Truncated receptors and uses thereof

Abstract

The present invention, in some aspects, is directed to engineered receptors comprising an extracellular ligand binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises a kinase domain or a kinase binding domain and a synthetic phosphorylatable domain

Description

Docket No.: 130492-867918 (2025-033-PCT)TRUNCATED RECEPTORS AND USES THEREOF

[0001] For countries that permit incorporation by reference, all patents, patent applications and publications cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted being prior art.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority from U.S. provisional application 63 / 726,852. filed December 2, 2024, the contents of w hich are incorporated by reference in their entirety.GOVERNMENT INTERESTS

[0004] This invention was made with government support under Grant No. N00014-21-4- 4006 awarded by the Office of Naval Research and Grant No. EB029483 aw arded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The application contains a Sequence Listing which has been submitted electronically in .xml format and is hereby incorporated by reference in its entirety. Said .xml copy, created on December 2, 2025, is named 'T30492-867918 (2025-033) Sequence Listing.xml” and is 83,592 bytes in size.FIELD OF THE INVENTION

[0006] This invention is directed to engineered receptor comprising an extracellular ligand binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises a kinase domain or a kinase binding domain and a synthetic phosphorylatable domain.- 1 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)BACKGROUND OF THE INVENTION

[0007] Mammalian cells rely on membrane receptors such as receptor tyrosine kinases (RTKs) and Janus kinase (JAK)-dependent receptors to sense extracellular cues and convert them into intracellular phosphory lation signals that control diverse cellular processes. Although these native receptor systems provide potent and specific responses, they are intrinsically wired into endogenous pathways, which can limit the ability to precisely redirect signaling to synthetic outputs or avoid undesired downstream programs. Synthetic receptor platforms have begun to address this by exploiting the modularity of receptor domains to rewire ligand recognition or downstream signaling, but many existing systems still depend on native transcriptional programs or are constrained to particular ligand-receptor pairs. Thus, there remains a need for engineered receptors that preserve the natural diversity and specificity of RTK and JAK receptor ectodomains, while decoupling and replacing their downstream effector functions. Provided herein are methods and compositions that address such and other needs.SUMMARY OF THE INVENTION

[0008] In some aspects, provided herein is an engineered receptor, wherein the engineered receptor comprises a native extracellular ligand binding domain and a truncated intracellular signaling domain, wherein the truncated intracellular signaling domain comprises a native kinase domain or a native kinase binding domain and a synthetic phosphor latable domain.

[0009] In some aspects, the engineered receptor comprises an engineered Receptor Tyrosine Kinase (RTK) or an engineered Janus Kinase (JAK)-dependent receptor. In some aspects, the native extracellular ligand binding domain comprises an amino acid sequence having at least about 85% sequence identity7to an extracellular ligand binding domain of an RTK receptor amino acid sequence or a JAK receptor amino acid sequence.

[0010] In some aspects, the native extracellular ligand binding domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:10-16 or SEQ ID NOs:52-56.

[0011] In some aspects, the native kinase domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:23-31.- 2 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)

[0012] In some aspects, the synthetic phosphorylatable domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:32-37.

[0013] In some aspects, the engineered receptor further comprises a transmembrane domain. In some aspects, the transmembrane domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 17-22.

[0014] In some aspects, the engineered receptor further comprises a signal peptide. In some aspects, the signal peptide comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-7 or SEQ ID NOs:47-51.

[0015] In some aspects, wherein the engineered receptor further comprises an epitope tag. In some aspects, the epitope tag comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 9.

[0016] In some aspects, the engineered receptor comprises a) a native extracellular ligand binding domain comprising an amino acid sequence having at least about 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 10-16; b) a transmembrane domain comprising an amino acid sequence having at least about 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 17-22; and c) a native kinase domain comprising an amino acid sequence having at least about 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:23-31.

[0017] In some aspects, the engineered receptor comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:38-46 or SEQ ID NOs:60-64.

[0018] In some aspects, the engineered receptor is a homodimer, a heterodimer, or a heteromultimer.

[0019] In some aspects, the receptor comprises a first receptor chain and a second receptor chain.

[0020] In some aspects, the first receptor chain comprises an amino acid sequence having at least about 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 61 and the second receptor chain comprises an amino acid sequence having at least about 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 62.- 3 -107176621.5DocketNo.: 130492-867918 (2025-033-PCT)

[0021] In some aspects, the first receptor chain comprises an amino acid sequence having at least about 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 63 and the second receptor chain comprises an amino acid sequence having at least about 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 64.

[0022] In some aspects, the extracellular ligand binding domain is specific for a cytokine or, a hormone, or a growth factor. In some aspects, the hormone is erythropoietin, or wherein the cytokine is TNF alpha, IFN-y, or IL-6. In some aspects, the engineered receptor comprises EpoRl-375. In some aspects, the grow th factor is VEGF.

[0023] In some aspects, the synthetic phosphorylatable domain comprises an immune tyrosine activation motif (IT AM) domain or a functional fragment thereof. In some aspects, the IT AM domain is from IgA or CD3Z.

[0024] In other aspects, provided herein is a nucleic acid encoding the engineered receptor described herein.

[0025] In other aspects, provided herein is a vector comprising the nucleic acid described herein.

[0026] In other aspects, provided herein is a host cell comprising the vector described herein.

[0027] In other aspects, provided herein is a nucleic acid encoding an engineered receptor, wherein the engineered receptor comprises a native extracellular ligand binding domain and a truncated intracellular signaling domain, wherein the truncated intracellular signaling domain comprises a native kinase domain or a native kinase binding domain and a synthetic phosphorylatable domain.

[0028] In some aspects, the engineered receptor comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: l-37.

[0029] In some aspects, the engineered receptor comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:38-46 or SEQ ID NOs:60-64.

[0030] In other aspects, provided herein is a vector comprising the nucleic acid described herein.

[0031] In other aspects, provided herein is a host cell comprising the vector described herein.- 4 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)

[0032] In other aspects, provided herein is a genetically engineered cell expressing and bearing on its cell surface the engineered receptor described herein.

[0033] In some aspects, the cell is a mesenchymal stem cell (MSC), a T cell, an ARPE cell, a macrophage, a neutrophil, a glial cell, an NKT, an NK cell, a Treg, or a B cell. In other aspects, the cell is a stem cell, an induced pluripotent stem cell (IPSC), an embry onic stem cell, or a derivate thereof.

[0034] In other aspects, provided herein is a synthetic signaling circuit, wherein the synthetic signaling circuit integrates the engineered receptor described herein into a cellular signaling pathway.

[0035] In some aspects, the cellular signaling pathway comprises a native signaling pathway or a synthetic signaling pathway.

[0036] In some aspects, the cellular signaling pathway comprises a secretory pathway, a translocation pathway or a degradation pathway.

[0037] In other aspects, provided herein is a method of engineering a synthetic receptor, the method comprising: a) providing a nucleic acid encoding a native receptor comprising an extracellular ligand-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a native kinase domain or a native kinase-binding domain; b) truncating the intracellular signaling domain at a C terminal position sufficient to ablate native downstream signaling while retaining the native kinase domain or the native kinase-binding domain; and c) appending, to the truncated intracellular signaling domain, a synthetic phosphorylatable domain configured to be phosphorylated by the native kinase domain or by a kinase recruited via the native kinase-binding domain, thereby producing the synthetic receptor. In some aspects, the native receptor is a receptor ty rosine kinase (RTK) or a Janus kinase (JAK)-dependent receptor.

[0038] In some aspects, the extracellular ligand-binding domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence of an RTK or a JAK receptor extracellular domain. In some aspects, the native extracellular ligand binding domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 10-16 or SEQ ID NOs:52-56. In some aspects, the native kinase domain comprises an amino acid sequence having at least about 85% sequence identity7to any one of the amino acid sequences set forth in SEQ ID NOs:23-31. In some aspects, the synthetic phosphorylatable domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:32-37, SEQ ID NO:59, and- 5 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)SEQ ID NOs:66-77. In some aspects, the transmembrane domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 17-22.BRIEF DESCRIPTION OF THE FIGURES

[0039] FIG. 1 shows an overview of the approach for engineering receptors with synthetic phosphorylation substrates to connect to circuits.

[0040] FIG. 2 shows validation of truncated ery thropoietin receptor fused to synthetic phosphorylation substrate.

[0041] FIG. 3 shows passive recruitment of synSub is supported with this truncated EpoR framework.

[0042] FIG. 4 shows active recruitment w orks with this configuration and enables communication with our circuit.

[0043] FIG. 5 shows expression of EpoR in primary MSCs.

[0044] FIG. 6 show s the direct fusion of this receptor enables a “sense-and-secrete’’ application.

[0045] FIG.7 shows the new receptor designs can be connected to our existing circuit infrastructure or native pathways.

[0046] FIG. 8 shows certain embodiments for synthetic designs and domains.

[0047] FIG. 9 - Expand ligand sensing capabilities with a TNF-a sensor module, (a) The FRB* / FKBP domains from the rapalog-sensing receptor pair were swapped for an scFv specific for TNF-a. (b) Schemetic and behavior for the cytokine sensor circuit. TNF-a binding induced synSub phosphorylation. HHH plots depict flow-cytometry data analyzed 12 h after treatment with 20 ng / mL TNF-a (+lig) or a carrier-only control (-lig). Values in each plot indicate mean phosphorylation (AU) ± SEM (n=3). (c) Dose response for cytokine circuit.

[0048] FIG. 10 - Engineering of Epo receptor by direct fusion of synthetic substrate, (a) Structure of engineered Epo receptor with direct fusion of synthetic CD3Z. (b) Subdomain and truncations for the native Epo receptor, (c) Phosphorylation level of the syntheticc CD3Z while fusing to different truncated EpoR.

[0049] FIG. 11 - Expanding the engineering of EpoR with diverse signal transduction ability, (a) The LZ -fused synSub could also get phosphorylated by the Epo-associated JAK2 through LZ recruitement. (b) Active recruitment configuration, where a SH2-fused CD3Z- 6 -107176621.5DocketNo.: 130492-867918 (2025-033-PCT) will be recruited to the receptor after the receptor-fused IgA gets phosphorylated, histograms showed the phosphorylation level of the CD3Z before and after adding the ligand EpO.

[0050] FIG. 12 - Engineering a phospho-signaling pathway for closed-loop therapeutic control, (a) HEK293T cells expressing a circuit that can sense TNF-a (green arrow) and respond by secreting IL- 10 (blue arrow) (top left) are placed in transwell coculture with activated T cells (bottom left) for 60 h, with media collected every 12 h to measure cytokine levels. T cell proliferation was assessed by EdU assay at 60 h. TNF-a and IL-10 time courses are shown for empty HEK293T cells (no circuit), constitutive IL- 10 expression driven by a non-receptor synKin driving phosphory lation of 2-step cascade (open loop), and the sense- and-respond circuit (closed loop). Each circle represents a different PBMC replicate (black line, mean values; shaded regions. ± SEM) (middle), (b) Quantitating T cell proliferation by EdU assay. CD4+ and CD8+ subsets are replotted as Andy Fluor™ 488 histograms (see Materials and Methods for details). The red gate is used to calculate the percentage of the population that is proliferative. Values indicate the mean ± SEM for n=3 replicates, (c) Endpoint cytokine secretion profdes. At the conclusion of the co-culture time course, concentrations for IL-10. TNF-a, and IFN-y were measured by ELISA for different circuits. Error bars show mean ± SEM for n=3.

[0051] FIG. 13 - Controlling membrane localization of SOAR using an engineered EpoR to gate Orai channel activation and cargo release, (a) Monitering Epo-dependent calcium influx using a pNFAT-based transcriptional reporter, (b) EPO-induced regulated secretion over 3h, a chromogranin A-fused mCherry was used as the reporter of secretion.

[0052] FIG. 14 - Predicted immunogenicity of cytokine control circuit components compared to other commonly used synthetic signaling parts. T Cell Class I pMHC Immunogenicity tool from the Immune Epitope Database (IEDB) was used to evaluate the immunogenicity scores of all 9-mer peptide sequences within the six engineered proteins in the cytokine control circuit, along with three commonly-used synthetic receptors, two proteases and three FDA-approved fully humanized monoclonal antibodies (mAbs). A higher immunogenicity score (hotter color) for a peptide indicates that its composition more closely resembles that of immunogenic peptides, and thus indicates a higher probability of eliciting an immune response. A summary of immunogenicity scores of all 9-mer peptide sequences within all synthetic components from this research and other parts is shown in the tables on the left, with the total number of 9-mer peptides, the percentage of 9-mer scores >0, and the percentage of 9-mer scores >0.328 calculated. TVMVp, tobacco vein mottling virus protease. TEVp, tobacco etch virus protease.- 7 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)

[0053] FIG. 15 - Signaling circuit behavior in therapeutically-relevant cells, (a) Behavior of receptor-coupled sensor circuit in human APRE-19 cells and human UC-MSCs. HHH plots depict flow-cytometry data of the complete sensor circuit from Figure 16 in different cell types with 200 nM ligand (+ lig) or a carrier-only control (- lig). Values in each plot indicate mean phosphory lation (AU) ± SEM (n=3). Phosphorylation fold-change values are next to each set of plots, (b) Behavior of the sense-and-response signaling circuit in ARPE-19 . PC expression, PC phosphorylation signal and EGFP level were measured. Numbers indicate the geometric mean values (AU) ± SEM (n=3).

[0054] FIG. 16 is a schematic diagram showing the design of additional engineered receptors.

[0055] FIG. 17 is a schematic diagram showing the design of an engineered synthetic Vascular Endothelial Growth Factor (VEGF) receptor.

[0056] FIG. ISA is a schematic diagram showing the truncation strategy to design an engineered synthetic VEGF receptor.

[0057] FIG. 18B is a collection of histogram plots showing engineered receptor expression and phosphorylation (pplation) levels of the engineered receptors.

[0058] FIG. 18C is a histogram plot showing receptor expression and bar graphs showing mean phosphorylation levels of the engineered receptor.

[0059] FIG. 19A is a schematic diagram showing the design of a screening approach to assess kinase activity against synthetic substrates.

[0060] FIG. 19B is a collection of histogram plots showing phosphorylation levels of a synthetic kinase in a screen for kinase activity7using the engineered kinase incorporated in the engineered synthetic VEGF receptor.

[0061] FIG. 20 is a schematic diagram showing the design of an engineered synthetic interferon gamma (IFN-y) receptor and histogram plots showing expression levels of the engineered receptor.

[0062] FIG. 21A is a schematic diagram showing the design of a heterotetrametric engineered synthetic IFN-y receptor.

[0063] FIG. 21B shows histogram plots showing phosphorylation levels (pplation Ivl), phospho-JAK2 (pJAK2) activation, and HA-APC alpha-chain expression of the engineered synthetic IFN-y receptor.

[0064] FIG. 22A is a schematic diagram showing the design of an engineered synthetic interleukin 6 (IL6) receptor and histogram plots showing expression levels of the engineered receptor.- 8 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)

[0065] FIG. 22B is a schematic diagram showing the design of an engineered synthetic IL6 receptor.

[0066] FIG. 22C shows histogram plots showing phosphorylation levels (pplation Ivl), phospho-JAK2 (pJAK2) activation, and HA-APC alpha-chain expression of the engineered synthetic IL6 receptor.DETAILED DESCRIPTION OF THE INVENTIONAbbreviations and Definitions

[0067] Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

[0068] The singular forms ‘'a”, "an" and "‘the” include plural reference unless the context clearly dictates otherwise. The use of the word "a" or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one.” and “one or more than one.”

[0069] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly “an example,” “exemplary” and the like are understood to be nonlimiting.

[0070] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0071] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.- 9 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)

[0072] The term about is used herein to mean approximately, roughly, around, or in the region of. When the term ‘“about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0073] The term “identity” or “sequence identity” as used herein with respect to polynucleotide or polypeptide sequences can refer to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window. Thus, “percentage of sequence identify” or “percent identity” can refer to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i. e. , gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identify. It w ould be understood that, when calculating sequence identify between a DNA sequence and an RNA sequence, T residues of the DNA sequence align with, and can be considered “identical” with, U residues of the RNA sequence. For purposes of determining “percent complementarity” of first and second polynucleotides, one can obtain this by determining (i) the percent identity between the first polynucleotide and the complement sequence of the second polynucleotide (or vice versa), for example, and / or (ii) the percentage of bases between the first and second polynucleotides that would create canonical Watson and Cnck base pairs.

[0074] Various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain embodiments. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein can be used. Alternatively, a variant amino acid sequence or polynucleotide sequence can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein. The variant amino acid sequence or polynucleotide sequence has the same function / activity of the disclosed sequence, or at least about 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,- 10 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)97%, 98%, or 99% of the function / activity of the disclosed sequence. Any polypeptide amino acid sequence disclosed herein not beginning with a methionine can typically further comprise at least a start-methionine at the N-terminus of the amino acid sequence. Any polypeptide amino acid sequence disclosed herein beginning with a methionine can optionally be considered without this methionine residue (i.e., a polypeptide sequence can be referred to in reference to the position-2 residue to the C-terminal residue of the sequence).

[0075] Aspects of the invention are drawn to an engineered receptor. For example, an “engineered receptor” or “recombinantly engineered receptor” can refer to a cell surface receptor that is generated by the hand of man using standard techniques for genetic recombination.

[0076] As used herein, the term “extracellular ligand binding domain” can refer to the part of the engineered receptor that is located outside of the cell membrane and can bind to an antigen, target or ligand. In some aspects, the extracellular ligand binding domain can be specific for a cytokine or a hormone. For example, the extracellular ligand binding domain can be specific for TNF alpha or erythropoietin.

[0077] In some aspects, the extracellular ligand binding domain can comprise a native extracellular binding domain. As used herein, the term “native extracellular binding domain” can refer to a specific region or sequence within a protein that naturally interacts with a ligand, substrate, or another molecule. A native extracellular binding domain can comprise a naturally occurring sequence or structure that allows it to interact with other specific molecules in a biological system. A native extracellular binding domain can be responsible for recognizing and binding to a particular target in a biological context, such as a receptor for a signaling molecule (e.g., cy tokine or hormone), or a binding site for a regulator}' factor.

[0078] In some aspects, the extracellular ligand binding domain can comprise an amino acid sequence according to Table 1 or a sequence at least 90% identical thereto.

[0079] As used herein, the term “transmembrane domain” can refer the portion of the engineered receptor that extends across the cell membrane and anchors the engineered receptor to cell membrane. The transmembrane domain can function to link extracellular and signal transduction domains.

[0080] In some aspects, the transmembrane domain can comprise an amino acid sequence according to Table 1. Table 2, or a sequence at least 90% identical thereto.

[0081] As used herein, the term “intracellular signaling domain” can refer to the part of the engineered receptor that is located inside of the cell membrane and is capable of transducing an effector signal.- 11 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)

[0082] In some aspects, the intracellular signaling domain comprises an amino acid sequence according to Table 3 or a sequence at least 90% identical thereto.

[0083] In some aspects, the intracellular signaling domain comprises a kinase domain or a kinase binding domain.

[0084] As used herein the term “kinase domain” can refer to the part of the engineered receptor that is responsible for the enzymatic kinase activity. In particular embodiments, the kinase domain comprises kinases covalently attached to them (e.g.. RTKs).

[0085] As used herein, the term “kinase binding domain” can refer to the part of the engineered receptor that interacts with a kinase enzyme. For example, the kinase binding domain allows the engineered receptor to specifically bind to a kinase, such as a JAK kinase. By binding to the kinase, the kinase binding domain can modulate (e.g., activate, inhibit) the kinase’s activity.

[0086] In some aspects, the kinase domain or the kinase binding domain comprises a native kinase or native kinase binding domain. For example, a native kinase domain or native kinase binding domain can comprise a naturally occurring sequence or structure that allows it to carry out its naturally occurring function.

[0087] In some aspects, the engineered receptor comprises a functional fragment of the kinase domain or the kinase binding domain. As used here, the term “functional fragment” can refer to a portion of a protein or domain (e.g., extracellular ligand binding domain, transmembrane domain, or intracellular signaling domain) that retains some or all of its activity as the full-length protein or domain from which the fragment is derived. Such functional fragments can comprise truncated fragments.

[0088] In some aspects, the engineered receptor can be a homodimer, a heterodimer, or a heteromultimer.

[0089] Aspects of the invention include a synthetic signaling circuit that integrates the engineered receptor described herein a cellular signaling pathway. A “signaling pathway” can refers to a series of molecular events that are triggered by a signal (often from outside the cell) and lead to a cellular response. Signaling pathways can be critical for regulating many cellular processes, such as growth, differentiation, metabolism, survival, and immune responses.

[0090] Aspects of the invention are also drawn to nucleic acids, such as a nucleic acid encoding the engineered receptor as described herein. As used herein, the term “nucleic acid” or nucleic acid molecule, synonymously referred to as “polynucleotide.” can refer to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or- 12 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) modified RNA or DNA. Nucleic acids can include, without limitation single- and doublestranded DNA. DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, nucleic acids can refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term nucleic acids also include DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, nucleic acids can refer to chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. Nucleic acids can also refer to relatively short nucleic acid chains, often referred to as oligonucleotides.

[0091] In some aspects, the nucleic acid encodes an engineered receptor as described herein. For example, such engineered receptor comprises an extracellular ligand binding domain, a transmembrane domain, and an intracellular signaling domain, as described herein, such as in Table 1 or Table 2.

[0092] Aspects of the invention are further drawn to vector(s) comprising the nucleic acid as described herein. As used herein, the term “vector” can refer to a replicon in which another nucleic acid segment can be operably inserted so as to bring about the replication or expression of the segment.

[0093] Still further, aspects of the invention are drawn to a cell, or a host cell, comprising the vector described herein. As used herein, the term “cell” or “host cell” can refer to a cell comprising a nucleic acid molecule of the invention. The "host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a “host cell” is a cell transfected with a nucleic acid molecule of the invention. In another embodiment, a “host cell” is a progeny or potential progeny of such a transfected cell. A progeny of a cell may or may not be identical to the parent cell, e.g., due to mutations or environmental influences that can occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome.

[0094] The term “expression” can refer to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-- 13 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) transcriptional and post-translational modifications. The expressed engineered receptor can be within the cytoplasm of a host cell, into the extracellular milieu such as the growth medium of a cell culture or anchored to the cell membrane.

[0095] Aspects of the invention are also drawn towards a genetically engineered cell expressing and bearing on its cell surface the engineered receptor as described herein. As used herein, the term “genetically engineered cell’' can refer to a cell that has been genetically modified by the addition of extra genetic material in the form of DNA or RNA to the total genetic material of the cell. According to embodiments herein, the engineered cells have been genetically modified to express an engineered receptor according to the invention.

[0096] Non-limiting examples of cells which can be utilized in the invention include a mesenchymal stem cell (MSC), a T cell, a retinal pigment epithelium (RPE) cell, an ARPE cell, a macrophage, a neutrophil, , a glial cell, an NKT, an NK cell, a Treg, or a B cell. In other aspects, the cell is a stem cell, an induced pluripotent stem cell (IPSC), an embry onic stem cell, or a derivate thereof. In some aspects, the cell is derived from a human cell or a human cell line.Engineered Receptors

[0097] Provided herein, in some aspects, are engineered receptors, wherein the engineered receptors comprise a native extracellular ligand binding domain and a truncated intracellular signaling domain, wherein the truncated intracellular signaling domain comprises a native kinase domain or a native kinase binding domain and a synthetic phosphor latable domain.

[0098] In an embodiment, the engineered receptor comprises a native extracellular ligand binding domain and a truncated intracellular signaling domain. In some aspects, the extracellular ligand binding domain and truncated intracellular signaling domain can be functionally integrated. As used herein, the term “functionally integrated” can refer to different components (e.g., the extracellular ligand binding domain, the transmembrane domain, and the intracellular signaling domain) working together in a cohesive and coordinated manner to achieve a specific function or purpose. For example, the extracellular ligand binding domain, the transmembrane domain, and the intracellular signaling domain are not operating in isolation, but rather are interconnected and cooperate effectively to perform a particular biological function.

[0099] For example, the engineered receptor can comprise an engineered Receptor Tyrosine Kinase or an engineered Janus Kinase (JAK)-dependent receptors.- 14 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)

[0100] Accordingly, such aspects of the invention can refer to genetically engineered Receptor Tyrosine Kinases. A receptor tyrosine kinase (RTK) is a type of cell surface receptor that, when activated by a signaling molecule (such as a grow th factor or hormone), triggers intracellular signaling pathways that regulate various cellular processes. Non-limiting examples of RTKs include EGFR, ErbB2-4, InsR, IGF1R, InsRR, PDGFRa.0. CSF1R, SCFR, VEGFR1-3, Flt3, FGFR1-4, PTK7, TrkA-C. Rorl-2, MuSK, Met, Ron, Axl, Mer, Tyro3, Tiel-2, EphAl-10, EphBl-6. Ret, Ryk, DDR1-2, Ros. LMR1-3, ALK, LTK, SuRTkl06, and Kit.

[0101] In some aspects, the engineered receptor comprises an engineered Janus Kinase (JAK)-dependent receptor. A JAK kinase comprises a Wpe of enzyme that plays a crucial role in the signaling pathways of cells, particularly in the immune system and other cell processes. The JAK family of enzymes are tyrosine kinases, meaning they add a phosphate group (phosphorylation) to specific proteins, which can modify the activity of those proteins and trigger downstream effects in the cell. Non-limiting examples of JAK receptors include interleukin 2-7, 9-13, 15. 19-23, and 26, interferon a, 0, and y, erythropoietin (EPO), thrombopoietin (TPO). granulocyte colony-stimulating factor (G-CSF), granulocytemacrophage colony-stimulating factor (GM-CSF), growth hormone (GH), prolactin, ci I i ary neurotrophic factor (CNTF), thymic stromal lymphopoietin (TSLP), leptin, oncostatin M (OSM), and leukemia inhibitory factor (LIF).

[0102] Thus, in some aspects, the engineered receptor may comprise a native extracellular ligand binding domain comprising an amino acid sequence having at least about 85%, 90% 95%, 99% or more sequence identify to an extracellular ligand binding domain of an RTK receptor amino acid sequence or a JAK receptor amino acid sequence.

[0103] In some aspects, the truncated intracellular signaling domain comprises a truncation of the native receptors (e.g., native RTK or native JAK-dependent receptors) to retain the native kinase domain / kinase binding domain and ablate the native downstream signaling domains.

[0104] In some aspects, the engineered receptor comprises a native extracellular ligand binding domain comprising an amino acid sequence having at least about 80%. 85%. 90%. 95%, or more sequence identify to any one of the amino acid sequences set forth in SEQ ID NOs: 10-16 or SEQ ID NOs:52-56. In some aspects, the engineered receptor comprises a native extracellular ligand binding domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 10-16 or SEQ ID NOs:52-56, comprising one or more amino acid substitutions. In some aspects, the engineered receptor comprises a native- 15 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) extracellular ligand binding domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 10-16 or SEQ ID NOs:52-56.

[0105] In some aspects, the engineered receptor comprises a transmembrane domain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 17-22. In some aspects, the engineered receptor comprises a transmembrane domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 17-22, comprising one or more amino acid substitutions. In some aspects, the engineered receptor comprises a transmembrane domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 17-22.

[0106] In some aspects, the engineered receptor comprises a native kinase domain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:23-31. In some aspects, the engineered receptor comprises a native kinase domain comprising an amino acid sequence set forth in any one of SEQ ID NOs:23-31, comprising one or more amino acid substitutions. In some aspects, the engineered receptor comprises a native kinase domain comprising an amino acid sequence set forth in any one of SEQ ID NOs:23-31.

[0107] In some aspects, the engineered receptor comprises: i) a native extracellular ligand binding domain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%. or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 10-16; ii) a transmembrane domain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 17-22; and iii) a native kinase domain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:23-31.

[0108] In some aspects, the engineered receptor comprises: i) a native extracellular ligand binding domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 10- 16; ii) a transmembrane domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 17-22; and iii) a native kinase domain comprising an amino acid sequence set forth in any one of SEQ ID NOs: 23-31.

[0109] In some aspects, the engineered receptor further comprises a signal peptide. In some aspects, the signal peptide comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: l-7 or SEQ ID NOs:47-51. In some aspects, the signal peptide- 16 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-7 or SEQ ID NOs:47-51, comprising one or more amino acid substitutions. In some aspects, the signal peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-7 or SEQ ID NOs:47-51.

[0110] In some aspects, the engineered receptor further comprises an epitope tag. In some aspects, the epitope tag comprises an amino acid sequence having at least about 80%, 85%, 90%. 95%. or more sequence identity to SEQ ID NO:8 or SEQ ID NO:9. In some aspects, the epitope tag comprises an amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO:9, comprising one or more amino acid substitutions. In some aspects, the epitope tag comprises an amino acid sequence set forth in SEQ ID NO: 8 or SEQ ID NO: 9.Table 1 : Exemplary engineered receptor sequences- 17 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)- 18 -107176621.5DocketNo.: 130492-867918 (2025-033-PCT)- 19-107176621.5Docket No.: 130492-867918 (2025-033-PCT)

[0111] In some aspects, the engineered receptor comprises an amino acid sequence having at least about 80%. 85%. 90%. 95%. or more sequence identity to the amino acid sequences set forth in SEQ ID NO: 38. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO: 38, comprising one or more amino acid substitutions.- 20 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO:38.

[0112] In some aspects, the engineered receptor comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequences set forth in SEQ ID NO: 39. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO: 39, comprising one or more amino acid substitutions. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO: 39.

[0113] In some aspects, the engineered receptor comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequences set forth in SEQ ID NO: 40. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO: 40, comprising one or more amino acid substitutions. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO:40.

[0114] In some aspects, the engineered receptor comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequences set forth in SEQ ID NO:41. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO: 41, comprising one or more amino acid substitutions. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO:41.

[0115] In some aspects, the engineered receptor comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequences set forth in SEQ ID NO: 42. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO: 42, comprising one or more amino acid substitutions. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO:42.

[0116] In some aspects, the engineered receptor comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequences set forth in SEQ ID NO: 43. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO:43, comprising one or more amino acid substitutions. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO:43.

[0117] In some aspects, the engineered receptor comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequences- 21 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) set forth in SEQ ID NO: 44. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO: 44, comprising one or more amino acid substitutions. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO:44.

[0118] In some aspects, the engineered receptor comprises an amino acid sequence having at least about 80%, 85%, 90%. 95%. or more sequence identity to the amino acid sequences set forth in SEQ ID NO: 45. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO:45, comprising one or more amino acid substitutions. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO:45.

[0119] In some aspects, the engineered receptor comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequences set forth in SEQ ID NO: 46. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO: 46, comprising one or more amino acid substitutions. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO:46.

[0120] In some aspects, the engineered receptor comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 60. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO: 60. comprising one or more amino acid substitutions. In some aspects, the engineered receptor comprises an amino acid sequence set forth in SEQ ID NO: 60.

[0121] In some aspects, the engineered receptor comprises a first receptor chain and a second receptor chain derived from an interferon-gamma receptor (IFNGR). In some aspects, the first receptor chain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequences set forth inSEQ ID NO: 61. In some aspects, the first receptor chain comprises an amino acid sequence set forth in SEQ ID NO:61, comprising one or more amino acid substitutions. In some aspects, the first receptor chain comprises an amino acid sequence set forth inSEQ ID NO: 61. In some aspects, the second receptor chain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 62. In some aspects, the second receptor chain comprises an amino acid sequence set forth in SEQ ID NO:62, comprising one or more amino acid substitutions. In some aspects, the second receptor chain comprises an ammo acid- 22 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) sequence set forth in SEQ ID NO:62. In some aspects, the engineered receptor comprises a first receptor chain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequences set forth in SEQ ID NO:61 and a second receptor chain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:62.

[0122] In some aspects, the engineered receptor comprises a first receptor chain and a second receptor chain derived from an interleukin 6 receptor (IL6-R). In some aspects, the first receptor chain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequences set forth in SEQ ID NO:63. In some aspects, the first receptor chain comprises an amino acid sequence set forth in SEQ ID NO: 63, comprising one or more amino acid substitutions. In some aspects, the first receptor chain comprises an amino acid sequence set forth in SEQ ID NO:63. In some aspects, the second receptor chain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 64. In some aspects, the second receptor chain comprises an amino acid sequence set forth in SEQ ID NO:64, comprising one or more amino acid substitutions. In some aspects, the second receptor chain comprises an amino acid sequence set forth in SEQ ID NO: 64. In some aspects, the engineered receptor comprises a first receptor chain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequences set forth in SEQ ID NO:63 and a second receptor chain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:64.

[0123] In some aspects, the engineered receptor comprises a first receptor chain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity- to any of the amino acid sequences set forth in SEQ ID NOs: 1-77 and a second receptor chain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any of the amino acid sequences set forth in SEQ ID NOs: 1-77.Table 2: Additional exemplary engineered receptor sequences- 23 -107176621.5DocketNo.: 130492-867918 (2025-033-PCT)-24-107176621.5Docket No.: 130492-867918 (2025-033-PCT)- 25 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)

[0124] In other aspects, the truncated intracellular signaling domain comprises a synthetic phosphorylatable domain. Described herein are engineered receptors comprising a truncated intracellular signaling domain functionally linked to synthetic phosphorylation substrates (e.g., synSub), thereby interfacing with intracellular phosphorylation circuits. This connects the receptor binding-associated phosphorylation activity' to the intracellular phosphorylation circuits. For example, such synthetic phosphorylatable domains can be constructed or engineered to be phosphorylated and thereafter carry out cellular functions. Non-limiting examples of synthetic phosphorylatable domains include an immune tyrosine activation motif (IT AM) domain or a functional fragment thereof. For example, the IT AM domain is from IgA or CD3Z.

[0125] Referring to FIG. 1, for example, the engineered receptor comprises a truncated intracellular signaling domain to replace native domains with synSub or other synthetic proteins. Ligand binding to the extracellular ligand binding domain results in phosphorylation of synSub, which subsequently recruits components from a synthetic signaling network. The synthetic signaling cascade results in designer effects and functions.

[0126] In some aspects, the synthetic phosphorylatable domain comprises an amino acid sequence according to Table 3 or a sequence at least 90% identical thereto.Table 3: Exemplary synthetic phosphorylatable domains- 26 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)

[0127] In some aspects, the engineered receptor comprises a truncated intracellular signaling domain comprising a synthetic phosphorylatable domain. In some aspects, the synthetic phosphorylatable domain comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:32-37, SEQ ID NO:59, or SEQ ID NOs:66-77. In some aspects, the synthetic phosphorylatable domain comprises an amino acid sequence set forth in any one of SEQ ID NOs:32-37, SEQ ID NO:59, or SEQ ID NOs:66-77, comprising one or more amino acid substitutions. In some aspects, the synthetic phosphorylatable domain comprises an amino acid sequence set forth in any one of SEQ ID NOs:32-37, SEQ ID NO:59, or SEQ ID NOs:66-77.

[0128] In some aspects, the receptor chain comprising a synthetic substrate comprises: i) an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence- 27 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) identity to the amino acid sequence set forth in SEQ ID NO: 32; ii) an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:33; and iii) an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 34.

[0129] In other aspects, the receptor chain comprising a synthetic substrate comprises: i) an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:35; ii) an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:36; and iii) an amino acid sequence having at least about 80%. 85%, 90%. 95%. or more sequence identity to the amino acid sequence set forth in SEQ ID NO:37.Synthetic Signaling Circuits

[0130] Also provided herein, in some aspects, are synthetic signaling circuits capable of integrating any of the engineered receptors described herein into a cellular signaling pathway.

[0131] A cell signaling pathway can be initiated by a signal molecule (also called a ligand), which could be a hormone, growth factor, neurotransmitter, or cytokine. These signals are often secreted by other cells or come from environmental changes. The signal molecules bind to specific receptors on the surface of the target cell or inside the cell. Once the receptor binds to the signal, it activates a series of intracellular signaling molecules. These molecules amplify the signal and propagate it through the cell. These signaling molecules often activate proteins inside the cell, like kinases, phosphatases, or transcription factors. A signaling pathway often involves a cascade of protein activations or molecular interactions, where one molecule activates another in a sequence. This cascade helps amplify the signal and ensures that the response is appropriately coordinated. Ultimately, the signaling pathway leads to a cellular response.

[0132] In some aspects, the cellular signaling pathway comprises a native signaling pathway or a synthetic signaling pathway.

[0133] In some aspects, the cellular signaling pathway comprises a secretory pathway, a translocation pathway or a degradation pathway. In some aspects, the secretory pathway is a constitutive secretory pathway or a regulated secretory pathway. In some aspects, the- 28 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) degradation pathway comprises a ubiquitin-proteosome degradation pathway or an autophagy -lysosome degradation pathway.Methods of Engineering Synthetic Receptors

[0134] Also provided herein, in some aspects, is a method of engineering a synthetic receptor. In some aspects, the synthetic receptor presen es the natural diversity and specificity of RTK and JAK receptor ectodomains and kinase or kinase-binding modules, while decoupling and replacing the native receptors' downstream effector sites with synthetic phosphorylatable substrates that interface directly with customizable intracellular phosphorylation circuits.

[0135] In some aspects, the method comprises: a) providing a nucleic acid encoding a native receptor comprising an extracellular ligand-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a native kinase domain or a native kinase- binding domain; b) truncating the intracellular signaling domain at a C terminal position sufficient to ablate native downstream signaling while retaining the native kinase domain or the native kinase-binding domain; and c) appending, to the truncated intracellular signaling domain, a synthetic phosphorylatable domain configured to be phosphorylated by the native kinase domain or by a kinase recruited via the native kinase-binding domain, thereby producing the synthetic receptor.

[0136] In some aspects, the native receptor is a receptor tyrosine kinase (RTK) or a Janus kinase (JAK)-dependent receptor. Thus, in some aspects, the extracellular ligand-binding domain comprises an amino acid sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to an amino acid sequence of an RTK or a JAK receptor extracellular domain.

[0137] In some aspects, the native extracellular ligand binding domain comprises an amino acid sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 10-16 and SEQ ID NOs:52-56.

[0138] In some aspects, the native kinase domain comprises an amino acid sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:23-31.

[0139] In some aspects, the synthetic phosphorylatable domain comprises an amino acid sequence having at least about 85%. 90%. 95%. 99% or more sequence identity to an amino- 29 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) acid sequence set forth in any one of SEQ ID NOs:32-37, SEQ ID NO:59, andSEQ ID NOs:66-77.

[0140] In some aspects, the transmembrane domain comprises an amino acid sequence having at least about 85%, 90%, 95%, 99% or more sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 17-22.Nucleic Acids, Vectors and Host Cells

[0141] Provided herein, in some aspects are nucleic acids encoding an engineered receptor, wherein the engineered receptor comprises a native extracellular ligand binding domain and a truncated intracellular signaling domain, wherein the truncated intracellular signaling domain comprises a native kinase domain or a native kinase binding domain and a synthetic phosphorylatable domain.

[0142] In some aspects, the nucleic acid encodes an engineered receptor comprising: i) a native extracellular ligand binding domain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 10-16; ii) a transmembrane domain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 17-22; and iii) a native kinase domain comprising an amino acid sequence having at least about 80%, 85%, 90%, 95%, or more sequence identity7to any one of the amino acid sequences set forth in SEQ ID NOs:23- 31.

[0143] In other aspects, the nucleic acid encodes an engineered receptor comprising an amino acid sequence having at least about 80%. 85%. 90%. 95%. or more sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:38-46 or SEQ ID NOs:60- 64. In some aspects, the nucleic acid encodes an engineered receptor comprising any one of the amino acid sequences set forth in SEQ ID NOs:38-46 or SEQ ID NOs:60-64.

[0144] Also provided herein, in some aspects, are vectors comprising the nucleic acid(s) described herein. In some aspects, the vector is configured for expression of any one of the engineered receptors described herein. In some aspects, the vector is a plasmid, minicircle, bacterial artificial chromosome, or other recombinant DNA construct comprising one or more expression cassettes operably linked to promoters, untranslated regions, polyadenylation signals, and optional regulatory elements suitable for transcription in a selected host cell. The vectors may further comprise selectable markers, reporter genes, insulators, scaffold / matrix- 30 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) attachment regions, or site-specific recombination sites to facilitate cloning, selection, stable maintenance, or controlled integration, and may be produced and qualified using standard manufacturing and quality control methods known in the art.

[0145] Also provided herein, in some aspects, are host cell(s) comprising the vector described herein. In some aspects, the host cell(s) may be engineered to stably or transiently maintain and / or express the nucleic acid(s) of interest under conditions suitable for cloning, production, assembly, or functional testing.

[0146] In some aspects, the host cell is a bacterial cell, such as Escherichia coli, Bacillus, Vibrio, or other genetically tractable species, optionally comprising chromosomal or plasmid- encoded selection markers and replication origins to facilitate propagation and manufacturing of vector DNA, and further configured to support high-yield plasmid production or expression of protein where applicable.

[0147] In some aspects, the host cell is a mammalian cell, such as a human embryonic kidney (HEK293) cell, Chinese hamster ovary (CHO) cell, or other commonly used expression line, optionally comprising episomal maintenance systems or landing pad loci for targeted integration, and cultured under standard conditions to permit expression, assembly, and evaluation of the engineered receptor described herein. In certain aspects, suitable host cells also include yeast, insect, or plant cells where use of the vector supports desired cloning, amplification, or heterologous expression, with routine adaptation of promoters, selectable markers, and culture conditions to the selected host.

[0148] Host cells are transfected and preferably transformed wi th the vectors according to the invention described above and grow n in conventional culture media, which have been suitably modified to induce promoters, select transformants or amplify genes encoding the desired sequences.

[0149] The term "transfection" refers to the uptake of an expression vector by a host cell, regardless of whether any coding sequences are actually expressed or not. Numerous transfection methods are known to the person skilled in the art, e.g. the CaPCfi process and electroporation. Successful transfection is generally seen if there are any signs of the functionality of the engineered receptor within the host cell.Embodiments

[0150] Embodiment 1: An engineered receptor, wherein the engineered receptor comprises a native extracellular ligand binding domain and a truncated intracellular signaling domain.- 31 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) wherein the truncated intracellular signaling domain comprises a native kinase domain or a native kinase binding domain and a synthetic phosphorylalable domain.

[0151] Embodiment 2: The engineered receptor of embodiment 1, wherein the engineered receptor further comprises a transmembrane domain.

[0152] Embodiment 3: The engineered receptor of embodiment 1, wherein the engineered receptor is a homodimer, a heterodimer, or a heteromultimer.

[0153] Embodiment 4: The engineered receptor of embodiment 1. wherein the extracellular ligand binding domain is specific for a cytokine or a hormone.

[0154] Embodiment 5: The engineered receptor of embodiment 4, wherein the hormone is erythropoietin, or wherein the cytokine is TNF alpha.

[0155] Embodiment 6: The engineered receptor of embodiment 5, wherein the engineered receptor comprises EpoRl-375.

[0156] Embodiment 7 : The engineered receptor of embodiment 1 , wherein the extracellular ligand binding domain comprises an amino acid sequence according to Table 1 or Table 3, or a sequence at least 90% identical thereto.

[0157] Embodiment 8: The engineered receptor of embodiment 1, w herein the truncated intracellular signaling domain comprises an amino acid sequence according to Table 1 or Table 3, or a sequence at least 90% identical thereto.

[0158] Embodiment 9: The engineered receptor of embodiment 1, wherein the engineered receptor comprises an engineered Receptor Tyrosine Kinase or an engineered Janus Kinase (JAK)-dependent receptor.

[0159] Embodiment 10: The engineered receptor of embodiment 1, wherein the synthetic phosphorylatable domain comprises an immune ty rosine activation motif (IT AM) domain or a functional fragment thereof.

[0160] Embodiment 11 : The engineered receptor of embodiment 10, wherein the IT AM domain is from IgA or CD3Z.

[0161] Embodiment 12: The engineered receptor of embodiment 1, wherein the synthetic substrate comprises an amino acid sequence according to Table 1 or Table 3, or a sequence at least 90% identical thereto.

[0162] Embodiment 13: A nucleic acid encoding the engineered receptor of embodiment 1.

[0163] Embodiment 14: A vector comprising the nucleic acid of embodiment 13.

[0164] Embodiment 15: A cell comprising the vector of embodiment 14.- 32 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)

[0165] Embodiment 16: A nucleic acid encoding an engineered receptor, wherein the engineered receptor comprises a native extracellular ligand binding domain and a truncated intracellular signaling domain, wherein the truncated intracellular signaling domain comprises a native kinase domain or a native kinase binding domain and a synthetic phosphorylatable domain.

[0166] Embodiment 17: The nucleic acid of embodiment 16. wherein the nucleic acid comprises an amino acid sequence according to Table 1 or Table 2. or a sequence at least 90% identical thereto.

[0167] Embodiment 18: A vector comprising the nucleic acid of embodiment 16.

[0168] Embodiment 19: A cell comprising the vector of embodiment 15.

[0169] Embodiment 20: A genetically engineered cell expressing and bearing on its cell surface the engineered receptor of embodiment 1.

[0170] Embodiment 21 : The genetically engineered cell of embodiment 20, w herein the cell is a mesenchymal stem cell (MSC).

[0171] Embodiment 22: A synthetic signaling circuit, wherein the synthetic signaling circuit integrates the engineered receptor of embodiment 1 into a cellular signaling pathway.

[0172] Embodiment 23: The synthetic signaling circuit of embodiment 22, wherein the cellular signaling pathw ay comprises a native signaling pathw ay or a synthetic signaling pathway.

[0173] Embodiment 24: The synthetic signaling pathway of embodiment 22, wherein the cellular signaling pathway comprises a secretory pathway, a translocation pathway or a degradation pathway.

[0174] Embodiment 25: The synthetic signaling circuit of embodiment 22, wherein the synthetic signaling circuit comprises a calcium-dependent biomolecule secretion circuit.

[0175] Embodiment 26: The synthetic signaling circuit of embodiment 22, wherein the synthetic signaling circuit comprises a TNF alpha / IL-10 signaling circuit.SEQUENCESNickname Residues SEQ ID- 33 -107176621.5DocketNo.: 130492-867918 (2025-033-PCT)Nickname Residues SEQ ID-34-107176621.5Docket No.: 130492-867918 (2025-033-PCT)Nickname Residues SEQ ID- 35 -107176621.5DocketNo.: 130492-867918 (2025-033-PCT)Nickname Residues SEQ ID-36-107176621.5DocketNo.: 130492-867918 (2025-033-PCT)Nickname Residues SEQ ID-37-107176621.5DocketNo.: 130492-867918 (2025-033-PCT)Nickname Residues SEQ ID-38-107176621.5DocketNo.: 130492-867918 (2025-033-PCT)Nickname Residues SEQ ID-39-107176621.5Docket No.: 130492-867918 (2025-033-PCT)Nickname Residues SEQID-40-107176621.5Docket No.: 130492-867918 (2025-033-PCT)Nickname-41 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)Nickname Residues SEQID-42-107176621.5Docket No.: 130492-867918 (2025-033-PCT)Nickname Residues SEQ ID- 43 -107176621.5DocketNo.: 130492-867918 (2025-033-PCT)Nickname Residues SEQ ID-44-107176621.5Docket No.: 130492-867918 (2025-033-PCT)Nickname Residues SEQ ID- 45 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)Nickname Residues SEQ IDEXAMPLES

[0176] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. Flowever, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.Example 1

[0177] The ability of cells to sense and respond to their environment often relies on extracellular facing membrane receptors that propagate a signal into the cells via phosphorylation cascades. We have established synthetic phosphorylation pathways that leverage these principles, enabling modular designs to build these signaling circuits. As described herein, we developed a framework for connecting receptor tyrosine kinases (RTKs) and Janus kinase (JAK) receptor scaffolds to our synthetic phosphorylation signaling networks. Fundamentally, this allows us to leverage the naturally diverse sensing capabilities of these receptors, then hijack their signaling to route through our synthetic phosphorylation circuits instead of the native signaling pathway. We accomplish this by performing serial truncations of the receptors to i) retain the native kinase binding domains, ii) ablate the native- 46 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) dow nstream signaling domains, and iii) install our synthetic phosphorylation substrates so they can interface with our intracellular phosphory lation circuits.

[0178] Receptor tyrosine kinases (RTKs) and Janus kinase (JAK)-dependent receptors (e.g., cytokine receptors) represent a diverse class of multi- and homomeric receptors that are used by mammalian cells to sense their environment and transmit this information into the cell via phosphorylation singling networks. The ectodomain of these proteins have evolved to be specific, diverse, and capable of binding to a wide array of cytokines and hormones. Typically, these receptors have a kinase domain on the cytoplasmic tail, or a binding site for an endogenous kinase (e.g., JAK2). Upon ligand binding, the kinase phosphorylates tyrosine (pY) residues on the tail of the receptor, which interact with endogenous phosphory lation signaling cascades and related downstream effects through SH2 domain recruitment.

[0179] We made the observation that the activity of most of the Y kinases we were working with are largely proximity-driven. See, Yang, Xiaoyu, et al. "Engineering synthetic phosphorylation signaling networks in human cells." bioRxiv (2023). This indicates that RTKs and receptors that utilize JAK-family kinases may have the capacity to phosphorylate not only native Y residues, but also synthetic Ys appended to the receptor. Given this capability, we can delete regions of the receptors responsible for native Y signaling output by truncating them at the C-terminus. We can then append our synthetic phosphorylation substrates to connect receptor binding-associated phosphorylation activity to our synthetic signaling networks, including converting a pY phosphorylation into a phosphothreonine (pT) or phosphoserine (pS), thereby expanding the available connections in our system. By doing this, we leverage the diverse ligand recognition and efficient trans-membrane activation capabilities of these naturally evolved receptors, but gain full control over their downstream outputs, alloyving us to make orthogonal circuit connections to transcription, secretion, condensate formation, and potentially cell motility (see Fig. 1).

[0180] Without wishing to be bound by theory, this approach will be applicable to essentially all RTKs and JAK receptors. A list of examples of RTKs include, but are not limited to, EGFR, ErbB2-4, InsR, IGF1R, InsRR, PDGFRa,(3, CSF1R, SCFR, VEGFR1-3. Flt3, FGFR1-4, PTK7. TrkA-C, Rorl-2. MuSK. Met, Ron, Axl, Mer, Tyro3. Tiel-2. EphAl- 10, EphBl-6, Ret, Ryk, DDR1-2, Ros, LMR1-3, ALK, LTK, SuRTklO6, and Kit (Lemmon & Schlessinger 2010). Examples of JAK receptors include, but are not limited to, interleukin 2-7, 9-13, 15, 19-23, and 26, interferon a, (3, and y, erythropoietin (EPO), thrombopoietin (TPO), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony- stimulating factor (GM-CSF), growth hormone (GH), prolactin, ciliary neurotrophic factor- 47 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)(CNTF), thymic stromal lymphopoietin (TSLP), leptin, oncostatin M (OSM), and leukemia inhibitor}’ factor (LIF) (Zhang et al. 2023 and Xue et al. 2023).

[0181] As a proof of principle, we engineered one of these receptors, the erythropoietin receptor (EpoR), with a series of truncations in the intracellular tail to retain only the minimal sequence required for JAK2 binding while removing the native Y residues, thus abolishing native protein docking and signal transduction via pY-SH2 interactions. Additionally, we appended the ITAM domain from CD3Z, which we previously used to engineer the synSub protein described in Yang et al. 2024 (see Fig. 2 A-B). This strategy enabled us to validate JAK2's capacity to phosphorylate synthetic Y residues while simultaneously preventing native signaling pathway activation. We then assessed phosphorylation levels of CD3Z upon Epo addition and calculated the fold-change activation before and after ligand addition (see Fig. 2C). Among the truncations, EpoRl-375 was identified as the optimal truncation, demonstrating a significant ligand-dependent increase in phosphorylation and favorable fold change (6.3x). We also confirmed that EpoRl-375 was non-functional unless exogenous JAK2 was provided via transgene expression in HEK293Ts, as JAK2 is not expressed in these cells (see Fig. 2D).

[0182] Next, we sought to demonstrate that the cytoplasmic synSub could also be phosph orylated by activated JAK2 through a leucine zipper (LZ)-mediated recruitment. To validate this, we engineered a ‘‘passive” recruitment circuit configuration, as illustrated in Fig. 3A. Our results confirmed that synSub phosphorylation occurred upon ligand addition, further validating recruitment-dependent activity of JAK2 associated with the truncated EpoR and underscoring the modular versatility7of leveraging recruitment mechanisms to achieve phosphorylation and circuit activation (FIG. 3B).

[0183] We sought to further extend this strategy with a more complex native signal transduction pathway. Without wishing to be bound by theory, a synSub recruited to the synthetic receptor via SH2-pY interactions could also be phosphorylated by JAK2. To validate this, we used domains from parts from our previous work, fusing the ITAM domain from IgA (FIG. 4A) to the tail of EpoRl-375. and appending the ITAM from CD3Z to tandem SH2 domain of ZAP70. We termed this design an “active recruitment” circuit configuration. We assayed phosphorylation of CD3Z before and after ligand addition, demonstrated both the pY -SH2-dependent recruitment and the JAK2-mediated phosphorylation of CD3Z (FIG. 4B).

[0184] Importantly, our receptor circuit is amenable to use in therapeutic cell types, which include but are not limited to mesenchymal stem / stromal cells (MSCs). We found excellent- 48 -107176621.5DocketNo.: 130492-867918 (2025-033-PCT) expression in primary MSCs (FIG. 5). Further work will confirm that ligand-inducible phosphorylation occurs in these and other primary cell types.Through this series of engineering efforts on Epo-mediated signal transduction, we demonstrated that the modular design of receptor elements enables the creation of customizable synthetic signaling networks. These networks can mimic, enhance, or entirely replace native signaling mechanisms, providing a flexible platform for constructing native- inspired sense-and-response circuits. These circuits can robustly detect physiologically relevant biomolecules, opening up new possibilities for advanced therapeutic applications that respond to specific disease conditions. For example, the direct fusion variant of this receptor from Fig. 1 enabled the development of a “sense-and-secrete” system where the receptor triggers a phosphorylation event that results in an increase in secretion (FIG. 6).

[0185] Other applications of how these receptor designs can be used include interfacing them with our existing phosphorylation circuit infrastructure such as forming condensates (FIG. 7A) or through connections to native pathway s (FIG. 7B). Importantly, this design enables the conversion of a phosphotyrosine (pY) to a phosphothreonine (pT) or phosphoserine (pS), which dramatically expands the diversity of downstream pathways, effects, and functions that can be leveraged.Certain applications of embodiments of the invention:

[0186] This technology is a step towards dramatically diversifying the sensing capabilities of our synthetic phosphorylation circuit engineering platform. Without wishing to be bound by theory, this technology will allow us to engineer cell-based circuitry that can be deployed in infused or encapsulated cell products, either as diagnostics or therapeutics that can sense and respond to a wide range of targets in vivo.

[0187] As described herein, we used a completely new approach by keeping the receptor scaffold but abolishing the native downstream signaling and instead relying on the creation of a synthetic phosphorylation event. Additionally, this approach now allows us to utilize the phosphorylated tyrosine (pY) to recruit a serine / threonine kinase, thereby enabling the conversion of a pY to phosphoserine (pS) or phosphothreonine (pT). This expansion of our circuit capabilities dramatically expands the diversity of native and synthetic functions we can build into our circuit, as serine and threonine phosphorylation represent a significant fraction of phosphorylation events used in biological systems.

[0188] Our approach merges the diverse sensing capabilities of native receptors with the modularity of our synthetic phosphorylation circuit and the widespread biological- 49 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) applications that rely on native phosphorylation. We can now connect nearly any extracellular output to a quantitatively engineerable user-defined behavior.References:

[0189] Yang et al. Engineering synthetic phosphorylation signaling networks in human cells. Science. 2025;387(6729):74-81.

[0190] Lemmon & Schlessinger Cell Signaling by Receptor Tyrosine Kinases. Cell. 2010

[0191] Zhang et al. Synthesis and clinical application of small-molecule inhibitors of Janus kinase. European Journal of Medicinal Chemistry. 2023.

[0192] Faustova et al. A synthetic biology approach reveals diverse and dynamic CDK response profiles via multisite phosphorylation ofNLS-NES modules. Science Advances. 2022.

[0193] Child et al. A cancer-derived mutation in the PSTAIRE helix of cyclin- dependent kinase 2 alters the stability of cyclin binding. Biochimica et Biophysica Acta Molecular Cell Research. 2010.

[0194] Xue et al. Evolving cognition of the JAK-STAT signaling pathway: autoimmune disorders and cancer. Signal Transduction and Targeted Therapy. 2023

[0195] Makri Pistikou et al. Engineering a scalable and orthogonal platform for synthetic communication in mammalian cells. Nature Communications. 2023.

[0196] Scheller et al. Generalized extracellular molecule sensor platform for programming cellular behavior. Nature Chemical Biology. 2018.Example 2

[0197] This example provides the engineering and implementation of synthetic phosphorylation-based sense-and-respond circuits for therapeutic applications. The work expands on ligand sensing capabilities by designing synthetic receptors for detecting specific physiological biomolecules, such as TNF-a and Epo, and coupling circuit activation to therapeutic cargo release and multiple reporter activation. This chapter highlights the modular and versatile nature of the engineered circuits, which were successfully integrated into primary human cells. Without wishing to be bound by theory, this demonstrates the use of these circuits for cell-based therapies that can be customized for specific disease contexts, offering real-time, programmable responses.- 50 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)

[0198] This example provides the results of efforts to engineer synthetic biological networks in more therapeutic-relevant applications. This section contains the following main objectives: (1) diversify the sensing ability of the system for physiologically relevant biomolecules, allowing the system to detect and respond to disease-specific environments through precise activation based on relevant biomarkers; (2) designing the network to couple its activation with the release of therapeutic cargo, or incorporating a reporter system that provides real-time feedback on the circuit’s activity upon biomolecule detection; (3) described herein is the successful implementation of the synthetic circuit into multiple primary human cells, a key step in demonstrating the feasibility of using these engineered cells for innovative cell-based therapies. This example will illustrate the versatility and modularity of the system, showing how it can be adapted for a wide range of applications, making it a platform for the development of new cell-based therapies with customizable, programmable functions tailored to specific diseases and patient needs.Engineering new receptors for sensing physiology-related biomolecules Expand ligand sensing capabilities with TNF-alpha sensor module

[0199] To demonstrate the configurability and modularity of our system, we sought to expand its sensing capabilities by engineering receptors capable of detecting new extracellular ligands. Without wishing to be bound by theory, the structure of our receptors would permit us to modify sensing specificity by swapping the extracellular dimerization domain. We identified an scFv (Abdolalizadeh, J. et al. Targeting cytokines: production and characterization of anti-TNF-a scFvs by phage display technology. Curr Pharm Des 19, 2839-2847 (2013)) that binds specifically to the cytokine TNF-a, which exists as a multimer (Tang, P., Hung M-C, null & Klostergaard, J. Human pro-tumor necrosis factor is a homotrimer. Biochemistry 35, 8216-8225 (1996).), and fused it to both receptor chains, replacing the FKBP and FRB* domains used for the sensor circuit (FIG. 9, panel A). This enabled the phospho-sensor circuit to activate in the presence of TNF-a (FIG. 9, panel B), demonstrating a nearly 6-fold induction in synSub phosphory lation upon treatment with 20 ng / mL TNF-a. We measured a dose response curve for our TNF-a sense-and-response circuit (FIG. 9, panel C). We found that this sensor circuit was highly linear (nH=1.07), with an EC50 (6. 14 ng / mL) similar to reported scFv interaction affinity.Engineering synthetic receptor for Epo sensing- 51 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)

[0200] Erythropoietin (Epo) is a crucial cytokine that regulates red blood cell production, primarily through its interaction with the erythropoietin receptor (EpoR)68. Conditions like hypoxia (Haase, V. H. Hypoxic regulation of erythropoiesis and iron metabolism. American Journal of Physiology - Renal Physiology 299, Fl (2010)) or tumors can lead to overproduction of Epo, causing an abnormal increase in red blood cell mass, as seen in certain diseases such as chronic hypoxia, renal tumors, and polycythemia. Upon binding of Epo to EpoR, the receptor undergoes a conformational change that activates intracellular signaling via the JAK / STAT pathway (Livnah, O. et al. Crystallographic evidence for preformed dimers of erythropoietin receptor before ligand activation. Science 283, 987-990 (1999)). In the native system, EpoR is pre-associated with JAK2 in an inactive state (Ferrao. R. D., Wallweber, H. J. & Lupardus, P. J. Receptor-mediated dimerization of JAK2 FERM domains is required for JAK2 activation. Elife 7, e38089 (2018)), with JAK2 remaining inhibited until Epo binding induces its activation. Once activated, JAK2 phosphorylates tyrosine residues on the intracellular tail of EpoR, which subsequently recruits downstream effectors through SH2 domain interactions.

[0201] Building on previous findings regarding the specificity of tyrosine kinases, we observed that their activities are largely proximity-driven. This indicates that JAK2, a key tyrosine kinase, may phosphorylate not only native ty rosine residues but also synthetic tyrosines appended to the C-terminal tail of EpoR (FIG. 10, panel A). To validate this, we engineered a series of truncations (FIG. 10, panel B) in the EpoR tail to retain only the minimal sequence required for JAK2 binding while removing the native Y residues, thus abolishing native protein docking and signal transduction via pY-SH2 interactions. Additionally, we appended the ITAM domain from CD3Z, which we previously engineered for the synSub system. We then assessed the phosphorylation levels of CD3Z upon ligand (Epo) addition and calculated the fold change in circuit activation before and after ligand binding (FIG. 10, panel C). Among the truncations, EpoRi-375 was identified as the optimal construct, demonstrating a significant ligand-dependent increase in phosphorylation and favorable fold change (4.8 x). This strategy enabled us to validate JAK2's capacity to phosphorylate synthetic tyrosine residues while simultaneously preventing the activation of native signaling pathways.

[0202] Next, we sought to demonstrate that the cytoplasmically localized synSub could also be phosphorylated by activated JAK2 through LZ recruitment. To validate this, we engineered a passive recruitment circuit configuration, as illustrated in FIG. 11, panel A. Our results confirmed that synSub phosphorylation occurred upon ligand addition, further- 52 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) validating the recruitment-dependent activity' of JAK2 associated with the truncated EpoR. These findings underscore the versatility of the system in leveraging recruitment mechanisms to achieve phosphory lation and circuit activation, supporting the modular nature of the engineered pathway.

[0203] Building on the phosphory lation-dependent recruitment mechanism, we sought to mimic a more complex native signal transduction pathway. Without wishing to be bound by theory, a substrate recruited to the receptor via SH2-pY interactions could also be phosphorylated by JAK2. To validate this, we fused the IT AM domain from IgA (FIG. 1 1 , panel B) to the tail of EpoRi-375 and built another construct by fusing IT AM from CD3Z with the tandem SH2 domain of ZAP70. This allow ed us to design an active recruitment circuit configuration. Upon ligand addition, we tested the phosphorylation of CD3Z, which demonstrated both the pY-SH2-dependent recruitment and the JAK2-mediated phosphorylation of CD3Z.

[0204] Through this series of engineering efforts on Epo-mediated signal transduction, we demonstrated that the modular design of receptor elements enables the creation of customizable synthetic signaling networks. These networks can mimic, enhance, or entirely replace native signaling mechanisms, providing a flexible platform for constructing native-inspired sense-and-response circuits. These circuits can detect physiologically relevant biomolecules, opening up new possibilities for advanced therapeutic applications that respond to specific disease conditions.Build therapeutic-relevant sense-and-response circuit with phosphorylation-based signaling networkEngineering and testing a closed-loop therapeutic control circuit

[0205] Next, we asked whether our framework could be used to engineer a circuit programmed to sense markers associated with inflammation and respond via secretion of a therapeutic factor. Because TNF-a is broadly involved in inflammation and is a marker for numerous inflammatory disorders, we elected to use TNF-a as the circuit’s input. To create a circuit that responds with an anti-inflammatory7output, we selected the cytokine IL- 10 due to its well-documented immunosuppressive properties, including the inhibition of CD28- costimulated T cell activation, proliferation, and cytokine production under inflammatory conditions (Moore, K. W., de Waal Malefyt, R., Coffman, R. L. & O’Garra, A. Interleukin- 10 and the interleukin- 10 receptor. Annu Rev Immunol 19, 683-765 (2001); Akdis, C. A. &- 53 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)Blaser, K. Mechanisms of interleukin- 10-mediated immune suppression. Immunology 103, 131-136 (2001)). Such a TNF-a sensing / IL-10 response circuit offers an excellent test-case for our system. For the past two decades, numerous studies and preclinical trials have been conducted using IL-10 to treat a variety of inflammatory disorders, including inflammatory bowel disease (Cardoso, A. et al. The Dy namics of Interleukin- 10- Afforded Protection during Dextran Sulfate Sodium-Induced Colitis. Front Immunol 9, 400 (2018)), and psoriasis (Ouyang. W. & O’Garra, A. IL-10 Family Cytokines IL-10 and IL-22: from Basic Science to Clinical Translation. Immunity 50, 871-891 (2019)). However, clinical trials have generally failed to significantly improve patient outcomes (ColombeL J. et al. Interleukin 10 (Tenovil) in the prevention of postoperative recurrence of Crohn’s disease. Gut 49, 42 (2001).Schreiber, S. et al. Safety’ and efficacy of recombinant human interleukin 10 in chronic active Crohn’s disease. Crohn’s Disease IL-10 Cooperative Study Group. Gastroenterology 1 19, 1461-1472 (2000)), which is likely due to an inability’ to achieve sufficiently high local concentrations of IL- 10 following systemic infusion. On the other hand, high systematic levels of IL-10 can lead to toxicity involving excessive B cell activation and antibody production, a mechanism that plays a significant role in systemic lupus erythematosus (SLE) pathology (Facciotti, F. et al. Evidence for a pathogenic role of extrafollicular, IL-10- producing CCR6+B helper T cells in systemic lupus ery thematosus. Proc Natl Acad Sci U S A 117, 7305-7316 (2020)). Furthermore, the immunosuppressive effect of IL-10 can lead to chronic viral infection without timely clearance (Brooks, D. G. et al. Interleukin- 10 determines viral clearance or persistence in vivo. Nat Med 12, 1301-1309 (2006)). As a way to mitigate these toxic effects, our goal yvas to develop a circuit that responds to TNF-a by proportional secretion of IL-10, thereby inhibiting T cell activation and TNF-a production and rapidly establishing a loyv setpoint for both cytokines.

[0206] Building upon the design of the optimized sense-and-respond circuit composition, we coupled our TNF-a sensor circuit to our sense-and-respond circuit and placed the expression of IL- 10 under control of the synTF cassette. To demonstrate circuit function, we used a transwell system to co-culture CD3 / CD28-activated human PBMCs with HEK293T cells transfected yvith the circuit for 60 h (FIG. 12. panel A). We sampled TNF-a and IL- 10 secretion from the culture every’ 12 h, and assayed T cell proliferation (FIG. 12, panel B) and IFN-y secretion (FIG. 12, panel C) at the end of the time course. In the coculture containing activated T cells and HEK293T cells with no circuit, we obser ed rapid TNF-a accumulation and robust T cell proliferation (FIG. 12. panel A top right). In the open-loop circuit (FIG. 12, panel A, middle right), which is configured with a synKin (no receptors) that- 54 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) constitutively activates a two-step circuit driving IL-10 expression, we observed continuous accumulation of IL-10 as well as substantial inhibition of TNF-a production and T cell proliferation. In contrast to the open-loop composition, the closed-loop circuit could dynamically sense TNF-a levels and adjust IL-10 production down early in the time course, but still suppressed T cell proliferation and reduced IFN-y secretion (FIG. 12, panel A, bottom right). As a demonstration that sustained circuit activity was responsible for the low setpoint of TNF-a and IL-10, we added imatinib mesylate (10 pM) midway through the time course at 36 h. We observed a -40% drop in IL-10 and 2-fold increase for TNF-a and IFN-y secretion compared to the uninhibited circuit, indicating that secreted cytokine suppression is driven by continuous circuit activity throughout the time course and is not an artifact of initial culture conditions (FIG. 12, panel C).Integrating phosphorylation circuits with calcium signaling for biomolecule secretion

[0207] We next engineered a sense-and-secrete system by combining a calciumdependent biomolecule secretion circuit, with ligand-induced circuit activation to trigger biomolecule release upon sensing specific extracellular environments. This approach leverages the rapid activation characteristics of the phosphorylation circuit, enabling fast and controlled secretion.

[0208] We utilized the Orai family of plasma membrane Ca2+channels, which are primarily responsible for store-operated calcium entry (SOCE) in response to depleted endoplasmic reticulum (ER) calcium stores. The ER transmembrane protein STIM (stromal interaction molecule) plays a crucial role in this process by sensing the reduced ER luminal Ca2+content. Upon depletion, STIM undergoes conformational changes that expose its STIM-Orai activating region (SOAR), which directly interacts with and opens Orai channels, facilitating the influx of extracellular calcium to restore ER calcium levels (Lunz, V., Romanin, C. & Frischauf, I. STIM1 activation of Orail. Cell Calcium 77, 29-38 (2019)).

[0209] We coupled this calcium influx mechanism with calcium-induced regulated secretion using a suite of proteins (Rao, S. K., Huynh, C., Proux-Gillardeaux, V., Galli, T. & Andrews, N. W. Identification of SNAREs involved in synaptotagmin Vll-regulated lysosomal exocytosis. J Biol Chem 279, 20471-20479 (2004)), including SYT7, STX4, RAB26, and RAB27B, to drive secretion. Without wishing to be bound by theory, phosphorylation-induced colocalization of SOAR with Orai channels could trigger calcium influx and, subsequently, cargo secretion from the cell. This design allows us to explore a- 55 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) new application of the phosphorylation circuit’s fast activation in driving dynamic biomolecule release in response to changes in the extracellular environment.

[0210] For this design, we used the direct-fusion configuration of the Epo-induced phosphorylation circuit. SOAR was fused to ZAP70 tSH2, which would be recruited to the membrane upon phosphorylation of the CD3Z domain in the receptor tail (FIG. 13, panel A). This membrane recruitment would, in turn, trigger calcium influx, resulting in a maximum detectable secretory response of 1.3x (FIG. 13, panel B). However, we observed elevated basal cytosolic Ca2+levels associated with the circuit, as indicated by our transcriptional assay. This elevated baseline potentially contributed to reduced basal secretion rates following ligand induction, limiting the cell’s capacity to accumulate secretory reporters before circuit activation. Future optimization of the EpoR-SOAR-Orai circuit should focus on minimizing basal Orai channel activation and preventing Ca2leakage to improve overall secretion performance.Translatabilitv of the synthetic phosphorylation network in therapeutically relevant cell types

[0211] As a final set of experimental demonstrations, we sought to assess the translatability of our synthetic phospho-signaling components. One reason we selected protein domain parts derived from human cell signaling pathways was to mitigate immunogenicity associated with deploying the circuits in an adoptive cell therapy setting. We analyzed all the protein components from the sense-and-respond circuit in FIG. 12, panel A using T Cell Class I pMHC Immunogenicity Tool, an established immunogenicity prediction tool (Dhanda, S. K. et al. IEDB-AR: immune epitope database-analysis resource in 2019. Nucleic Acids Res 47, W502-W506 (2019)) used to predict the relative chance a peptide / MHC complex will elicit an immune response. We compared our signaling components to several widely used synthetic receptors (Roybal, K. T. et al. Precision Tumor Recognition by T Cells With Combinatorial Antigen- Sensing Circuits. Cell 164, 770-779 (2016)), protease parts used for engineering post-translational circuits (Fink, T. & Jerala. R. Designed protease-based signaling networks. Cun Opin Chem Biol 68, 102146 (2022)), chimeric antigen receptors, and FDA-approved fully humanized monoclonal antibodies (mAbs) (Paul, S. et al. Cancer therapy with antibodies. Nat Rev Cancer 24, 399-426 (2024)) as references (FIG. 14). We evaluated the immunogenicity scores of all 9-mer peptide sequences within the six engineered proteins in the cytokine control circuit, along with the synthetic receptors, proteases and mAbs and set a cutoff threshold value of 0.328 as the mean- 56 -107176621.5DocketNo.: 130492-867918 (2025-033-PCT)+ 2xSD of the immunogenicity scores for all the peptides in the three chosen FDA-approved mAbs. Proteins with a percentage of peptides scoring higher than 0.328 are more likely to be more immunogenic compared to these mAbs. While certain regions of our synthetic proteins (e.g., the TM domains), showed high predicted immunogenicity, our phosphorylation circuit components had average scores that were similar to the other parts. It should be possible to further de-immunize our part set using a combination of computational analysis and functional testing, thereby improving the safety profile of cell therapy products engineered using our proteins. This should be aided by the modularity of our design scheme set, which allows us to swap out highly immunogenic parts for less reactive parts that retain similar biophysical properties (e.g., new protein-protein interaction domains that bind with equivalent, tunable affinity).

[0212] We also tested our circuits for their ability to function in therapeutically relevant cell types. We introduced circuits into human umbilical-cord-derived mesenchymal stem cells (hUC-MSCs) (Chamberlain, G., Fox, J., Ashton, B. & Middleton, J. Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing. Stem Cells 25, 2739-2749 (2007)), which have been historically one of the most frequently investigated therapeutic cell types, and ARPE-19 cells (Dunn, K. C., Aotaki-Keen, A. E., Putkey, F. R. & Hjelmeland, L. M. ARPE-19, ahuman retinal pigment epithelial cell line with differentiated properties. Exp Eye Res 62, 155-169 (1996)), which have been approved for clinical trials involving in vivo therapeutic production (Bashor, C. J., Hilton, I. B., Bandukwala, H., Smith, D. M. & Veiseh, O. Engineering the next generation of cell-based therapeutics. Nat Rev Drug Discov 21, 655-675 (2022)). Our phospho-sensor circuit ported well into both cell ty pes, yielding 9.7x and 6. Ox fold-change inductions in response to AP21967 as assayed by flow cytometry (FIG. 15, panel A). We also tested the sense and respond circuit in ARPE-19 cells and showed that the full pathway response works, exhibiting a 7.3x fold-change induction (FIG. 15, panel B). These results demonstrate that, with little optimization, our synthetic signaling circuits can function in cell types that can be translated to clinical settings.Example 3

[0213] This example describes the design of additional engineered receptors having sensing capabilities for inflammatory cytokines such as interferon gamma (IFN-y) and interleukin 6 (IL-6) and the angiogenic growth factor Vascular Endothelial Growth Factor- 57 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)(VEGF). The design of the following engineered receptors followed a similar approach as disclosed in Example 1 and Example 2, above. Briefly, the cytoplasmic domains of the receptors were systematically truncated and fused to a synthetic substrate and / or one or more leucine zipper domains to facilitate recruitment of other substrates.Synthetic Receptor Tyrosine Kinase Engineering

[0214] A sy nthetic VEGFR2 receptor was engineered by truncating the carboxyl- terminal region of the receptor through a truncation series to determine cut sites that removed endogenous signaling residues while preserving ligand-inducible phosphorylation activity (FIGs. 17 and 18A). A construct including VEGFR2 amino acid residues 1-1212 fused to a synthetic substrate exhibited inducible receptor activity upon ligand addition (FIG. 18B). The selected synVEGFR2 variant, when gated on receptor expression, demonstrated ligandinducible phosphorylation output at approximately 3.3-fold over a negative control consisting of synEPOR co-expressed with an inactive kinase (FIG. 18C). Although the phosphorylation signal was relatively weak, kinase activity against other synthetic substrates confirmed that the kinase domain was active with the selected truncation (FIGs. 19A and 19B). A soluble kinase effector version was generated by truncating away autoinhibitory domains upstream of residue 806 and retaining residues necessary for activity identified in the truncation screen, producing VEGFR2[806-1212] fused to a flexible linker and leucine zipper. Kinase screening indicated that the effector kinase on synVEGFR2 was active with the selected carboxyl terminus truncation, and that CD3^, the synthetic substrate used in initial synVEGFR2 prototypes, was the least compatible substrate among those tested (FIG. 19B). Heterotetrametric Receptor Engineering svnlFN-y R

[0215] A heterotetrameric interferon-gamma receptor pathway was engineered comprising two chains, IFNGR1 (alpha chain) and IFNGR2 (beta chain) (FIGs. 20 and 21 A). The full-length beta chain (IFNGR2) was used with a fused synthetic substrate, and IFNGR1 was truncated to residues 1-375 to mitigate activation of endogenous signaling pathways, with a synthetic substrate also fused to IFNGR1. This configuration preserved BOX1 and BOX2 sites to enable effector kinase activity and utilized JAK1 in addition to JAK2. Initial validation used a sensitive stain for the beta chain and showed expression at least 50-fold above background in both uninduced and induced conditions. The full pathway was then expressed with JAK1, and alpha chain expression was more sensitively assessed; despite weak alpha chain expression, JAK2 activation and downstream phosphorylation were clearly above background (>50-fold), indicating sufficient alpha chain expression for liganddependent activation of JAK2 (FIG. 21B).- 58 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)Heterotetrametric Receptor Engineering svnIL6R

[0216] A heterotetrameric interleukin-6 receptor circuit was engineered consisting of GP130 and IL6Ra, with GP130 truncated to residues 1-714 to reduce off-target signaling (FIG. 22A). Expression of the IL6Ra chain was validated and observed to be well above background, with expression varying with ligand induction (FIG. 22A). The full pathway including JAK1 was then tested (FIG. 22B). The engineered pathway exhibited clear kinase effector activity above background, which was more apparent from population-level means and distribution shapes; weak GP130 expression was observed and was identified as a likely contributor to the modest inducible behavior (FIG. 22C).EQUIVALENTS

[0217] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.- 59 -107176621.5

Claims

Docket No.: 130492-867918 (2025-033-PCT)CLAIMSWhat is claimed:

1. An engineered receptor, wherein the engineered receptor comprises a native extracellular ligand binding domain and a truncated intracellular signaling domain, wherein the truncated intracellular signaling domain comprises a native kinase domain or a native kinase binding domain and a synthetic phosphorylatable domain.

2. The engineered receptor of claim 1, wherein the engineered receptor comprises an engineered Receptor Tyrosine Kinase (RTK) or an engineered Janus Kinase (JAK)- dependent receptor.

3. The engineered receptor of claim 1 or claim 2. wherein the native extracellular ligand binding domain comprises an amino acid sequence having at least about 85% sequence identity to an extracellular ligand binding domain of an RTK receptor amino acid sequence or a JAK receptor amino acid sequence.

4. The engineered receptor of any one of claims 1 to 3, wherein the native extracellular ligand binding domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:l Ct- 16 or SEQ ID NOs:52-56.

5. The engineered receptor of any one of claims 1 to 4, wherein the native kinase domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:23-31.

6. The engineered receptor of any one of claims 1 to 5, wherein the synthetic phosphorylatable domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one ofSEQ ID NOs:32-37, 59, and SEQ ID NOs:66-77.

7. The engineered receptor of any one of claims 1 to 6. wherein the engineered receptor further comprises a transmembrane domain.

8. The engineered receptor of claim 7, wherein the transmembrane domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 17-22.- 60 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)9. The engineered receptor of any one of claims 1 to 8, wherein the engineered receptor further comprises a signal peptide.

10. The engineered receptor of claim 9, wherein the signal peptide comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: l-7 or SEQ ID NOs:47-51.

11. The engineered receptor of any one of claims 1 to 10, wherein the engineered receptor further comprises an epitope tag.

12. The engineered receptor of claim 11, wherein the epitope tag comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO:8 orSEQ ID NO:

913. The engineered receptor of any one of claims 1 to 12, wherein the engineered receptor comprises a) a native extracellular ligand binding domain comprising an amino acid sequence having at least about 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:10-16; b) a transmembrane domain comprising an amino acid sequence having at least about 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 17-22; and c) a native kinase domain comprising an amino acid sequence having at least about 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:23-31.

14. The engineered receptor of any one of claims 1 to 13, wherein the engineered receptor comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:38-46 or SEQ ID NQs:60- 64.

15. The engineered receptor of any one of claims 1 to 14, wherein the engineered receptor is a homodimer, a heterodimer, or a heteromultimer.

16. The engineered receptor of any one of claims 1 to 15, wherein the receptor comprises a first receptor chain and a second receptor chain.- 61 -107176621.5Docket No.: 130492-867918 (2025-033-PCT)17. The engineered receptor of claim 16, wherein the first receptor chain comprises an amino acid sequence having at least about 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:61 and the second receptor chain comprises an amino acid sequence having at least about 85% or more sequence identity7to the amino acid sequence set forth in SEQ ID NO:62.

18. The engineered receptor of claim 16, wherein the first receptor chain comprises an amino acid sequence having at least about 85% or more sequence identity to the amino acid sequence set forth in SEQ ID NO:63 and the second receptor chain comprises an amino acid sequence having at least about 85% or more sequence identity' to the amino acid sequence set forth in SEQ ID NO:64.

19. The engineered receptor of any one of claims 1 to 18, wherein the extracellular ligand binding domain is specific for a cytokine, a hormone, or a growth factor.

20. The engineered receptor of claim 19, wherein the hormone is erythropoietin, or wherein the cytokine is TNF alpha, IFN-y, or IL-6.

21. The engineered receptor of claim 19 or claim 20, wherein the engineered receptor comprises EpoRi-375.

22. The engineered receptor of claim 19, wherein the grow th factor is VEGF.

23. The engineered receptor of any one of claims 1 to 22, wherein the synthetic phosphorylatable domain comprises an immune tyrosine activation motif (ITAM) domain or a functional fragment thereof.

24. The engineered receptor of claim 23, wherein the ITAM domain is from IgA or CD3Z.

25. A nucleic acid encoding the engineered receptor of any one of claims 1 to 24.

26. A vector comprising the nucleic acid of claim 25.

27. A host cell comprising the vector of claim 26.

28. A nucleic acid encoding an engineered receptor, wherein the engineered receptor comprises a native extracellular ligand binding domain and a truncated intracellular signaling domain, wherein the tmncated intracellular signaling domain comprises a- 62 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) native kinase domain or a native kinase binding domain and a synthetic phosphorylatable domain.

29. The nucleic acid of claim 28. wherein the engineered receptor comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-37.

30. The nucleic acid of claim 28 or claim 29, wherein the engineered receptor comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:38-46 or SEQ ID NOs:60-64.

31. A vector comprising the nucleic acid of any one of claims 28 to 30.

32. A host cell comprising the vector of claim 31.

33. A genetically engineered cell expressing and bearing on its cell surface the engineered receptor of any one of claims 1 to 24.

34. The genetically engineered cell of claim 33, wherein the cell is a mesenchymal stem cell (MSC), a T cell, an ARPE cell, a macrophage, a neutrophil, a glial cell, an NKT, an NK cell, a Treg. or a B cell.

35. The genetically engineered cell of claim 33, wherein the cell is a stem cell, an induced pluripotent stem cell (IPSC), an embryonic stem cell, or a derivate thereof.

36. A synthetic signaling circuit, wherein the synthetic signaling circuit integrates the engineered receptor of any one of claims 1 to 24 into a cellular signaling pathway.

37. The synthetic signaling circuit of claim 36, wherein the cellular signaling pathway comprises a native signaling pathway or a synthetic signaling pathway.

38. The synthetic signaling pathway of claim 36 or claim 37, wherein the cellular signaling pathway comprises a secretory pathway, a translocation pathway or a degradation pathway.

39. A method of engineering a synthetic receptor, the method comprising: a) providing a nucleic acid encoding a native receptor comprising an extracellular ligand-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a native kinase domain or a native kinase-binding domain;- 63 -107176621.5Docket No.: 130492-867918 (2025-033-PCT) b) truncating the intracellular signaling domain at a C terminal position sufficient to ablate native downstream signaling while retaining the native kinase domain or the native kinase-binding domain; and c) appending, to the truncated intracellular signaling domain, a synthetic phosphorylatable domain configured to be phosphorylated by the native kinase domain or by a kinase recruited via the native kinase-binding domain, thereby producing the synthetic receptor.

40. The method of claim 39, wherein the native receptor is a receptor tyrosine kinase (RTK) or a Janus kinase (JAK)-dependent receptor.

41. The method of claim 39 or claim 40, wherein the extracellular ligand-binding domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence of an RTK or a JAK receptor extracellular domain.

42. The method of any one of claims 40 to 41. wherein the native extracellular ligand binding domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:10- 16 or SEQ ID NOs:52-56.

43. The method of any one of claims 40 to 42, wherein the native kinase domain comprises an amino acid sequence having at least about 85% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs:23-31.

44. The method of any one of claims 40 to 43, wherein the synthetic phosphorylatable domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs:32-37, SEQ ID NO:59, and SEQ ID NOs:66-77.

45. The method of any one of claims 40 to 44. wherein the transmembrane domain comprises an amino acid sequence having at least about 85% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 17-22.- 64 -107176621.5

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