35368^ ^^ r A r A r A r A r B r B r B r T r T r V r V r V O A r A r A r A r C r p t B BCalcitonin^receptor^^ P30988^ ^^^ ^ Calcitonin^gene^related^ Q16602^ ^^ p E s G r 6 C C C t C t C t C t C t C t C t C t C t C t C 1 C 2 C r G t C c C c CchemotacƟc^receptor^2^^^^ ^ CorƟcotropin^releasing^ P34998^ ^^ f C f C r C r C r C r C r C r C r D r D D D D r E E t f N r N r F 1 F 2 F 3 F 4 Preceptor^1^^^^^ ^ Proteinase^acƟvated^ P55085^ ^^ r P r P r G G G G a s G a s G G r r G p G r G r S r r r r r r rreceptor^8^^^ ^ Follicle^sƟmulaƟng^ P23945^ ^^ h L c h T G h P r r G a G e G r G r G r G r G r U n l N r P c G r P c G r P c Greceptor^26^^ ^ Probable^G^protein^ Q9NS67^ ^^ c 1 5 e r P c P c P c G r P G G r G r G r P c r G r G r G r G r P c P O p r Pcoupled^receptor^75^^ ^ G^protein^coupled^ Q96P69^ ^^ r P c G r G r P c G r P c P c G i P c G r P c P c P c P c P c P c P c G r P c Pcoupled^receptor^153^^^ ^ Probable^G^protein^ Q9UJ42^ ^^ c G r P c G r P c P c G r G r G r L c c L c c L c c P c c m c m c m cmember^G^^^ ^ Mas^related^G^protein^ Q96LB2^ ^^ c m c m c m c m P P p R p G r 5 G r 5 G r 5 P c P c P c F F F F F F F F F FProtein^smoothened^^ Q99835^ ^^^ ^ Histamine^H1^receptor^^^ P35367^ ^ ^^ ^ H H H H r H r H r O r O C r C r L 1 L 2 O 1 L r L r L r L r L r L r h h h Ahormone^receptor^^^^^^^ ^ MelanocorƟn^receptor^3^^ P41968^ ^ ^^ ^ 1 1 N 1 N 2 N r N r N r N r N r N r N r N r N r P r N t N t L o o oopsin^3^^^^^^ ^ Short^wave^sensiƟve^ P03999^ ^^ o R O O R c V r 2 1 P h h p P r G s t K P p P a T h U P r P P P r P r P r Preceptor^EP2^subtype^^^^^ ^ Prostaglandin^E2^ P43115^ ^^ r P r P r P T r P P P P P P P P R R R R S t S t S t S t S t S p S p S p S p S pSuccinate^receptor^1^^^ Q9BXA5^ ^^^ ^ Substance^P^receptor^^^^^ P25103^ ^ ^^ ^ S N T m T m T m T m T m T m T m T m T m T m T m T m T m T m T m T m T m T m P t Ptype^2^member^36^^^^ ^ Taste^receptor^type^2^ P59533^ ^^ m T m T m T m T m T m T m T m T m T m T r T r P a T r T r T r T r P c p r V p VpolypepƟde^receptor^2^^^^^^ ^ Vomeronasal^type^1^ Q9GZP7^ ^^ r V r V r V r V r P r C 1

[0080] In some embodiments, the activated GPCR binds native G-proteins in a cell. In some embodiments, the GPCR is fused to an output module (described below). It will be understood that in any embodiment, there is an “output” intracellular signal from the activated synthetic receptor, but the output signal is not necessarily from an "output module". Output Module

[0081] The synthetic receptors of the disclosure can further comprise an optional output module. An output module can be linked to the synthetic receptor and generates a signal upon activation of the receptor. Thus, in some embodiments, the synthetic receptor comprises, in an amino terminus to carboxy terminus direction, a compound of formula (II): [input module] – [G-protein coupled receptor (GPCR)] – [output module] (II).

[0082] In some embodiments, the synthetic receptor comprises, in an amino terminus to carboxy terminus direction, a compound of formula (IIa): [polypeptide inhibitor of a GPCR] – [ABM] – [amino acid linker] – [GPCR] – [output module] (IIa).

[0083] In some embodiments, the synthetic receptor comprises, in an amino terminus to carboxy terminus direction, a compound of formula (IIb):[ABM] – [polypeptide inhibitor of a GPCR] – [amino acid linker] – [GPCR] – [output module] (IIb).

[0084] In some embodiments, the synthetic receptor comprises, in an amino terminus to carboxy terminus direction, a compound of formula (IIc): [polypeptide inhibitor of a GPCR] – [ABM] – [GPCR] – [output module] (IIc).

[0085] In some embodiments, the synthetic receptor comprises, in an amino terminus to carboxy terminus direction, a compound of formula (Id): [ABM] – [polypeptide inhibitor of a GPCR] – [GPCR] – [output module] (IId).

[0086] In some embodiments, the output module is covalently linked to the carboxy terminus of the GPCR, which comprises the intracellular carboxy-terminal tail of the synthetic receptor when expressed in a cell. Thus, when expressed in a cell, the output module is attached to the intracellular carboxy-terminal tail of the synthetic receptor. Spark Output Module

[0087] In some embodiments, the output module comprises a fusion polypeptide comprising, in an amino to carboxy terminal direction, a) a light-oxygen-voltage-sensing (LOV) domain, b) a tobacco etch virus protease recognition sequence (TEVcs), and c) a transcription factor (TF). For convenience, the fusion polypeptide output module is referred to as a “SPARK” (Specific Protein Association tool giving transcriptional Readout with rapid Kinetics) output module. SPARK modules are described in WO 2021 / 062063 and US 2023 / 0067225 A1, Kim et al., eLife , 2017 (DOI: 10.7554 / eLife; and Kim et al., eLife , 2019 (DOI: 10.7554 / eLife, which are incorporated by reference herein. In the SPARK module, the LOV domain “cages” the TEV cleavage site to reduce background. In some embodiments, the SPARK fusion polypeptide output module is covalently linked to the carboxy terminus of the GPCR in the synthetic receptors of the disclosure.

[0088] In some embodiments, the output module is expressed in a cell of interest. When expressed in cells, the SPARK output module interacts with an arrestin (for example, humanbeta-arrestin or rat beta-arrestin)-TEV protease (TEVp) fusion protein. The arrestin-TEVp fusion protein is typically expressed in a cell of interest that is transfected or transduced with a nucleic acid molecule encoding the SPARK output module. See, Kim et al., eLife , 2019 (DOI: 10.7554 / eLife. When a GPCR is activated, its cytoplasmic C-terminal tail becomes phosphorylated. Arrestin binds this phosphorylated GPCR tail as well as the crevice on the intracellular side of the GPCR transmembrane core that is formed in the active GPCR conformation. See, e.g., Sheerer et al., Current Opinion in Structural Biology, Volume 45, August 2017, Pages 160-169. Upon activation of the GPCR, the arrestin-TEV protease fusion protein is recruited to and binds the cytoplasmic surface of the GPCR via a binding site on the arrestin molecule. When a GPCR is activated, the GPCR cytoplasmic C-terminal tail becomes phosphorylated. Arrestin binds this phosphorylated GPCR tail as well as the crevice on the intracellular side of the GPCR transmembrane core that is formed in the active GPCR conformation. See, e.g., Sente, A. et al. Molecular mechanism of modulating arrestin conformation by GPCR phosphorylation. Nat. Struct. Mol. Biol.25, 538–545 (2018). In some embodiments, arrestin binding to the GPCR results in proximity induced proteolytic cleavage and release of the sequestered transcription factor (TF) in the SPARK output module. The released TF can translocate to the nucleus and bind to its cognate transcription initiation DNA sequence, thereby activating gene expression. In some embodiments, the cleavage site is caged and blue light or other wavelength light is used to uncage the cleavage site so that it is accessible for cleavage by the protease. Other output modules can also be used. For example, in some embodiments, Tango is used as an output module. See, Barnea et al., PNAS, 105, 1, 64-69, 2008 (DOI: 10.1073 / pnas.0710487105). In some embodiments, ChaCha is used as an output module. See, Kipniss et al., Nature Communications, 8, Article number 2212, 2017 (DOI: 10.1038 / s41467-017-02075-1).

[0089] In some embodiments, the arrestin-TEVp fusion protein further comprises a bioluminescent polypeptide that emits blue light. In some embodiments, the fusion protein comprises, in an amino terminal to carboxy terminal direction, a bioluminescent polypeptide - an arrestin - a TEVp though other orientations are possible as desired. When expressed in cells, the bioluminescent polypeptide-arrestin-TEVp fusion protein can emit blue light, which, within proximity, results in a conformation change in the LOV domain and allows the TEVp to contact and cleave the TEV cleavage site in the output module. In some embodiments, thebioluminescent polypeptide comprises a fluorescent protein (e.g., Blue Fluorescent Protein (BFP) or a luciferase or nanoluciferase (e.g., NanoLuc® commercially available from Promega).

[0090] As above, after activation of the GPCR, the TEVp component of the bioluminescent polypeptide-arrestin-TEVp fusion protein cleaves the SPARK output module at the TEVcs sequence, which release the carboxy terminal TF. The released TF can translocate to the nucleus and bind to its cognate transcription initiation DNA sequence, thereby activating gene expression.

[0091] The gene whose transcription or expression is activated can be an endogenous gene, a transgene (e.g., a heterologous gene that is introduced into the genome of a cell) or a gene present on a plasmid or expression vector transfected into a cell. In some embodiments, the gene encodes a chimeric antigen receptor (CAR), an antibody (for example a therapeutic antibody) or fragments thereof (e.g., an scFv, FAB, or a VH), a monobody, a chemokine, a chemokine receptor, a cytokine, a cytokine receptor, a differentiation factor, a growth factor, a growth factor receptor, a hormone, a hormone receptor, a metabolic enzyme, a proliferation inducer, a receptor, a small molecule 2ndmessenger synthesis enzyme, a T cell receptor, a transcription activator, a transcription repressor, a transcriptional activator, a transcriptional repressor, a translation regulator, a translational activator, a translational repressor, an activating immunoreceptor, an apoptosis inhibitor, an apoptosis inducer, an immunoactivator, an immunoinhibitory, or an inhibiting immunoreceptor.

[0092] In some embodiments, the TEV protease is a wild-type TEV protease. In some embodiments, wild-type TEV protease comprises the amino acid sequence of SEQ ID NO:1 (EC number 3.4.22.44, CAS number 139946-51-3, see UniProtKB: P04517). In some embodiments, the protease is a low-affinity protease, for example a TEV protease having a carboxy-terminal truncation. In some embodiments, the low affinity protease has a Km of greater than 300 microMolar. In some embodiments, the protease is a C-terminally truncated, low-affinity wild- type TEV (TEVǻ219, or TEVǻ) protease. In some embodiments, the TEV protease is a TEVǻ220-242 protease described in U.S. Patent Publication 2018 / 0201657. In some embodiments, the C-terminally truncated, low-affinity wild-type TEV protease comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the TEV protease has increased catalytic activity compared to a wild-type TEV protease (SEQ ID NO:1; EC number 3.4.22.44,CAS number 139946-51-3, see UniProtKB: P04517) or a C-terminally truncated wild-type TEV protease (e.g., TEVǻ219, or TEVǻ). In some embodiments, the improved TEV protease comprises an amino acid sequence differing from wild-type TEV at one or more positions selected from T30, S31, S153, and N177. In some embodiments, the modified TEV protease comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to wild-type TEV (SEQ ID NO:1) and comprises one or more mutations selected from T30A, T30I, S31W, S153N, N177Y, or a double T30A / S153N mutation. In some embodiments, the modified TEV protease comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a C-terminally truncated wild- type TEV protease (e.g., TEVǻ219, or TEVǻ) and comprises one or more mutations selected from T30A, T30I, S31W, S153N, N177Y, or a double T30A / S153N mutation. See, e.g., WO 2021 / 062063.

[0093] In some embodiments, the output module is selected from a sequence in a row of Table 8 below. In some embodiments, the output module comprises a sequence having at least, or greater than or equal to, 90% sequence identity (e.g., at least, or greater than or equal to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity) to a sequence selected from a row of Table 8 below. Table 8. Exemplary Output Modules. O N S O ( S OModule^2^ LYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSH (Specific Embodiments

[0094] In some embodiments, the synthetic receptor comprises a combination of a peptide inhibitor selected from a row of Table 1, an ABM selected from a row of Table 2, a linker selected from a row of Table 4 or a linker selected from a row of Table 6, and a GPCR selected from a row of Table 7. In some embodiments, the synthetic receptor comprises a combination of a peptide inhibitor selected from a row of Table 1, an ABM selected from a row of 2, and a GPCR selected from a row of Table 7. In some embodiments, the synthetic receptor comprises a combination of a peptide inhibitor selected from a row of Table 1, an ABM selected from a row of Table 2, a linker selected from a row of Table 4, and a GPCR selected from a row of Table 7. In some embodiments, the synthetic receptor comprises a combination of a peptide inhibitor selected from a row of Table 1, an ABM selected from a row of Table 2, a linker selected from a row of Table 6, and a GPCR selected from a row of Table 7.

[0095] In some embodiments, the synthetic receptor comprises a combination of a peptide inhibitor selected from a row of Table 1, an ABM selected from a row of 2, a GPCR selected from a row of Table 7, and an output module selected from a row of Table 8. In some embodiments, the synthetic receptor comprises a combination of a peptide inhibitor selected from a row of Table 1, an ABM selected from a row of Table 2, a linker selected from a row of Table 4, a GPCR selected from a row of Table 7, and an output module selected from a row of Table 8. In some embodiments, the synthetic receptor comprises a combination of a peptide inhibitor selected from a row of Table 1, an ABM selected from a row of Table 2, a linker selected from a row of Table 6, a GPCR selected from a row of Table 7, and an output module selected from a row of Table 8.

[0096] In some embodiments, the full length synthetic receptor comprises a sequence selected from a row of Table 9 below. In some the full length synthetic receptor comprisesa sequence having at least, or greater than or equal to, 70, 75, 80, 85, or 90% sequence identity (e.g., at least, or greater than or equal to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity) to a sequence selected from a row of Table 9 below. Table 9. Exemplary full length synthetic receptor fusion protein sequences Full^PAGER^ Full^PAGER^receptor^sequence^(amino^acid^sequence)^ SEQ^ID^ r V T HYSREEILGRNCRFLQGPETDRATVRKIRDAIDNQTEVTVQLINYTKSGKKFWNLFHLQPMRDQKGDVQYFIGVQLDGTERVRDAAEREAVMLVKKTAEE T T GDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQ G G GWRIYRETENRARELAALQGSETPGKGGGSSSSSERSQPGAEGSPETPPGRCCRCCRAPRLLQAYSWKEEEEEDEGSMESLTSSEGEEPGSEVVIKMPMVD G PG protein Activation

[0097] In another aspect, the synthetic receptor does not comprise a heterologous output module of the disclosure. Rather, when expressed in a cell, the synthetic receptor activates an endogenous heterotrimeric G protein. In some embodiments, the synthetic receptor comprises, from amino to carboxy terminus, a compound of formula (I), (Ia), (Ib), (Ic) or (Id).

[0098] In some embodiments, the endogenous G protein is selected from the group consisting of GĮi, GĮs, GĮ12 / 13, and GĮq. The Gs and Gi families regulate adenylyl cyclase activity (where Gs stimulates cAMP production in the cell and Gi inhibits production of cAMP in the cell), Gq activates phospholipase Cȕ and G12 / 13 can activate small GTPase families. The Gq family consists of four members: Gq, G11, G14, and G15 / 16 (and the respective Į subunits are GĮq, GĮ11, GĮ14, and GĮ15 / 16). Additional subtypes include Go, Gz, Ggust, Gs-L, and Gs-S, among others.

[0099] Thus, in some embodiments, activation of the synthetic receptor by an antigen of interest results in activation of an endogenous G protein, leading to different signal transduction pathways depending on the particular GPCR / G protein class or subclass combination selected. Therefore, the synthetic receptors of the provide the advantage that they can be usedto control endogenous cellular pathways and functions using non-native extracellular signals (e.g., antigens or proteases) that are designed to activate a particular G protein. In some embodiments, Gi activation can lead to, for example, decreased levels of cAMP, phosphorylation of AKT and ERK, and activation of PLC-beta and K+ channels. Gs activation leads to increased levels of cAMP, phosphorylation of PKA, CREB, and ERK, and expression of CREB controlled genes. In some embodiments, Gq activation leads to calcium-mobilization, PLC-beta activation, diacylglycerol (DAG) and inositol triphosphate (IP3) production, and expression of NF-kB regulated genes. In some embodiments, G12 activation leads to the activation of small GTPases including Rho and Ras and can activated members of the MAPK family. The above pathways can be activated by the different G protein classes, depending on which GPCR is being activated. Nucleic acids

[0100] In another aspect, the disclosure provides nucleic acid molecules (e.g., polynucleotides, e.g., DNA or RNA) that encode a synthetic receptor of the disclosure. In some embodiments, the nucleic acid molecule comprises a sequence that encodes a synthetic receptor fusion protein of formula (I), (Ia), (Ib), (Ic) or (Id). In some embodiments, the nucleic acid molecule comprises a sequence that encodes a synthetic receptor fusion protein of formula (II), (IIa), (IIb), (IIc) or (IId). Vectors

[0101] In another aspect, the disclosure provides a recombinant vector comprising a nucleic acid molecule of the disclosure. Vectors typically comprise one or more regulatory sequences operably linked to a nucleic acid sequence (e.g., a coding region or open reading frame) encoding a synthetic receptor of the disclosure. In some embodiments, the vector is an expression vector that is used to transcribe an RNA encoding a synthetic receptor of the disclosure. The vector can also include sequences that regulate transcription of the coding region (e.g., promoters, enhancers, transcription terminators) and translation of the protein (e.g., translation initiation codons or Kozak sequences) and well as sequences that control location of the translated protein in the cell (e.g., nuclear localization signals).Cells

[0102] In another aspect, the disclosure provides a genetically modified cell comprising a nucleic acid molecule, vector, and / or synthetic receptor of the disclosure. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell or transformed mammalian cell line. In some embodiments, the cell is a human cell or transformed human cell line. In some embodiments, the cell is a primary cell (i.e., isolated from a subject or not transformed). In some embodiments, the cell is an immune cell. Exemplary immune cells include, but are not limited to, cytotoxic T cells, helper T cells, regulatory T cells, NK cells, macrophages, dendritic cells, B cells, microglia, and peripheral blood mononuclear cells (PBMCs). In some embodiments, the cell is selected from fibroblasts, hematopoietic cells, neurons, pancreatic cells, muscle cells, bone cells, hepatocyte, pancreatic cells, epithelial cells, endothelial cells, or cardiomyocytes. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or is a cell differentiated from an iPSC.

[0103] In some embodiments, the genetically modified cell comprises a synthetic receptor fusion protein of formula (I), (Ia), (Ib), (Ic) or (Id). In some embodiments, the cell comprises a synthetic receptor fusion protein of formula (II), (IIa), (IIb), (IIc) or (IId). Methods of Use

[0104] In another aspect, the disclosure provides methods of using the synthetic receptor fusion proteins of the disclosure. In some embodiments, the method comprises (i) providing a cell comprising a synthetic receptor fusion protein comprising a GPCR, optionally a linker amino acid sequence, and a polypeptide antagonist such that polypeptide antagonist binds and occupies the orthosteric site of the GPCR, (ii) contacting the synthetic receptor fusion protein with a ligand for the orthosteric site, thereby displacing the polypeptide antagonist, and (iii) measuring an output signal from the synthetic receptor fusion protein. See, e.g., FIG.1A-D and FIG.3B. In some embodiments, an antigen binding module (ABM), for example but not limited to a nanobody or as otherwise described herein, that specifically binds an antigen of interest is included in the linker between the GPCR and the polypeptide antagonist. See, e.g., FIG, 1C. In the absence of the specific antigen, the peptide antagonist remains bound and the GPCR remainsinactive. In the presence of a specific antigen for the ABM (which could be for example, a surface antigen on a neighboring cell (see, FIG.1D), the ABM binds the antigen and the polypeptide antagonist is displaced from the orthosteric site, thereby allowing a ligand (e.g., a drug) to bind and the GPCR to be activated. Any output module for measuring activation of the receptor can be used and detected in the methods. Methods for Activating Transcription of a Target Gene

[0105] In one aspect, the disclosure provides a method of activating transcription of a target gene in a cell. In some embodiments, the method comprises (i) providing a cell expressing the synthetic receptor fusion protein on the target cell surface, wherein the synthetic receptor fusion protein further comprises a protease cleavage site linked to a transcription factor, and wherein the cell also expresses an arrestin-TEV protease (TEVp) fusion protein; (ii) contacting the synthetic receptor fusion protein with a ligand for the orthosteric site, thereby displacing the polypeptide antagonist, activating the GPCR, and inducing binding of the arrestin portion of the arrestin-TEVp fusion protein, and cleaving the protease cleavage site with the TEVp, thereby releasing the transcription factor to activate gene expression in the cell, and (iii) measuring the gene expression of the cell. In some embodiments, the synthetic receptor fusion protein further comprises a LOV domain that protects the protease cleavage site and blue light is applied to uncage (displace) the protection of the cleavage site, allowing for cleavage of the protease cleavage site by the protease. In some embodiments, transcription of the target gene is determined by detecting or measuring expression of the mRNA, for example, by Northern analysis, RT-PCR, and RNA sequencing (RNA-seq). In some embodiments, transcription of the target gene is determined by detecting or measuring expression of a protein encoded by the mRNA, for example, by Western analysis, or by detecting the expression of an antibody that binds to the protein. In yet other embodiments, transcription of the target gene is determined by flow cytometry (for example, but not limited to, for membrane protein transgenes, for example chimeric antigen receptors (CARs). In some embodiments, ELISA can be used to detect the expression of proteins that are secreted (for example but not limited to, cytokines, chemokines, growth factors, and antibodies).

[0106] In some embodiments, the method comprises (i) providing a cell expressing on the target cell surface a synthetic receptor fusion protein comprising a GPCR, optionally a linkeramino acid sequence, an antigen binding module (ABM) and a polypeptide antagonist such that polypeptide antagonist binds and occupies the orthosteric site of the GPCR, wherein the synthetic receptor fusion protein further comprises a protease cleavage site linked to a transcription factor, wherein the target cell also expresses an arrestin-TEV protease (TEVp) fusion protein; (ii) contacting the cell with an antigen that binds the ABM of the synthetic receptor thereby displacing the polypeptide antagonist from the GPCR, and cleaving the protease cleavage site with the TEVp, thereby releasing the transcription factor to activate gene expression in the cell, and (iii) measuring the gene expression of the cell. In some embodiments, the synthetic receptor fusion protein further comprises a LOV domain that protects the protease cleavage site and blue light is applied to uncage the protection of the cleavage site, allowing for cleavage of the protease cleavage site by the protease.

[0107] In some embodiments, the blue light that is used to illuminate the cell is provide by an external energy source that is exogenous to the cell. In some embodiments, the external energy source emits light having a wavelength in the range of 400 nm-500 nm (e.g., 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500 nm). In some embodiments, the cells are illuminated with exogenous blue light at an intensity of from about 0.1 to 5 mW / cm2(e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.0, 1.0, 1.1, 1.2, 1.3, 14,1.5, 1.6, 1.7, 1.8, 1.9, 20. 3.0, 4.0 or 5.0 mW / cm2). In some embodiments, the cells are illuminated with exogenous blue light for about 5 to 30 minutes.

[0108] To practice the method, a cell can be transfected or transduced with (i) a nucleic acid molecule (i) encoding a synthetic receptor fusion protein of the disclosure and a nucleic acid molecule (ii) encoding the arrestin-TEVp fusion protein. In some embodiments, the cell is transfected with both nucleic acid molecules (i) and (ii) simultaneously. In some embodiments, the cell is transfected or transduced with nucleic acid molecules (i) and (ii) at different times. For example, in some embodiments, the cell is transfected with nucleic acid molecule (ii) encoding the arrestin-TEVp fusion protein first, such that the cell stably or transiently expresses the arrestin-TEVp fusion protein prior to being transfected or transduced with nucleic acid molecule (i) encoding a synthetic receptor fusion protein of the disclosure. In any of the above embodiments, the cell can also comprise a third nucleic acid encoding a target transgene whose expression is controlled by a transcription factor linked to the synthetic receptor fusion proteinand released upon activation of the receptor. For example, the third nucleic acid can comprise a promoter operably linked to a nucleic acid that is expressed upon binding of the transcription factor to the promoter. The transgene can encode any RNA or polypeptide as desired. In some embodiments, the encoded RNA or polypeptide is detectable or produces a detectable signal. In other embodiments, the RNA or polypeptide is a therapeutic RNA or polypeptide.

[0109] After the fusion proteins are expressed by the cell, the cell is contacted with an antigen that binds to the ABM, which activates the GPCR and results in binding of the arrestin-TEVp fusion protein to the GPCR, bringing the TEVp into close proximity to the TEVcs present in the synthetic receptor SPARK output module. Illumination of the cell with blue light results in a conformational change in the LOV domain, which exposes the caged TEVcs. Cleavage of the TEVcs by TEVp results in release of the TF, which translocates to the cell nucleus and binds its cognate transcription factor binding site (e.g., in a heterologous expression cassette or in native genomic DNA), thereby activating transcription of the target gene.

[0110] In some embodiments, the blue light that is used to illuminate the cell is produced intracellularly by an energy source that is endogenous to the cell. For example, in some embodiments the blue light is provided by a heterologous polypeptide or protein that emits blue light at an intensity sufficient to activate a SPARK output module of the disclosure. Representative blue light intensities include 0.1 to 5 mW / cm2(e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.0, 1.0, 1.1, 1.2, 1.3, 14,1.5, 1.6, 1.7, 1.8, 1.9, 20.3.0, 4.0 or 5.0 mW / cm2) at a wavelength in the range of 400 nm-500 nm (e.g., 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500 nm).

[0111] In some embodiments, the heterologous protein that emits blue light is selected from a photoprotein, such as a bioluminescent protein or a fluorescent protein (e.g., Blue Fluorescent Protein (BFP)). In some embodiments, the heterologous protein comprises a bioluminescent protein. In some embodiments, the bioluminescent protein comprises a luciferase or nanoluciferase (e.g., NanoLuc® commercially available from Promega). As is known in the art, bioluminescent proteins are enzymes that emit light in the presence of a substrate. For example, luciferase enzymes from fireflies and beetles use D-luciferin as a substrate, luciferase enzymes from sea pansies use coelenterazine as the substrate, and nanoluciferase uses the substrate furimazine, among other substrates.

[0112] Thus, in any of the methods or embodiments described herein, the protein that emits blue light can be selected from the group consisting of a luciferase, nanoluciferase, and a fluorescent protein. In some embodiments, the protein emits blue light when contacted with a substrate.

[0113] In some embodiments, the heterologous protein that emits blue light can be used to active a SPARK output module of the disclosure via bioluminescence energy transfer (BRET) or fluorescence resonance energy transfer (FRET) without the need for external or exogenous illumination with blue light. Thus, in some embodiments, the arrestin-TEVp fusion protein further comprises a photoprotein that emits blue light. In this embodiment of the method, no external illumination with blue light is required. Thus, after binding of antigen and activation of the synthetic receptor, the photoprotein-arrestin-TEVp fusion protein binds to the GPCR, and blue light emitted by the photoprotein in the presence of the photoprotein enzyme’s substrate induces allosteric changes in the blue light-sensitive LOV domain, thereby exposing the previously LOV-caged TEVcs. As above, subsequent cleavage of the TEVcs by TEVp results in release of the TF, which translocates to the cell nucleus and binds its cognate transcription factor binding site (e.g., in a heterologous expression cassette or in native genomic DNA), thereby activating transcription of the target gene.

[0114] In some embodiments, the heterologous polypeptide comprises a nanoluciferase comprising an amino acid sequence having at least 90% sequence identity (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity) to VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVI IPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAV FDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA (SEQ ID NO:215.

[0115] The target gene whose transcription or expression is activated can be an endogenous gene, a transgene (e.g., a heterologous gene that is introduced into the genome of a cell) or a gene present on a plasmid or expression vector transfected into a cell. In some embodiments, the target gene is present in the genomic DNA of a cell. In some embodiments, the target gene encodes a chimeric antigen receptor (CAR), a therapeutic antibody or fragments thereof, a chemokine, a chemokine receptor, a cytokine, receptor, a differentiation factor, agrowth factor, a growth factor receptor, a hormone, a hormone receptor, a metabolic enzyme, a proliferation inducer, a receptor, a small molecule 2ndmessenger synthesis enzyme, a T cell receptor, a transcription activator, a transcription repressor, a transcriptional activator, a transcriptional repressor, a translation regulator, a translational activator, a translational repressor, an activating immunoreceptor, an apoptosis inhibitor, an apoptosis inducer, an immunoactivator, an immunoinhibitory, or an inhibiting immunoreceptor. In some embodiments, the target gene is a reporter gene that encodes a protein that emits a detectable signal. In some embodiments, the open reading frame of the reporter gene is operably linked to a nucleic acid sequence that binds the TF released by the cleavage of the TEVcs after activation of a synthetic receptor of the disclosure.

[0116] Thus, in some embodiments, the cell is transfected or transduced with a nucleic acid molecule encoding the synthetic receptor fusion protein, a nucleic acid molecule encoding the arrestin-TEVp fusion protein, and a nucleic acid molecule comprising the target gene operably linked to a promoter that binds the TF.

[0117] In some embodiments, the cell is transfected or transduced with one or more of the nucleic acid molecules in vitro, ex vivo, or in vivo. Methods for Activating a G Protein in a Cell In another aspect, the disclosure provides methods for activating a G protein in a target cell. In some embodiments, the G protein is an endogenous G protein. As noted above, in some embodiments, the synthetic receptor fusion protein comprises a GPCR, and upon activation by an antigen, the GPCR binds a G protein that initiates a signal transduction pathway in a cell expressing the synthetic receptor. Thus, in some embodiments, the method comprises providing a cell (e.g., a target cell) expressing a synthetic receptor fusion protein on the cell surface. In some embodiments, the synthetic receptor comprises a compound of formula (I), (Ia), (Ib), (Ic) or (Id). The cell is then contacted with an antigen that binds the ABM of the synthetic receptor. Antigen binding to the ABM results in binding of the GPCR to the G protein, thereby activating the G protein.

[0118] In some embodiments, the target cell is transfected or transduced with a nucleic acid encoding the synthetic receptor fusion protein. In some embodiments, the target cell is transfected or transduced with the nucleic acid in vitro, ex vivo, or in vivo.

[0119] In some embodiments, the G protein is selected from the group consisting of GĮi, GĮs, GĮ12 / 13, and GĮq, or a subtype or subclass thereof. Methods of Using a Synthetic Receptor Fusion Protein

[0120] In another aspect, the disclosure provides methods for using the synthetic receptor fusion proteins of the disclosure. In some embodiments, the method comprises providing a cell expressing a synthetic receptor fusion protein, wherein the synthetic receptor fusion protein is activated by either (i) binding of antigen to the ABM (thereby removing a polypeptide inhibitor), or (ii) proteolytic cleavage of an amino acid linker sequence in the input module. In the case of (i), binding of antigen to the ABM displaces a peptide inhibitor bound to the GPCR and activates the synthetic receptor. In the case of (ii), proteolytic cleavage releases a peptide inhibitor bound to the GPCR and activates the synthetic receptor.

[0121] In some embodiments, the cell comprises a synthetic receptor comprising a polypeptide of formula (I), (Ia), (Ib), (Ic) or (Id). Following activation, the GPCR binds to a G protein, thereby activating the G protein and initiating a signal transduction pathway in the cell. In some embodiments, the G protein is an endogenous G protein. In some embodiments, the G protein is selected from the group consisting of GĮi, GĮs, GĮ12 / 13, and GĮq, or a subtype or subclass thereof.

[0122] In some embodiments, the cell comprises a synthetic receptor fusion protein of formula (II), (IIa), (IIb), (IIc) or (IId). In some embodiments, the method comprises contacting the synthetic receptor fusion protein with blue light, wherein binding of antigen in the presence of blue light results in proteolytic cleavage of a protease cleavage site in the intracellular domain of the synthetic receptor and release of a TF. After release of the TF from the synthetic receptor, the TF can translocate to the nucleus and activate transcription of a target gene.Pharmaceutical Compositions

[0123] The disclosure also provides pharmaceutical compositions or formulations comprising a synthetic receptor fusion protein described herein. The pharmaceutical compositions and formulations can be combined with a pharmaceutically acceptable carrier for administration to a subject or patient.

[0124] In some embodiments, pharmaceutical composition comprises a compound of formula (I), (Ia), (Ib), (Ic) or (Id). In some embodiments, pharmaceutical composition comprises a compound of formula (II), (IIa), (IIb), (IIc) or (IId). In some embodiments, the pharmaceutical composition comprises a genetically modified cell comprising a nucleic acid molecule, vector, and / or synthetic receptor of the disclosure. In some embodiments, the genetically modified cell is a mammalian cell, such as a human cell. In some embodiments, the genetically modified cell is a genetically modified primary cell (i.e., isolated from a subject). In some embodiments, the cell is autologous to the subject, and is genetically modified ex vivo after isolating the cell from the subject.

[0125] In some embodiments, the genetically modified cell comprises a synthetic receptor fusion protein of formula (I), (Ia), (Ib), (Ic) or (Id). In some embodiments, the cell comprises a synthetic receptor fusion protein of formula (II), (IIa), (IIb), (IIc) or (IId). Methods for Preventing or Treating Disease

[0126] In another aspect, provided herein is a method for preventing or treating a disease in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition of the present disclosure to the subject.

[0127] In some embodiments, the method comprises administering a genetically modified cell comprising a synthetic receptor fusion protein of the disclosure, wherein the cell expresses a therapeutic protein following activation of the synthetic receptor fusion protein by an antigen or ligand. Examples of useful therapeutic proteins include chimeric antigen receptors (CAR), cytokines and cytokine receptors, chemokines and chemokine receptors, growth factors and growth factor receptors, hormones and hormone receptors, and other immune system moleculesand receptors (e.g., expressed by B cells, T cells, monocytes, dendritic cells, and / or natural killer cells). In some embodiments, the antigen or ligand is a soluble molecule such as cytokine, chemokine, growth factor, or hormone. In some embodiments, the antigen or ligand is an immunosuppressant molecule or T cell inhibitor, and the therapeutic protein is an activator of an immune response, which provides a method to convert immunoinhibitory signals into immune activation signals that are useful to treat cancer, for example. Examples of immunosuppressive cytokines include, but are not limited to, TGF-beta, IL-10, and VEGF. In some embodiments, a synthetic receptor polypeptide herein is activated upon the binding of an immunosuppressive cytokine and triggers expression of a pro-inflammatory therapeutic protein. Exemplary proinflammatory proteins include but are not limited to IL-2, IL-15, or TNF-alpha, optionally targeted to be expressed in a tumor.

[0128] The compositions and methods of the present disclosure are suitable for any disease that is amenable to prevention or amelioration of disease-associated symptoms by a pharmaceutical composition of the disclosure. Non-limiting examples of diseases include X- linked severe combined immune deficiency, sickle cell anemia, thalassemia, hemophilia, neoplasia, cancer, age-related macular degeneration, schizophrenia, trinucleotide repeat disorders, fragile X syndrome, prion-related disorders, amyotrophic lateral sclerosis, drug addiction, autism, Alzheimer’s disease, Parkinson’s disease, cystic fibrosis, blood and coagulation diseases and disorders, inflammation, immune-related diseases and disorders, metabolic diseases and disorders, liver diseases and disorders, kidney diseases and disorders, muscular / skeletal diseases and disorders, neurological and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, and ocular diseases. The compositions and methods of the present disclosure can also be used to prevent or treat any combination of suitable diseases.

[0129] In some embodiments, the subject is treated before any symptoms of the disease develop. In some embodiments, the subject has symptoms of the disease. In some embodiments, treatment results in a reduction or elimination of the symptoms of the disease.

[0130] In some embodiments, treatment includes administering compositions of the present disclosure directly to a subject. As a non-limiting example, pharmaceutical compositions of the present disclosure can be delivered directly to a subject (e.g., by local injection or systemicadministration). In some embodiments, a genetically modified cell of the disclosure is administered or transplanted to the subject. The genetically modified cell can be administered or transplanted with a pharmaceutically acceptable carrier. In some embodiments, progeny of the genetically modified cell are administered or transplanted into the subject. In some embodiments, the cell is an autologous cell that is obtained from a subject or patient and is genetically modified ex vivo to express a synthetic receptor of the disclosure before being administered to the same subject or patient. In some embodiments, the cell is an allogenic cell obtained from a different individual than the subject or patient. In some embodiments, the allogenic cell is obtained from an individual who has an identical or partial human leukocyte antigen (HLA)-match with the subject or patient. In some embodiments, the allogenic cell is genetically modified ex vivo to express a synthetic receptor of the disclosure before being administered to the same subject or patient. Procedures for transplantation and administration of genetically modified cells will be known to one of skill in the art.

[0131] Pharmaceutical compositions of the present disclosure, including genetically modified cells of the present disclosure, may be administered as a single dose or as multiple doses, for example two doses administered at an interval of about one month, about two months, about three months, about six months or about 12 months. Other suitable dosage schedules can be determined by a medical practitioner.

[0132] Prevention or treatment can further comprise administering agents and / or performing procedures to prevent or treat concomitant or related conditions. As non-limiting examples, it may be necessary to administer drugs to suppress immune rejection of transplanted cells, or prevent or reduce inflammation or infection. A medical professional will readily be able to determine the appropriate concomitant therapies. Further Applications

[0133] The disclosure provides new types of engineered cell therapies that may be useful to fulfill the unmet need for more effective and less toxic treatments for a wide range of diseases, including cancer, autoimmune diseases, and neurodegenerative diseases. Engineered cells comprising the synthetic receptors of the disclosure provide cells that can (i) respond to solubleantigens (e.g., in tumor microenvironment); (ii) turn on transgene expression or G protein activation, (iii) activate cell signaling when cleaved by proteases (e.g., tumor specific proteases), and (iv) provide high modularity that allows rapid development against new targets.

[0134] The disclosure also provides new types of gene therapies for a wide range of diseases, including cancer, autoimmune diseases, and neurodegenerative diseases. The synthetic receptors of the disclosure may be useful to directly modulate GPCR activity in response to disease specific biomarkers, and provide more local and specific targeting effects than administering small molecules that bind and activate GPCRs.

[0135] The disclosure provides new techniques and methods for tissue engineering to create more effective treatments or products for a wide range of diseases or for other biotechnology applications.

[0136] The disclosure allows new types of biosensors to be made to fulfill the unmet need for facile development of sensitive sensors for various antigens of interest by taking advantage of the modular nature of the synthetic receptors. These new biosensors could be used to screen for antigens of interest in samples / products, to screen for new binders specific for an antigen of interest, to screen for antagonists, agonists or inhibitors of receptors of interest, or to develop a synthetic receptor against an antigen of interest. These new biosensors could also be used by clinics to measure antigens of interest in clinical samples from patients. EXAMPLES Example 1.

[0137] Here we present a generalizable, programmable synthetic receptor platform for customized cell responses. PAGER, for Programmable Antigen-gated G protein-coupled Engineered Receptor, can be programmed in a single step to respond to a wide variety of extracellular antigens, simply by swapping in a nanobody binder against the antigen of interest (between the GPCR component and the antagonist peptide component of PAGER as shown in FIG.1C-D). In addition, through selection of the appropriate PAGER scaffold (Gi-PAGER, Gs- PAGER, Gq-PAGER, G12-PAGER, or PAGER-SPARK) it is possible for PAGER to either control endogenous G protein signaling or to expression of a transgene of choice (e.g., afluorescent protein, luciferase, therapeutic antibody, etc.). Furthermore, PAGER is designed to be controllable by an external drug (e.g. salvinorin B), so that it can be turned on and off at will. We have reduced many of our claims to practice, showing data for PAGERs designed to respond to a wide variety of soluble (GFP, mCherry, VEGF, HGF, TNFĮ, IL-17, IL-23, sIL-6R, CCL2, Sars-CoV-2 spike protein, and uPA; FIG.6) and cell-surface (EGFR, HER2, CD38, and PD-L1; FIG.6) antigens, by driving intracellular transgene expression or Gi / Gs / Gq / G12 activation (FIG. 9A-B and FIG.10B-D). PAGER is a powerful, versatile, and simple / robust platform for programming a variety of cell types to provide customized responses to a wide range of natural and unnatural extracellular cues.

[0138] Mammalian cell engineering is leading to advances in therapeutics, basic research, and beyond. Synthetic (non-natural) receptors allow for the programming of mammalian cells to sense a target antigen in their extracellular environment and respond in a precisely tailored manner. Early strategies to develop such synthetic receptors focused on mutation of natural receptors, via site-directed mutagenesis or directed evolution, to alter ligand-specificity. This approach is generally time, labor, and resource intensive and needs to be repeated for every new synthetic receptor being made. An alternative approach alters ligand specificity by replacing the sensing domains of native receptors with other ligand-binding domains such as single-chain antibody variable fragments (scFvs) or nanobodies (single-domain camelid antibody fragments). This approach generally creates programmable receptors that can be easily engineered due to the modularity of the ligand-binding domain. Existing technologies for programmable synthetic receptors capable of target antigen-induced cell signaling include Chimeric Antigen Receptors (CARs), synthetic Notch receptor (synnotch), synthetic intramembrane proteolysis receptors (SNIPRs), Modular Extracellular Sensor Architecture (MESA) receptors, and generalized extracellular molecule sensor (GEMS) receptors.

[0139] Despite their considerable contributions to advancing mammalian cell engineering, particularly with regards to therapeutics, these technologies still have some constraints. One prevailing limitation of existing synthetic receptors is their inability to respond to soluble antigens. Existing synthetic receptors that are capable of sensing soluble antigens require two antigen recognition domains to do so, largely restricting their modularity and functionality. Moreover, although some existing synthetic receptors can drive antigen-dependent transgeneexpression, the repertoire of endogenous signaling outputs for existing synthetic receptors is somewhat limited as well.

[0140] Given the status of the field, we believe that there is one class of cell surface receptors that has been overlooked for use in developing programmable synthetic receptors: G protein- couple receptors (GPCRs). GPCRs are essential cell surface receptors that mediate responses to diverse extracellular signals, including hormones, neurotransmitters, peptides, light, and odorants. Ligand binding induces a conformational change in the GPCR that, in turn, activates heterotrimeric G proteins and downstream intracellular signaling cascades. We set out to capitalize on the inherent ability of GPCRs to transduce signals across the cell membrane to develop a new class of programmable synthetic receptors based on GPCRs.

[0141] To develop this new GPCR-based class of programmable synthetic receptors, we introduced modular antigen gating on GPCR scaffolds through fusion of an extracellular nanobody and a receptor auto-inhibitory domain. Binding to antigen relieves auto-inhibition and enables receptor activation by an orthogonal drug, thus generating Programmable Antigen-gated G protein-coupled Engineered Receptors (PAGERs). We created PAGERs responsive to more than a dozen biologically and therapeutically important soluble and cell surface antigens, in a single step, from corresponding nanobody binders. Different PAGER scaffolds permit antigen binding to drive transgene expression or endogenous Gi, Gs, Gq, or G12 activation, leading to control of cytosolic Ca2+, lipid signaling, cAMP, and downstream signaling events. Due to PAGER's simplicity, modular design, and generality, it has the potential to transform new areas of therapeutics and basic research.

[0142] In order to develop programmable synthetic receptors based on GPCRs, we introduced modular antigen gating on GPCR scaffolds through fusion of an extracellular nanobody and a receptor auto-inhibitory domain. If binding of the auto-inhibitory domain to the GPCR and binding of the nanobody to an antigen could be engineered to be mutually exclusive, then antigen binding would relieve auto-inhibition and enable receptor activation by an orthogonal drug, thus generating Programmable Antigen-gated G protein-coupled Engineered Receptors (PAGERs) (FIG. 1A-E). PAGER activation could then drive transgene expression using existing methodologies, such as SPARK (Kim et al., eLife, 2017, DOI: 10.7554 / eLife.30233; Kim et al., eLife, 2019, DOI: 10.7554 / eLife.43826), Tango (Barnea et al., PNAS, 105, 1, 64-69, 2008, DOI:10.1073 / pnas.0710487105), or ChaCha (Kipniss et al., Nature Communications, 8, Article number 2212, 2017, DOI: 10.1038 / s41467-017-02075-1) among others.

[0143] The GPCR of choice to use as the scaffold for such a synthetic receptor would ideally meet two requirements. First, the GPCR would be activatable by an orthogonal, non-toxic, bio- available small molecule agonist. Second, the GPCR would have a known peptide antagonist that could be genetically fused to the GPCR to auto-inhibit it. Based on these requirements, we focused in on Designer Receptors Exclusively Activated by Designer Drugs (DREADDs; Armbruster et al., PNAS, vol.104, no.12, 5163–5168, 2007, DOI: 10.1073 / pnas.0700293104; Roth, Neuron, Volume 89, Issue 4, P683-694, 2016, doi: 10.1016 / j.neuron.2016.01.040; Vardy et al., Neuron, Volume 86, Issue 4, P936-946, 2015, DOI: 10.1016 / j.neuron.2015.03.065) as candidate GPCRs to use. DREADDs are GPCRs that have been engineered, through single or double point mutations, to not bind any native ligands, but can still be activated by orthogonal small molecule agonists. DREADDs were made using the Kappa opioid receptor and the muscarinic M1-5 acetylcholine receptors. The wild type version of these receptors also have known peptide or protein antagonists; though these antagonists have never been tested on the DREADD receptors, we reasoned these may work to antagonize the DREADDs as well. Thus, the DREADD receptors potentially fulfill both requirements to use as the GPCR scaffold to build programmable synthetic receptors. We chose to use the Kappa opioid receptor DREADD (KORD) as the GPCR core for building the first PAGER due to the small size and simplicity of its peptide antagonists.

[0144] We decided to use SPARK to couple GPCR activation to transgene expression in engineered PAGERs due to its generally lower background and higher signal to noise compared to Tango or Cha-cha. In SPARK, activated GPCR and arrestin interaction drives together the low-affinity tobacco etch virus protease (TEVp) and its recognition sequence (TEVcs), leading to cleavage and transcription factor (TF) release only when light or the NanoLuc substrate furimazine is also present. Different GPCRs can function differently in SPARK assays due to inherent differences in arrestin coupling. So, in order to build a KORD-based PAGER using SPARK, we first needed to assess if KORD activation can be coupled to transgene activation using SPARK (FIG.2A-B). Activation of KORD-SPARK by the orthogonal small molecule agonist salvinorin B (Sal B) resulted in robust transgene expression of a firefly luciferasereporter but only when either exogenous light (FIG.2C) or the NanoLuc substrate furimazine (FIG. 2D) was simultaneously provided. These results demonstrate that SPARK can be used to turn KORD activation into transgene expression and that KORD could potentially serve as the core to a GPCR-based synthetic receptor that converts antigen detection into transgene expression.

[0145] To continue towards building a KORD-based PAGER, we next needed to screen for antagonist peptides that could be genetically fused to KORD and inhibit Sal B-dependent activation of the receptor. Since we ultimately wanted to include a genetically fused antigen- specific nanobody for antigen gating of the receptor, we decided to include a GFP-specific nanobody (LaG17) from the beginning and screen for peptide antagonists in the context of the nanobody (even though the nanobody will not be used yet). From N-terminus to C-terminus, we genetically fused individual peptide antagonists directly to the GFP-specific nanobody, which was fused to the N-terminus of KORD-SPARK via a flexible GS linker that also contained an ALFA epitope tag. Antagonist-nanobody-GPCR constructs are referred to as PAGERs, while Antagonist-nanobody-GPCR-SPARK constructs are referred to as PAGER-TFs (FIG.3A-B). This PAGER-TF containing the LaG17 GFP-specific nanobody is referred to as GFP(LaG17)- PAGER-TF. To create mutually exclusive of the antagonist to the GPCR and the nanobody to the antigen, we reasoned that putting the antagonist as close as possible to the nanobody might promote steric clash when the antigen binds that would displace the antagonist. Candidate peptide antagonists that were screened were dynorphin analogs that utilize the N-terminus of the peptide to bind the orthosteric binding pocket of KOR. Therefore, in order to maintain function, these peptides had to have a free and intact N-terminus and thus could only be fused at the extreme N-terminus of PAGER-TF. For this reason, it was also necessary that the signal peptide that was used to promote efficient and proper receptor targeting leave no scar on the N-terminus of the peptide antagonist; hence, we chose to use the IL-2 signal peptide. Several of the screened peptides were able to antagonize Sal B activation in the context of GFP-PAGER-TF (FIG.3C).

[0146] One example of a desirable peptide antagonist would not only prevent Sal B activation of PAGER-TF, but it would also be reversible such that its removal would permit Sal B activation again. We then screened peptide antagonists for their ability to prevent Sal B activation of GFP(LaG17)-PAGER-TF in the absence of GFP antigen, but that could permit SalB activation of GFP(LaG17)-PAGER-TF in the presence of GFP antigen. We screened top candidate peptide antagonists in the context of GFP(LaG17)-PAGER-TF using both surface- expressed GFP antigen in trans in a co-culture assay (FIG.4A) and using soluble recombinant GFP. Several peptide antagonists responded to both surface-expressed (FIG.4B-C) and soluble GFP (FIG.5A-B), but only when Sal B and light / furimazine were also present as we intended. Peptide antagonists based off the dynorphin analog called arodyn appeared to be the most promising (FIG.5B). We believe that the longer arodyn peptides have higher affinity for KORD and therefore antagonize better but are also more difficult to be displaced by GFP antigen (FIG. 5B).

[0147] For PAGERs to have programmable antigen-specificity, the antigen-specific nanobody in these PAGERs can be highly modular. In this way, simply swapping the nanobody for another one against a different antigen would easily create a new functional receptor capable of sensing and responding to the new antigen of interest. We attempted to quickly and easily make PAGER- TFs against many different antigens of various types, sizes, and shapes, including growth factors, cytokines, chemokines, receptor tyrosine kinases, other cancer-expressed surface receptors, a viral protein, and a protease, again by simply swapping out the nanobody in PAGER-TF for published nanobodies against each new antigen of interest. In this way, we successfully made PAGER-TFs against GFP, mCherry, VEGF, HGF, TNFĮ, IL-17, IL-23, sIL-6R, CCL2, EGFR, HER2, CD38, PD-L1, Sars-CoV-2 spike protein, and uPA (FIG.6); each PAGER was responsive to its cognate antigen.

[0148] Having successfully developed PAGER-TFs against many different antigens, we wondered if we could create PAGERs that responded to protease activity by inserting a protease cleavage site between the nanobody and the GPCR; we refer to these receptors as Protease- PAGERs or Protease-PAGER-TFs (FIG.7A). This way, in the absence of protease activity, Protease-PAGER-TF would be inhibited by the fused peptide antagonist; in the presence of protease activity, however, the protease would cut its cleavage site in the PAGER receptors resulting in release of the peptide antagonist and rendering the GPCR once again activatable by the orthogonal drug (FIG.7B). We first used TEV protease as a model protease to test. GFP(LaG2)-PAGER-TF (without TEVcs) was responsive to GFP but non-responsive to TEV protease (FIG. 7C). Including a TEVcs site into this PAGER though (making GFP(LaG2) / TEV-PAGER-TF), made the PAGER not only responsive to GFP but also TEV protease (FIG.7D). We further showed that a Thrombin-PAGER-TF could be made in the same way by including a thrombin cleavage site into the linker between the nanobody and the GPCR (FIG.7E). Thus, PAGERs can be easily reprogrammed to be responsive to different protease simply by changing the protease cleavage site that is included in the receptor.

[0149] Next, to expand on the limited repertoire of endogenous signaling outputs available for existing synthetic receptors, we sought to use the native function of GPCRs to couple antigen recognition to heterotrimeric G protein activation using PAGER (referred to as G protein- PAGERs; FIG. 8A). In order to develop PAGERs that couple to each of the four major G protein classes (GĮi, GĮs, GĮq, and GĮ12), we needed to make new PAGERs using different GPCRs that natively activate each of the G protein classes. For this, we took advantage of the muscarinic DREADDs that had already been developed to be activated by an orthogonal drug and couple to each of the major G protein classes. We screened muscarinic toxins in GFP(LaG2) / TEV-G protein-PAGERs to identify antagonists that would create functional PAGERs for each G protein class. Screening was done using the TRUPATH BRET assay (Olsen et al., Nature Chemical Biology, 16, pages 841–849, 2020, DOI: 10.1007 / 978-1-0716-2473-9_13) to measure PAGER- driven G protein activation. Final GFP(LaG2) / TEV-G protein-PAGERs (FIG. 8B) could activate each of their respective heterotrimeric G protein effectors in response to TEV protease activity (FIG. 9A). In addition, mCherry(LaM6)-G protein-PAGERs could activate each of their respective G protein effectors in response to mCherry (FIG.9B).

[0150] Heterotrimeric G protein activation results in downstream cell signaling events including changes in concentration of second messengers; GĮi inhibits cAMP levels, GĮs stimulates cAMP levels, GĮq stimulates DAG production and increases in intracellular calcium, and GĮ12 activates RhoA (FIG.10A). Thus, G protein-PAGERs also drive antigen-dependent control of these downstream intracellular signaling events. GFP(LaG2)-Gi-PAGER reduces cAMP levels in response to GFP (FIG.10B, left), while GFP(LaG2)-Gs-PAGER increases cAMP levels in response to GFP (FIG.10B, right). mCherry(LaM6)-Gq-PAGER stimulates DAG production in response to GFP (FIG. 10C), while mCherry(LaM6)-Gq-PAGER increases intracellular calcium levels in response to mCherry (FIG. 10C).

[0151] PAGER presents an exciting prospect for overcoming the limitations of existing synthetic receptor platforms, potentially leading to a new wave of breakthroughs in therapeutics and fundamental research. PAGER is highly modular modularity, can respond to surface and soluble antigens, can drive antigen-dependent transgene expression or antigen-dependent G protein activation, and is chemogenetically controlled by an orthogonal drug. These advantageous features of PAGER set the stage for a myriad of potential applications in cell- based therapies and beyond. Methods and Materials

[0152] Plasmid constructs and cloning. Constructs used for transient expression in HEK293T cells were cloned into the pAAV viral vector. For stable expression, the constructs were cloned into the pCDH viral vector. For all constructs, standard cloning procedures were used. PCR fragments were amplified using Q5 polymerase (NEB). Vectors were digested with NEB restriction enzymes and ligated to gel-purified PCR products using T4 ligation, Gibson, NEB HiFi, or Golden Gate assembly. Ligated plasmids were introduced into competent XL1-Blue, NEB5-alpha, or NEB Stable bacteria via heat shock transformation.

[0153] HEK293T cell culture. HEK293T cells were obtained from ATCC (tested negative for mycoplasma) and cultured as monolayers in complete growth media: Dulbecco’s Modified Eagle Medium (DMEM, Corning) containing 4.5 g / L glucose and supplemented with 10% Fetal Bovine Serum (FBS, VWR), 1% (v / v) GlutaMAX (Gibco), and 1% (v / v) Penicillin-Streptomycin (Corning, 5000 units / mL of penicillin and 5000 ^g / mL streptomycin), at 37°C under 5% CO2. For experimental assays, cells were grown in 6-well, 12-well, 24-well, or 96-well plates pretreated with 20 μg / mL human fibronectin (Millipore) for at least 10 min at 37°C.

[0154] HEK293T cell transient transfection. A 1 mg / mL solution of PEI Max (Polysciences, catalog no.24765) was prepared for transient transfection as follows. Polyethylenimine (PEI, 500 mg) was added to 450 mL of Milli-Q H20 in a 500 mL glass beaker while stirring with a stir bar. Concentrated HCL was added dropwise to the solution until the pH was less than 2.0. The PEI solution was stirred until PEI was dissolved (~2-3 hours). Concentrated NaOH was then added dropwise to the solution until the pH was 7.0. The volume of the solution was thenadjusted to 500mL, filter-sterilized through a 0.22-^m membrane, and frozen in aliquots at - 20°C. Working stocks were kept at 4°C for no more than 1 month.

[0155] For transient transfection, HEK293T cells were grown in 6-well, 12-well, or 24-well plates pretreated with 20 μg / mL human fibronectin (Millipore) for at least 10 min at 37°C. Cells were grown to a confluency of ~70-90% prior to transfection. DNA transfection complexes were made by mixing DNA and 1 mg / mL PEI solution in serum-free DMEM at a 1 ^g DNA: 5 ^L PEI (1 mg / mL): 100 ^L serum-free DMEM. Complexes were allowed to form for 20 min at room temperature. After 20 min, complexes were diluted in complete DMEM up to the growth volume per well size (2.5 mL for 6-well, 1 mL for 12-well, and 500 ^L for 24-well). The entire well volume of the HEK293T cells was replaced with the diluted complexes and allowed to transfect cells at 37°C for 5-24 hours. Complete transfection protocols including amounts of DNA and length of transfection are described for each experiment below.

[0156] Firefly Luciferase Reporter PAGER-SPARK experiments. HEK293T cells were plated in human fibronectin-coated 6-well dishes at a density of 750,000 cells per well and allowed to grow overnight (~18 hours) at 37°C until they reached ~70-90% confluency. After ~18 hours, the cells were transfected with 350 ng of the indicated Antagonist-Nanobody-GPCR-eLOV- TEVcs-Gal4 (PAGER-TF) receptor plasmid, 100 ng of NanoLuc-ȕarrestin2-TEVp plasmid, and 150 ng of UAS-Firefly Luciferase (FLuc) plasmid. Cells were transfected for 5 hours at 37°C. After 5 hours of transfection, cells from each well were lifted and resuspended in 6 mL of complete DMEM to make an ~400,000 cells per mL single cell suspension, and 100 ^L of cell suspension (~40,000 cells) was plated per well in a human fibronectin-coated white, clear bottom 96-well plate in triplicate. Plates were wrapped in aluminum foil to protect them from light and incubated at 37°C overnight (~18 hours). After ~18 hours, cells should be stimulated.

[0157] Stimulation should take place in a dark room under red light (red light does not open LOV domain). Stimulation solutions were optimized for each given antigen and PAGER receptor. Unless otherwise indicated, PAGERs were stimulated as follows: GFP(LaG17 / LaG2 / LaG16)-PAGERs were stimulated with 1 ^M GFP, 1 ^M salvinorin B, and 1x furimazine; mCherry(LaM6)-PAGERs were stimulated with 1 ^M mCherry, 1 ^M salvinorin B, and 1x furimazine; VEGF(Nb35)-PAGERs were stimulated with 500 nM VEGF, 500 nM salvinorin B, and 1x furimazine; HGF(Nb1E2)-PAGERs were stimulated with 250 nM HGF, 250nM salvinorin B, and 1x furimazine; TNFĮ(Ozoralizumab)-PAGERs were stimulated with 500 nM TNFĮ, 250 nM or 500 nM salvinorin B, and 1x furimazine; IL17(Sonelokimab)-PAGER was stimulated with 500nM IL-17, 100 nM salvinorin B, and 1x furimazine, IL23(Nb22E11)-PAGER was stimulated with 250 nM IL-23, 500 nM salvinorin B, and 1x furimazine; sL6R(Voberilizumab)-PAGER was stimulated with 500 nM sIL-6R, 500 nM salvinorin B, and 1x furimazine; CCL2(Nb8E10)-PAGER was stimulated with 1 ^M CCL2, 500 nM salvinorin B, and 1x furimazine; EGFR(NbEgB4)-PAGERs were stimulated with 500 nM EGFR ECD, 100nM or 250 nM or 500 nM salvinorin B, and 1x furimazine; HER2(Nb2Rs15d)-PAGERs were stimulated with 500 nM HER2 ECD, 500 nM or 1 ^M salvinorin B, and 1x furimazine; CD38(NbMU375)-PAGER was stimulated with 1 ^M CD38 ECD, 500 nM salvinorin B, and 1x furimazine; PDL1(KN035)-PAGER was stimulated with 1 ^M PD-L1 ECD, 500 nM salvinorin B, and 1x furimazine; SarsCoV2-RBD(NbF2)-PAGER was stimulated with 200 nM Sars-CoV-2 Spike protein, 500 nM or 1 ^M salvinorin B, and 1x furimazine; uPA(Nb4)-PAGER was stimulated with 500 nM uPA, 250 nM or 500 nM salvinorin B, and 1x furimazine.

[0158] For stimulations, growth media was removed from the 96-well plate by flicking off and dabbing excess on paper towel. To initiate stimulation, 100 ^L stimulation solution was added to each well for a total of 15 min. After 15min, stimulation solution was removed by flicking off and dabbing excess on a paper towel, and 100uL of complete DMEM was added back to each well. Plates were again wrapped in aluminum foil and placed in 37°C incubator for 8 hours. After 8 hours post-stimulation, media was removed from 96-well plate by flicking off and dabbing excess on paper towel. Wells were washed once with 125 ^L DPBS, and then 50 uL of 1x Bright-Glo (2x diluted 1:1 in DPBS; Promega) was added to each well and incubated for 1 min. After 1 min, firefly luciferase luminescence was measured using a Tecan Infinite M1000 Pro plate reader using the following parameters: 1000 msec acquisition time, green-1 filter (520- 570 nm), 25°C linear shaking for 10 sec.

[0159] In some experiments where indicated, exogenous ambient room white light was used to uncage the LOV domain instead of furimazine-dependent NanoLuc BRET. In these experiments, furimazine was not included in the stimulation solutions; all else remained the same.

[0160] In some experiments where indicated, salvinorin B or antigen dose response curves were analyzed. In these experiments, the concentrations of salvinorin B or antigen were includedin the stimulation solutions at different concentrations according to those indicated; all else remained the same.

[0161] HEK293T co-culture for trans assays. For trans assays using HEK293T cells, cells were cultured in 6-well and 12-well plates as described above. Receiver cells in 12-well plates were transfected with 140 ng of the indicated pAAV-Antagonist-Nanobody-GPCR-eLOV-TEVcs- Gal4 receptor plasmid, 40 ng of pAAV-NanoLuc-ȕarrestin2-TEVp plasmid, and 60 ng of pAAV- UAS-Firefly Luciferase (FLuc) plasmid. Sender cells in 6-well plates were transfected with 2 ^g of pAAV-GFP-PDGFR transmembrane domain (surface-expressed GFP). Cells were transfected for 5 hours in a 37°C incubator. After 5 hours, cells were lifted with trypsin, washed with DPBS, and resuspended in 6.25 mL or 2.5 mL of complete DMEM per well for 6-well and 12-well plates, respectively. Sender and receiver cells were mixed at a 4:1 sender:receiver ratio and then 100uL of cell mixtures were plated into 96-well white, clear-bottom microplates at a density of 40,000 cells / well. Plates were wrapped in aluminum foil and incubated for ~18 hr in a 37°C incubator, then the stimulations and luciferase reporter assay were performed as described above.

[0162] TRUPATH G protein activation BRET assay. HEK293T cells were plated in human fibronectin-coated 6-well dishes at a density of 1,250,000 cells per well and allowed to adhere and grow for 2-4 hours at 37°C. After ~2-4 hours, the cells were transfected with 250 ng of the indicated G protein-PAGER receptor plasmid, 250 ng of the corresponding GĮ-RLuc8 TRUPATH plasmid (GĮi1-RLuc8, GĮsS-RLuc8, GĮq-RLuc8, or GĮ12-RLuc8), 250 ng of Gȕ3 TRUPATH plasmid, and 250 ng GȖ9-GFP2 TRUPATH plasmid. Cells were incubated at 37° and transfection was allowed to proceed for ~20-24 hours. After transfection, cells from each well were lifted and resuspended in 6 mL of complete DMEM to make an ~400,000 cells per mL single cell suspension, and 100 ^L of cell suspension (~40,000 cells) was plated per well in a human fibronectin-coated white, clear bottom 96-well plate in triplicate. Plates were incubated at 37°C for ~20-24 hours. For protease activation of G protein-PAGERs, cells were treated with 1^M TEV protease for 90 min followed by stimulation with various concentrations of CNO and 20^M CTZ400a (substrate for TRUPATH assay) for 5 min before reading out BRET. For antigen activation of G protein-PAGERs, cells were treated with 1^M mCherry for 15 min followed by stimulation with various concentrations of CNO and 20^M CTZ400a for 5 min before reading out BRET. BRET was readout using a Tecan Infinite M1000 Pro plate readerusing the following parameters: Filter 1 Magenta (370 to 450 nm), 500 ms integration time; Filter 2 Green (510 to 540 nm), 500 ms integration time; 25°C. Data is presented as NET BRET and displayed as scatter plots with variable slope (four parameter) non-linear regression lines.

[0163] GloSensor cAMP assay. HEK293T cells were plated in human fibronectin-coated 6- well dishes at a density of 1,250,000 cells per well and allowed to adhere and grow for 2-4 hours at 37°C. After ~2-4 hours, the cells were transfected with 250 ng of the indicated GFP(LaG2)-G protein-PAGER receptor plasmid and 625 ng of the Glo-22F cAMP biosensor-encoding pcDNA3 plasmid. Cells were incubated at 37° and transfection was allowed to proceed for ~20- 24 hours. After transfection, cells from each well were lifted and resuspended in 6 mL of complete DMEM to make an ~400,000 cells per mL single cell suspension, and 100 ^L of cell suspension (~40,000 cells) was plated per well in a human fibronectin-coated white, clear bottom 96-well plate in triplicate. Plates were incubated at 37°C for ~20-24 hours. Prior to stimulation, cells were washed once with 125 ^L HBSS containing 20 mM HEPES pH 7.4 and then treated with 100 ^L of 5 mM D-luciferin in HBSS containing 20 mM HEPES pH 7.4 for ~3 hours in the dark at room temperature. For measuring GĮi activity, the D-luciferin was removed and then the cells were treated with 50 ^L of 5 mM D-luciferin, 1^M GFP, and various concentrations of CNO in the dark at room temperature for 15 min followed by addition of 50 ^L of 200nM isopreteronol (final concentration 100nM) for 30 min before reading out luminescence. For measuring GĮs activity, the D-luciferin was removed and then the cells were treated with 50 ^L of 5 mM D-luciferin and 1^M GFP for 15 min in the dark at room temperature, followed by stimulation with various concentrations of CNO in the dark at room temperature for 2 min before reading out luminescence. Luminescence was measured using a Tecan Infinite M1000 Pro plate reader using the following parameters: 1000 msec acquisition time, green-1 filter (520-570 nm), 25°C linear shaking for 10 sec. Data is displayed as scatter plots with variable slope (four parameter) non-linear regression lines.

[0164] Lentivirus generation and stable cell line generation. To generate lentivirus, HEK293T cells were cultured in T25 flasks and transfected at ~70% confluency with 2.5 ^g of the pCDH lentiviral transfer vector of interest and packaging plasmids psPAX2 (1.25 ^g) and pMD2.g (1.25 ^g) with 25 μL of polyethyleneimine (PEI, 1 mg / mL; Polysciences). Approximately 72 hours post-transfection, the cell medium was collected and centrifuged for 5 min at 300 x g toremove cell debris. Media containing lentivirus was used immediately for transduction or was aliquoted into 0.5 mL aliquots, flash-frozen in liquid nitrogen, and stored at –80°C for later use. Frozen viral aliquots were thawed at 37°C prior to infection.

[0165] HEK293T cells were plated on 6-well human fibronectin-coated plates. When cells reached ~70-90% confluency, cells were transduced with lentivirus for 1-3 days. The cells were then lifted and replated om a T25 flask, and stably expressing cells were selected for in complete DMEM containing 1^g / mL puromycin for at least 1 week. Cells were split and expanded when they reached ~80-90% confluency. Cells were maintained under this puromycin selection until the time of experiments. Construct expression was confirmed by flow cytometry, immunofluorescence imaging, or functional characterization.

[0166] Fluorescence imaging. Confocal imaging was performed on a Zeiss AxioObserver inverted confocal microscope with 10x and 20x air objectives, and 40x and 63x oil-immersion objectives, outfitted with a Yokogawa spinning disk confocal head, a Quad-band notch dichroic mirror (405 / 488 / 568 / 647), and 405 (diode), 491 (DPSS), 561 (DPSS) and 640 nm (diode) lasers (all 50 mW). The following combinations of laser excitation and emission filters were used for various fluorophores: GFP (491 laser excitation; 528 / 38 emission), mCherry / Alexa Fluor 568 (561 laser excitation; 617 / 73 emission), Alexa Fluor 647 (647 excitation; 680 / 30 emission), and differential interference contrast (DIC). Acquisition times ranged from 100 to 500 ms. All images were collected and processed using SlideBook (Intelligent Imaging Innovations).

[0167] Fluorescent DAG assay. An mCherry-based fluorescent DAG biosensor was made by C-terminally tagging mCherry to the C1(PKCȖ) from Addgene plasmid #21205 (Oancea et al., J Cell Biol, 140 (3): 485–498, 1998, DOI: 10.1083 / jcb.140.3.485) and cloning into the pCDH lentivirus backbone. HEK 293T cells stably co-expressing GFP(LaG16)-Gq-PAGER and C1(PKCȖ)-mCherry were incubated in 1:1000 anti-ALFA–AlexaFluor647 and 1 ^M EGFP for 3 min. Cells were then located under the microscope and time-lapse images were obtained every 4 s, and 1 mL of 200 nM CNO was added (to a final concentration of 133 nM) between the first and the second frame. Images at the first time frame (t=0) and 15th time frame (t=60s) were used for analysis. Images were analyzed using ImageJ software. Region of interests (ROIs) were manually added to images and the mean of cytosolic mCherry fluorescence in each cell wasquantified. The difference in mean fluorescence compared to the initial fluorescence (ǻF / F0) was calculated and used for statistical comparison.

[0168] GCaMP6s calcium assay. HEK 293T cells stably co-expressing mCherry(LaM6)-Gq- PAGER and GCaMP6s were incubated in 1:1000 anti-ALFA–AlexaFluor647 and 2 ^M mCherry for 3 min. Cells were then located under the microscope and time-lapse images were obtained every 4 s, and 1 mL of 200 nM CNO was added (to a final concentration of 133 nM) between the first and the second frame. Images at the first time frame (t=0) and 15th time frame (t=60s) were used for analysis. Images were analyzed using ImageJ software. Region of interests (ROIs) were manually added to images and the mean of cytosolic mCherry fluorescence in each cell was quantified. The difference in mean fluorescence compared to the initial fluorescence (ǻF / F0) was calculated and used for statistical comparison.

[0169] Quantification and Data Analysis. All graphs were created using GraphPad Prism 9.

[0170] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.Informal Sequence Listing SEQ^ID^NO:1:^^sp|P04517|POLG_TEV^Genome^polyprotein^OS=Tobacco^etch^virus^OX=12227^ PE=1^SV=1.^ MALIFGTVNANILKEVFGGARMACVTSAHMAGANGSILKKAEETSRAIMHKPVIFGEDYI^ TEADLPYTPLHLEVDAEMERMYYLGRRALTHGKRRKVSVNNKRNRRRKVAKTYVGRDSIV^ EKIVVPHTERKVDTTAAVEDICNEATTQLVHNSMPKRKKQKNFLPATSLSNVYAQTWSIV^ RKRHMQVEIISKKSVRARVKRFEGSVQLFASVRHMYGERKRVDLRIDNWQQETLLDLAKR^ FKNERVDQSKLTFGSSGLVLRQGSYGPAHWYRHGMFIVRGRSDGMLVDARAKVTFAVCHS^ MTHYSDKSISEAFFIPYSKKFLELRPDGISHECTRGVSVERCGEVAAILTQALSPCGKIT^ CKRCMVETPDIVEGESGESVTNQGKLLAMLKEQYPDFPMAEKLLTRFLQQKSLVNTNLTA^ CVSVKQLIGDRKQAPFTHVLAVSEILFKGNKLTGADLEEASTHMLEIARFLNNRTENMRI^ GHLGSFRNKISSKAHVNNALMCDNQLDQNGNFIWGLRGAHAKRFLKGFFTEIDPNEGYDK^ YVIRKHIRGSRKLAIGNLIMSTDFQTLRQQIQGETIERKEIGNHCISMRNGNYVYPCCCV^ TLEDGKAQYSDLKHPTKRHLVIGNSGDSKYLDLPVLNEEKMYIANEGYCYMNIFFALLVN^ VKEEDAKDFTKFIRDTIVPKLGAWPTMQDVATACYLLSILYPDVLRAELPRILVDHDNKT^ MHVLDSYGSRTTGYHMLKMNTTSQLIEFVHSGLESEMKTYNVGGMNRDVVTQGAIEMLIK^ SIYKPHLMKQLLEEEPYIIVLAIVSPSILIAMYNSGTFEQALQMWLPNTMRLANLAAILS^ ALAQKLTLADLFVQQRNLINEYAQVILDNLIDGVRVNHSLSLAMEIVTIKLATQEMDMAL^ REGGYAVTSEKVHEMLEKNYVKALKDAWDELTWLEKFSAIRHSRKLLKFGRKPLIMKNTV^ DCGGHIDLSVKSLFKFHLELLKGTISRAVNGGARKVRVAKNAMTKGVFLKIYSMLPDVYK^ FITVSSVLSLLLTFLFQIDCMIRAHREAKVAAQLQKESEWDNIINRTFQYSKLENPIGYR^ STAEERLQSEHPEAFEYYKFCIGKEDLVEQAKQPEIAYFEKIIAFITLVLMAFDAERSDG^ VFKILNKFKGILSSTEREIIYTQSLDDYVTTFDDNMTINLELNMDELHKTSLPGVTFKQW^ WNNQISRGNVKPHYRTEGHFMEFTRDTAASVASEISHSPARDFLVRGAVGSGKSTGLPYH^ LSKRGRVLMLEPTRPLTDNMHKQLRSEPFNCFPTLRMRGKSTFGSSPITVMTSGFALHHF^ ARNIAEVKTYDFVIIDECHVNDASAIAFRNLLFEHEFEGKVLKVSATPPGREVEFTTQFP^ VKLKIEEALSFQEFVSLQGTGANADVISCGDNILVYVASYNDVDSLGKLLVQKGYKVSKI^ DGRTMKSGGTEIITEGTSVKKHFIVATNIIENGVTIDIDVVVDFGTKVVPVLDVDNRAVQ^ YNKTVVSYGERIQKLGRVGRHKEGVALRIGQTNKTLVEIPEMVATEAAFLCFMYNLPVTT^ QSVSTTLLENATLLQARTMAQFELSYFYTINFVRFDGSMHPVIHDKLKRFKLHTCETFLN^ KLAIPNKGLSSWLTSGEYKRLGYIAEDAGIRIPFVCKEIPDSLHEEIWHIVVAHKGDSGI^ GRLTSVQAAKVVYTLQTDVHSIARTLACINRRIADEQMKQSHFEAATGRAFSFTNYSIQS^ IFDTLKANYATKHTKENIAVLQQAKDQLLEFSNLAKDQDVTGIIQDFNHLETIYLQSDSE^ VAKHLKLKSHWNKSQITRDIIIALSVLIGGGWMLATYFKDKFNEPVYFQGKKNQKHKLKM^ REARGARGQYEVAAEPEALEHYFGSAYNNKGKRKGTTRGMGAKSRKFINMYGFDPTDFSY^ IRFVDPLTGHTIDESTNAPIDLVQHEFGKVRTRMLIDDEIEPQSLSTHTTIHAYLVNSGT^ KKVLKVDLTPHSSLRASEKSTAIMGFPERENELRQTGMAVPVAYDQLPPKNEDLTFEGES^ LFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGV^ FKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSS^ MVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVP^ KNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVFMSKPEEPFQPVKEATQLMNELVYSQG^ EKRKWVVEALSGNLRPVAECPSQLVTKHVVKGKCPLFELYLQLNPEKEAYFKPMMGAYKP^LNMKAAMGALYKGKKKEALSELTLDEQEAMLKASCLRLYTGKLGIWNGSLKAELRPIEKV^ ENNKTRTFTAAPIDTLLAGKVCVDDFNNQFYDLNIKAPWTVGMTKFYQGWNELMEALPSG^ WVYCDADGSQFDSSLTPFLINAVLKVRLAFMEEWDIGEQMLRNLYTEIVYTPILTPDGTI^ IKKHKGNNSGQPSTVVDNTLMVIIAMLYTCEKCGINKEEIVYYVNGDDLLIAIHPDKAER^ LSRFKESFGELGLKYEFDCTTRDKTQLWFMSHRALERDGMYIPKLEEERIVSILEWDRSK^ EPSHRLEAICASMIEAWGYDKLVEEIRNFYAWVLEQAPYSQLAEEGKAPYLAETALKFLY^ TSQHGTNSEIEEYLKVLYDYDIPTTENLYFQSGTVDAGADAGKKKDQKDDKVAEQASKDR^ DVNAGTSGTFSVPRINAMATKLQYPRMRGEVVVNLNHLLGYKPQQIDLSNARATHEQFAA^ WHQAVMTAYGVNEEQMKILLNGFMVWCIENGTSPNLNGTWVMMDGEDQVSYPLKPMVENA^ QPTLRQIMTHFSDLAEAYIEMRNRERPYMPRYGLQRNITDMSLSRYAFDFYELTSKTPVR^ AREAHMQMKAAAVRNSGTRLFGLDGNVGTAEEDTERHTAHDVNRNMHTLLGVRQ^ ^ SEQ^ID^NO:2:^^Amino^acid^sequence^of^the^truncated^TEV:^ GESLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTLQQH LIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKHWIQTKD GQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWGGHKVF MV^

Claims

WHAT IS CLAIMED IS:

1. A synthetic receptor fusion protein comprising, from amino terminus to carboxy terminus, formula (I): [input module] – [G-protein coupled receptor (GPCR)] (I), wherein the input module comprises: (i) a polypeptide inhibitor of the GPCR; and optionally (ii) an antigen binding molecule (ABM), and optionally (iii) an amino acid linker between the input module and the GPCR.

2. The synthetic receptor fusion protein of claim 1, wherein the synthetic receptor comprises, in an amino terminus to carboxy terminus direction, a compound of formula (Ia) [polypeptide inhibitor of a GPCR] – [ABM] – [amino acid linker] – [GPCR] (Ia); a compound of formula (Ib) [ABM] – [polypeptide inhibitor of a GPCR] – [amino acid linker] – [GPCR] (Ib); a compound of formula (Ic): [polypeptide inhibitor of a GPCR] – [ABM] – [GPCR] (Ic); or a compound of formula (Id) [ABM] – [polypeptide inhibitor of a GPCR] – [GPCR] (Id).

3. The synthetic receptor fusion protein of claim 1 or 2, further comprising an output module, wherein the synthetic receptor fusion protein comprises formula (II): [input module] – [G-protein coupled receptor (GPCR)] – [output module] (II).

4. The synthetic receptor fusion protein of claim 3, wherein the synthetic receptor comprises, in an amino terminus to carboxy terminus direction, a compound of formula (IIa) [polypeptide inhibitor of a GPCR] – [ABM] – [amino acid linker] – [GPCR] – [output module] (IIa); a compound of formula (IIb):[ABM] – [polypeptide inhibitor of a GPCR] – [amino acid linker] – [GPCR] – [output module] (IIb); a compound of formula (IIc): [polypeptide inhibitor of a GPCR] – [ABM] – [GPCR] – [output module] (IIc); or a compound of formula (Id): [ABM] – [polypeptide inhibitor of a GPCR] – [GPCR] – [output module] (IId).

5. The synthetic receptor fusion protein of any one of claims 1 to 4, wherein the polypeptide inhibitor binds to the GPCR in the absence of the antigen and does not bind the GPCR in the presence of the antigen.

6. The synthetic receptor fusion protein of any one of claims 1-5, wherein the polypeptide inhibitor is an orthosteric binding peptide.

7. The synthetic receptor fusion protein of claim 6, wherein the orthosteric binding peptide is an antagonist or inverse agonist.

8. The synthetic receptor fusion protein of any one of claims 1 to 7, wherein the ABM is selected from the group consisting of a nanobody, scFv, Fab, VH, monobody, a designed ankyrin repeat protein (DARPin), and a miniprotein.

9. The synthetic receptor fusion protein of claim 8, wherein the ABM binds an antigen expressed on the surface of a cell, an antigen bound to a solid support, or a soluble antigen.

10. The synthetic receptor fusion protein of claims 1 to 9, wherein the amino acid linker comprises a protease cleavage site.

11. The synthetic receptor fusion protein of claim 3 or 4, where the output module comprises a polypeptide comprising a) a light-oxygen-voltage-sensing (LOV) domain, b) a tobacco etch virus protease recognition sequence (TEVcs), and c) a transcription factor (TF), and the polypeptide is covalently linked to the carboxy terminus of the GPCR.

12. The synthetic receptor fusion protein of claim 11, wherein antigen binding to the ABM results in recruitment of an arrestin-TEV protease fusion protein to the GPCR and proteolytic cleavage of the TEVcs and release of the TF.

13. The synthetic receptor fusion protein of any one of claims 1 to 12, wherein the GPCR is selected from a GPCR in Table 7.

14. A nucleic acid encoding a synthetic receptor fusion protein of any one of claims 1 to 13.

15. A vector comprising the nucleic acid of claim 14.

16. A cell comprising (i) the synthetic receptor fusion protein of any one of claims 1 to 13, (ii) the nucleic acid of claim 14 and / or (iii) the vector of claim 15.

17. The cell of claim 16, wherein the cell is a eukaryotic cell.

18. The cell of claim 17, wherein the cell is a human cell.

19. The cell of claim 18, wherein the cell is a primary cell.

20. A cell comprising (i) the synthetic receptor fusion protein of any one of claims 1 or 5 to 13, or a nucleic acid encoding (i), wherein the GPCR binds to a G protein endogenous to the cell in the presence of antigen.

21. The cell of claim 20, wherein the endogenous G protein is selected from the group consisting of Gi, Gs, Gq / 11, and G12 / 13.

22. A method of activating transcription of a target gene in a cell, the method comprising: (i) providing a cell expressing a synthetic receptor fusion protein of claim 11 on the cell surface, wherein the cell comprises an arrestin-TEV protease (TEVp) fusion protein; (ii) contacting the cell with an antigen that binds the ABM; (iii) contacting the cell with blue light, resulting in proteolytic cleavage of the TEVcs and release of the TF, thereby activating transcription of the target gene.

23. The method of claim 22, wherein the blue light is exogenous to the cell.

24. The method of claim 22, wherein the blue light is produced intracellularly by a heterologous bioluminescent protein.

25. The method of claim 22, wherein the arrestin-TEVp fusion protein further comprises a bioluminescent protein that emits blue light.

26. The method of claims 24 or 25, wherein the bioluminescent protein comprises a luciferase or nanoluciferase.

27. The method of any one of claims 22 to 26, wherein the target gene is a reporter gene present in the cell.

28. The method of any one of claims 22 to 27, wherein the cell is transfected or transduced with a nucleic acid encoding the synthetic receptor fusion protein, a nucleic acid encoding the arrestin-TEVp fusion protein, and a nucleic acid comprising the target gene operably linked to a promoter that binds the TF.

29. The method of claim 28, wherein the cell is transfected or transduced with one or more of the nucleic acids in vitro, ex vivo, or in vivo.

30. A method for activating a G protein in a target cell, the method comprising: (i) providing a target cell expressing a synthetic receptor fusion protein of any one of claims 1 or 2 on the cell surface; (ii) contacting the target cell with an antigen that binds the ABM; wherein antigen binding to the ABM results in binding of the GPCR to the G protein, thereby activating the G protein.

31. The method of claim 30, wherein the G protein is selected from the group consisting of Gi, Gs, Gq / 11, and G12 / 13.

32. The method of claims 30 or 31, wherein before step (i), the target cell is transfected or transduced with a nucleic acid encoding the synthetic receptor fusion protein.

33. The method of claim 32, wherein before step (i), the target cell is transfected or transduced with the nucleic acid in vitro, ex vivo, or in vivo.

34. A method of using the synthetic receptor fusion protein of any one of claims 1 to 13, comprising providing a cell expressing the synthetic receptor fusion protein, wherein the synthetic receptor fusion protein is activated by either (i) binding of antigen to the ABM, or (ii) proteolytic cleavage of an amino acid linker sequence in the input module.

35. The method of claim 34, wherein binding of antigen displaces the peptide inhibitor and activates the GPCR or in the case of (ii), proteolytic cleavage releases a peptide inhibitor bound to the GPCR and activates the synthetic receptor.

36. The method of claim 34, further comprising contacting the synthetic receptor fusion protein with blue light, wherein binding of antigen in the presence of blue light results in proteolytic cleavage of the TEVcs and release of the TF.

37. The method of claim 34, wherein binding of antigen results in activation of an endogenous G protein.

38. The method of claim 37, wherein the G protein is selected from the group consisting of Gi, Gs , Gq / 11, and G12 / 13.

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