Synthetic receptors for engineered ligands

Abstract

The present disclosure relates generally to new synthetic cellular receptors that bind engineered ligands and have selectable specificities and activities. The disclosure also provides compositions and methods useful for producing such receptors, nucleic acids encoding same, host cells genetically modified with the nucleic acids, as well as methods for modulating an activity of a cell and / or for the treatment of various health conditions or diseases, such as cancers.

Description

Attorney Docket No. 048536-729001WOSYNTHETIC RECEPTORS FOR ENGINEERED LIGANDSSTATEMENT REGARDING FEDERALLY SPONSORED R&D

[0001] This invention was made with government support under CA239143 awarded by the National Institutes of Health. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 691,880, filed on September 6, 2024. The disclosure of the above-referenced application is herein expressly incorporated by reference it its entirety, including any drawings.INCORPORATION OF THE SEQUENCE LISTING

[0003] The material in the accompanying Sequence Listing is hereby incorporated by reference into this application. The accompanying Sequence Listing XML file, named 048536- 72900 !WO_SequenceListing_ST26. XML, was created on September 5, 2025, and is approximately 172,032 bytes in size.FIELD

[0004] The present disclosure relates generally to new synthetic cellular receptors that bind engineered ligands and have selectable specificities and / or activities. The disclosure also provides compositions and methods useful for producing such receptors, nucleic acids encoding same, host cells genetically modified with the nucleic acids, as well as methods for modulating an activity of a cell and / or for the treatment of various health conditions or diseases, such as cancers.BACKGROUND

[0005] An important problem which limits the development of engineered cell therapies in humans is the ability to regulate therapeutic gene expression and activity of engineered cells. For example, the first generation of chimeric antigen receptor T cells (CAR-T) lack the ability to modulate or turn off CAR-T activity when needed; other problems include off-target activity and off-tumor / on-target activity. One possible solution to these problems is to utilize synthetic receptors that are capable of modifying gene expression and / or cellular behavior.

[0006] However, despite recent advances in mammalian synthetic biology, there remains aAttorney Docket No. 048536-729001WO lack of modular synthetic receptors that can robustly respond to soluble ligands and in turn activate bespoke cellular functions. Such receptors would have extensive clinical potential to regulate the activity of engineered therapeutic cells, but to date only receptors against cellsurface targets have approached clinical translation (Teng F. et al., Sig Transduct Target Ther 9, 1-25, 2024). Therefore, there remains an urgent need for the development of novel immunotherapies and more tools in order to provide synthetic receptors with a wider range of more advantageously regulatable characteristics, thereby extend the reach of these therapeutics to more diseases and to treat more patients.SUMMARY

[0007] The present disclosure generally relates to, inter alia, novel chimeric polypeptides, e.g., synthetic intramembrane proteolysis receptors (SNIPRs) containing an extracellular ligandbinding domain (ECD) having a binding affinity for an engineered ligand, e.g., an engineered bioorthogonal ligand (orthoLigand). In some embodiments of the disclosure, the ECD of chimeric polypeptides and SNIPRs disclosed herein includes a first monomer polypeptide of a designed a / p heterodimer (LHD) and the engineered ligand includes a second monomer polypeptide of the designed LHD heterodimer. In some embodiments, further provided are (i) recombinant nucleic acids encoding such chimeric polypeptides and / or SNIPRs, (ii) recombinant cells that have been engineered to express a chimeric polypeptide or a SNIPR as disclosed herein. Also provided are pharmaceutical compositions including a recombinant nucleic acid and / or a recombinant cell as disclosed herein.

[0008] In one aspect, provided herein are various chimeric polypeptides including, from N- terminus to C-terminus: (a) an extracellular ligand-binding domain (ECD) including a first monomer polypeptide of a designed a / p heterodimer (LHD) capable of selectively binding to an engineered ligand, wherein the engineered ligand includes a second monomer polypeptide of the designed LHD heterodimer; (b) a linking polypeptide; (c) a transmembrane domain (TMD) from a Type 1 transmembrane receptor including one or more ligand-inducible proteolytic cleavage sites; and (d) an intracellular domain (ICD) including a transcriptional regulator, wherein binding of the engineered ligand to the extracellular binding domain induces cleavage at the one or more ligand-inducible proteolytic cleavage sites.

[0009] Non-limiting exemplary embodiments of the chimeric polypeptides (e.g., polypeptideAttorney Docket No. 048536-729001WO constructs) of the disclosure include one or more of the following features. In some embodiments, the first and the second monomer polypeptides are capable of non-covalently interact to form the designed LHD heterodimer. In some embodiments, wherein the ECD does not substantially bind a naturally occurring ligand. In some embodiments, the engineered ligand is a bioorthogonal ligand. In some embodiments, the engineered ligand is a soluble ligand. In some embodiments, the second monomer polypeptide of the designed LHD heterodimer is fused to one or more designed oligomeric scaffolds. In some embodiments, second monomer polypeptide of the designed LHD heterodimer is capable of binding to the first monomer polypeptide of the designed LHD heterodimer with specific geometry, affinity, and / or valency as determined by EC50 and Emax, respectively.

[0010] In some embodiments, the first monomer polypeptide of a designed LHD heterodimer includes an amino acid sequence selected from SEQ ID NOS: 1-5, and the second monomer polypeptide of the designed LHD heterodimer includes an amino acid sequence selected from SEQ ID NOS: 6-10. In some embodiments, the linking polypeptide includes: (i) a polypeptide hinge domain; (ii) at least about 80% sequence identity to a Notch juxtamembrane domain (JMD); (iii) at least about 80% sequence identity to a Notch JMD wherein the LIN-12-Notch repeat (LNR) and / or a heterodimerization domain (HD) of a Notch receptor has been deleted; (iv) at least about 80% sequence identity to a ROBO1 JMD including at least one fibronectin repeat; or (v) a polypeptide having about 2 to about 40 amino acids.

[0011] In some embodiments of the present disclosure, the chimeric polypeptides of the present disclosure do not include a LIN-12-Notch repeat (LNR) and / or a heterodimerization domain (HD) of a Notch receptor. In some embodiments, the chimeric polypeptides of the present disclosure further include a stop-transfer-sequence (STS) in between the transmembrane domain and the intracellular domain. In some embodiments, the linking polypeptide includes a hinge domain capable of promoting oligomer formation of the chimeric polypeptide via intermolecular disulfide bonding. In some embodiments, the hinge domain is derived from a CD8a hinge domain, a CD28 hinge domain, a PD-1 hinge domain, a CTLA4 hinge domain, an 0X40 hinge domain, an IgGl hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, and an IgG4 hinge domain, or a functional variant of any thereof. In some embodiments, the hinge domain includes one or more mutations at cysteine residues that form the intermolecular disulfide bonds.Attorney Docket No. 048536-729001WG

[0012] In some embodiments of the present disclosure, the ligand-inducible proteolytic cleavage site is a y-sccrctasc cleavage site. In some embodiments, the transcriptional regulator includes a DNA-binding domain and an effector domain, wherein the effector domain selected from the group consisting of a transcription activating domain, a transcription repressor domain, or an epigenetic effector domain. In some embodiments, the effector domain of the transcriptional regulator includes a transcription activating domain selected from the group consisting of herpes simplex virus protein 16 (HSV VP 16) activation domain, an activation domain consisting of four tandem copies of VP 16 (VP64), a p65 activation domain of NFKB, an Epstein-Barr virus R transactivator activation domain (Rta), a tripartite activator consisting of VP64, and Rta activation domains (VPR), and a histone acetyltransferase core domain of the human ElA-associated protein p300 (p300 HAT core activation domain).

[0013] In some embodiments, the effector domain of the transcriptional regulator includes a transcription repressor domain selected from the group consisting of a Kruppel associated box repression domain (KRAB); a Repressor Element Silencing Transcription Factor repression domain (REST); a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins repression domain (WRPW); a DNA (cytosine-5)-methyltransferase 3B repression domain (DNMT3B); and an HP1 alpha chromoshadow repression domain. In some embodiments, the effector domain of the transcriptional regulator includes an epigenetic effector domain selected from the group consisting of a DNA methyltransferase DNMT (DNMT1, DNMT3), HAT1, GCN5, PCAF, MLL, SET, DOTI, SUV39H, G9a, KAT2A / B, EZH1 / 2, TET1 / 2, a SIRT family protein effector domain, a histone deacetylase, LSD1, and a KDM family protein effector domain. In some embodiments, the effector domain includes a domain from a human or humanized polypeptide.

[0014] In some embodiments, the DNA-binding domain (DBD) of the transcriptional regulator includes a GAL4-DBD, a TetR-DBD, a zinc finger (ZF) DBD, or a zinc-finger homeodomain (ZFHD) DBD. In some embodiments, the intracellular domain further includes a nuclear transport signal sequence.

[0015] In another aspect, provided herein are various recombinant nucleic acid molecules including nucleic acid sequences encoding a chimeric polypeptide as disclosed herein. Nonlimiting exemplary embodiments of the recombinant nucleic acid molecules include one or more of the following features. In some embodiments, the nucleotide sequence is incorporated into anAttorney Docket No. 048536-729001WO expression cassette or an expression vector. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, or a retroviral vector. In some embodiments, the recombinant nucleic acid further includes a response element, wherein the response element includes: (a) a cognate target sequence to which the DBD of the transcriptional regulator binds; (b) an engineered responsive promoter operably linked to the cognate target sequence; and (c) a polynucleotide of interest. In some embodiments, the polynucleotide of interest encodes a regulatory RNA, a regulatory protein, a therapeutic protein, or a reporter molecule. In some embodiments, the reporter molecule is a fluorescent protein, a herpes simplex virus type 1 (HSV- 1) thymidine kinase (TK), or a gas vesicle protein. In some embodiments, the therapeutic protein is a recombinant antigen-specific receptor. In some embodiments, the recombinant antigenspecific receptor is an engineered T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the regulatory RNA is a siRNA, shRNA, or miRNA. In some embodiments, the polynucleotide of interest encodes a protein, a regulatory RNA, or an antisense oligonucleotide.

[0016] In another aspect, some embodiments of the disclosure relate to a recombinant cell including: (a) a chimeric polypeptide as disclosed herein; and / or (b) a recombinant nucleic acid as disclosed herein. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some embodiments, the immune cell is a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, a cytotoxic T cell, or other T cell. In some embodiments, the recombinant cell of the disclosure further includes an engineered response element including i) a cognate target sequence to which the DBD of the transcriptional regulator binds, ii) a promoter sequence, wherein the cognate target sequence is operably linked to the 5' end of the promoter sequence, and iii) a polynucleotide of interest operably linked to the promoter sequence, wherein binding of the transcriptional regulator to the cognate target sequence modulates transcription initiation of a polynucleotide of interest. In some embodiments, the chimeric polypeptide is encoded by a first nucleic acid molecule and the engineered response element is encoded by a second nucleic acid molecule. In some embodiments, the engineered response element is present in a nucleic acidAttorney Docket No. 048536-729001WO vector, plasmid, DNA minicircle, minichromosome, or host chromosome.

[0017] In another aspect, some embodiments disclosed herein relate to methods for making a recombinant cell, wherein the method includes (a) providing a cell capable of protein expression; and (b) transducing the cell with a recombinant nucleic acid as disclosed herein to produce the recombinant cell. In some embodiments, the methods include a recombinant nucleic acid including a nucleotide sequence that encodes a chimeric polypeptide as disclosed herein, and the methods further include (c) transducing the cell with a second recombinant nucleic acid that encodes a response element, wherein the response element includes: (i) a cognate target sequence to which the DBD of the transcriptional regulator binds; (ii) an engineered responsive promoter operably linked to the cognate target sequence; and (iii) a polynucleotide of interest.Accordingly, in a related aspect, also provided herein are recombinant cells produced by the methods of the disclosure. In a further related aspect, some embodiments of the disclosure provide cell cultures that include at least one recombinant cell of the disclosure and a culture medium.

[0018] In another aspect, some embodiments of the disclosure relate to a composition, e.g., a pharmaceutical composition, including a pharmaceutically acceptable carrier and one or more of the following: (a) a chimeric polypeptide of the disclosure; (b) a recombinant nucleic acid molecule of the disclosure; and (c) a recombinant cell of the disclosure. In some embodiments, the composition includes a recombinant nucleic acid as disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle. In some embodiments, In some embodiments, the composition includes a recombinant cell of the disclosure and a pharmaceutically acceptable carrier.

[0019] In yet another aspect, some embodiments of the disclosure relate to various methods for modulating an activity of a target cell in a subject, the methods including administering to the subject an effective amount of a chimeric polypeptide as disclosed herein; a recombinant nucleic acid as disclosed herein; and / or an effective number of a recombinant cells as disclosed herein, wherein the administered chimeric polypeptide, recombinant nucleic acid and / or recombinant cells confers a modulation of an activity of the target cell in the subject. Non-limiting exemplary embodiments of the disclosed methods for modulating an activity of a target cell include one or more of the following features. In some embodiments, the target cell is a pathogenic cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the modulation of theAttorney Docket No. 048536-729001WO activity the target cell results in the death of the target cell.

[0020] In yet another aspect, some embodiments of the disclosure relate to various methods for modulating an activity of a cell, the methods including: (a) providing a recombinant cell as disclosed herein; and (b) contacting the recombinant cell with the engineered ligand, wherein binding of the engineered ligand to the extracellular ligand-binding domain results in cleavage of a ligand-inducible proteolytic cleavage site and release of the intracellular domain, wherein the release of the intracellular domain results in modulation of an activity of the recombinant cell. Non-limiting exemplary embodiments of the disclosed methods for modulating an activity of a cell include one or more of the following features. In some embodiments, the contacting is carried out in vivo, ex vivo, or in vitro. In some embodiments, the release of the intracellular domain results in binding of the transcriptional regulator of the released intracellular domain to a cognate target sequence, which results in modulation of the expression initiation of a polynucleotide of interest, which results in modulation of an activity of the recombinant cell. In some embodiments, the activity of the cell to be modulated is selected from the group consisting of: expression of a selected gene, proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of a molecule, cellular adhesion, and cytolytic activity. In some embodiments, the transcriptional regulator modulates expression of an endogenous gene product. In some embodiments, the transcriptional regulator modulates expression of a heterologous gene product. In some embodiments, the gene product is selected from the group consisting of chemokine, a chemokine receptor, a chimeric antigen receptor, a cytokine, a cytokine receptor, a differentiation factor, a growth factor, a growth factor receptor, a hormone, a metabolic enzyme, a pathogen-derived protein, a proliferation inducer, a receptor, an RNA guided nuclease, a site-specific nuclease, a T cell receptor, a toxin, a toxin derived protein, a transcriptional regulator, a transcriptional activator, a transcriptional repressor, a translational regulator, a translational activator, a translational repressor, an activating immuno-receptor, an antibody, an apoptosis inhibitor, an apoptosis inducer, an engineered T cell receptor, an immunoactivator, an immuno-inhibitor, an immune cell receptor, and an inhibiting immuno-receptor. In some embodiments, the released transcriptional regulator modulates differentiation of the cell, and wherein the cell is an immune cell, a stem cell, a progenitor cell, or a precursor cell.

[0021] In yet another aspect, some embodiments of the disclosure relate to various methods for the prevention and / or treatment of a health condition in a subject in need thereof, the methodsAttorney Docket No. 048536-729001WO including administering to the subject a composition including an effective amount of a chimeric polypeptide as disclosed herein; a recombinant nucleic acid as disclosed herein; and / or an effective number of recombinant cells as disclosed herein, wherein the administered chimeric polypeptide, the recombinant nucleic acid, and / or the recombinant cell confers a treatment of the health condition in the subject.

[0022] In another aspect, some embodiments of the disclosure provide various kits for the practice of the methods disclosed herein. Some embodiments of the disclosure relate to kits for modulating an activity of a cell, inhibiting a target cell, or treating a health condition in a subject in need thereof, the kits including one or more of the following: (a) a chimeric polypeptide as disclosed herein; (b) a recombinant nucleic acid as disclosed herein; (c) a recombinant cell as disclosed herein; and a pharmaceutical composition as disclosed herein.

[0023] Some embodiments of the disclosure relate to kits for modulating an activity of a cell, the kits including one or more of the following: (a) a chimeric polypeptide as disclosed herein; (b) a recombinant nucleic acid as disclosed herein; and (c) an engineered response element including: (i) a cognate target sequence to which the DBD of the transcriptional regulator binds; (ii) an engineered responsive promoter operably linked to the cognate target sequence; and (iii) a polynucleotide of interest; wherein binding of the transcriptional regulator to the cognate target sequence modulates transcription initiation of the polynucleotide of interest.

[0024] In another aspect, some embodiments of the disclosure relate to uses of one or more of the following for the prevention and / or treatment of a health condition: (a) a chimeric polypeptide as disclosed herein, (b) a recombinant nucleic acid as disclosed herein, (c) a pharmaceutical composition as disclosed herein.

[0025] In yet another aspect, some embodiments of the disclosure relate to uses of one or more of the following for the manufacturing for the prevention and / or treatment of a health condition: (a) a chimeric polypeptide as disclosed herein, (b) a recombinant nucleic acid as disclosed herein, (c) a pharmaceutical composition as disclosed herein

[0026] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.

[0027] Each of the aspects and embodiments described herein are capable of being usedAttorney Docket No. 048536-729001WO together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

[0028] Throughout this specification, various patents, patent applications and other types of publications (e.g. , j ournal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIGS. 1A-1E summarize the engineering of receptors for soluble cellular communication and disease sensing and illustrate that SNIPR mechanism depends on ligand type. FIG. 1A: Clinical and research applications for engineered receptors that are capable of soluble ligand detection, prgm, program; TF, transcription factor. FIG. IB: Left, activation of a TGF[3-responsive SNIPR or synNotch driving a BFP reporter circuit in primary human CD3+T cells after addition of recombinant human TGF01 (n = 2 technical replicates). Statistics calculated using two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons test; depicted significance corresponds to the comparison of SNIPR versus synNotch. Right, representative histograms of reporter expression level at the indicated concentrations of TGF0. ****p < 0.0001. MFI, mean fluorescence intensity. FIG. 1C: Induction (left) and representative histograms (right) of a VEGF-sensing SNIPR^BFP circuit in primary human T cells through recombinant human VEGF (n = 3 technical replicates). The indicated significance corresponds to the comparison of SNIPR versus synNotch; two-way ANOVA with Sidak’s multiple comparisons test. FIG. ID: Receptor-mediated induction of a BFP reporter gene in primary human CD3+T cells using recombinant human FGF2 (left) or IFNy (right) (n = 2 technical replicates). FIG. IE: Left, structural model of the de-novo-designed LHD heterodimer used to establish a bio-orthogonal receptor-ligand pair. Right, fold activation over background of an orthogonal ligand-responsive SNIPR or synNotch driving a BFP reporter circuit in Jurkat T cells through the introduction of orthoLigand Cl -active (w = 3 technical replicates). Depicted significance corresponds to the comparison of SNIPR versus synNotch; two-way ANOVA with Sidak’s multiple comparisons test.

[0030] FIGS. 2A-1G summarize the results of experiments demonstrating the soluble SNIPR tunability and specificity. FIG. 2A: BFP reporter induction in a panel of SNIPR variants bearingAttorney Docket No. 048536-729001WO scFvs targeted against TGFp (left) or VEGF (right) in VHVL or VLVH orientation in primary human T cells, n = 3 (left) and 2 (right) technical replicates. FIG. 2B: BFP reporter gene induction of primary human T cells expressing SNIPRs containing a set of unique Transcription Activation Domains (TADs) (n = 2 technical replicates). FIG. 2C: BFP reporter induction by conventional or hinge-mutant (hm) TGF and VEGF SNIPR or untransduced T cells (n = 3 technical replicates). Statistics represent difference between SNIPR and hmSNIPR variants computed via two-way ANOVA with Sidak’s multiple comparisons test. FIG. 2D: BFP reporter activation by TGFp (left) and VEGF (right) SNIPRs in primary T cells from three different donors (n = 3 technical replicates per donor). FIG. 2E: Activation of TGFP SNIPR and VEGF SNIPR T cells by their matched or mismatched ligands, respectively (n = 3 technical replicates). Statistics calculated using unpaired two-tailed Welch’s t-test. FIG. 2F: TGFp SNIPR activation in primary human T cells using recombinant active TGFp, recombinant latent TGFp, or recombinant latent TGFp re-activated via heat exposure (n = 3 technical replicates). FIG. 2G: TGFP SNIPR activation in primary human T cells using recombinantly expressed human TGFpi, P2, or P3 protein isoforms (n = 3 technical replicates).

[0031] FIGS. 3A-3F summarize the results of experiments demonstrating that SNIPRs access a distinct activation mechanism. FIG. 3A: Schematic of potential activation pathways for soluble or conventional SNIPRs, along with chemical inhibitors to perturb these reactions. The conventional mechanism of proteolytically regulated receptors depends on ligand-mediated engagement of cell surface proteases (conventionally ADAM10, which is inhibited by the small molecule GI 254023x). The extracellularly truncated receptor becomes a substrate for y- Secretase, whose activity is blocked by DAPT. A proposed alternative pathway depends on endocytic activation of the receptor, where the terminal y-Secretase cleavage step may be mediated by endosomal acidification, which is inhibited by chloroquine. FIG. 3B: Relative activation of primary human T cells by endogenous ligands, or Jurkat T cells by synthetic ligands, in the presence of the chemical inhibitors described in FIG. 3A. T cells expressing soluble SNIPRs or conventional SNIPRs, a conventional synNotch receptor or a proteolysisindependent chemically inducible promoter were selected as a representative panel. In the present disclosure, the term conventional synNotch receptor refers to synNotch receptors containing both the Notch transmembrane domain and also all or part of the Notch NRR region including the portion containing the S2 cleavage site. The term conventional SNIPR refers toAttorney Docket No. 048536-729001WG synNotch receptors containing the gamma secretase-cleavable transmembrane domain from Notch or a related protein substrate, but no LNRs from the Notch receptor. In some embodiments, the conventional SNIPRs of the disclosure containing the gamma secretase- cleavable transmembrane domain from Notch or a related protein substrate, but no NRR region from the Notch receptor. Data are mean of n = 3 technical replicate measurements after 24 h of incubation normalized to the activation of an inhibitor-free control sample. FIG. 3C: Confocal maximum intensity projection images of Jurkat T cells expressing mCherry-fused TGFp SNIPRs in the presence of recombinant TGFpi labelled with AF647. Data are representative of n = 25 cells (at 30 minutes after ligand exposure) or n = 27 cells (at 24 h after ligand exposure) across three independent experiments. Scale bar, 5 pm. FIG. 3D: Left, confocal maximum intensity projection images of TGFp SNIPR Jurkat T cells 24 h after exposure to AF647-labelled TGFpi, costained with LysoTracker. Scale bars, 20 pm (left); 5 pm (right). Right, Pearson’s correlation coefficient between TGFpi and LysoTracker. n = 10 cells; mean ± s.d. Statistics computed using Welch’s unpaired two-tailed / -test. FIG. 3E: Left, confocal images of HeLa cells expressing an orthoSNIPR fused to GFP incubated with mCherry-labelled C6-101 A orthoLigand and stained with LysoTracker marker for 30 min before imaging. Scale bar, 50 pm. Right, Pearson’s correlation coefficient between C6-101 A orthoLigand and LysoTracker. n = 4 images per time point with at least 10 cells per image; mean± s.e.m. Statistics computed using Welch’s unpaired two-tailed / -test. FIG. 3F: Top, schematic of dimerSNIPR architecture. A DmrA homodimerizing domain was inserted into a TGFP-responsive SNIPR between the juxtamembrane domain and the transcription factor. The activation assay was performed in the absence of the extracellular TGFP ligand. Bottom, activation of primary dimerSNIPR T cells by titration of the small molecule API 903 (n = 2 technical replicates).

[0032] FIGS. 4A-4F summarize the results of experiments to investigate the activation mechanism of soluble SNIPRs. FIG. 4A: Left, Schematic of a potential model of mechanosensitive soluble SNIPR activation wherein the mechanical force is applied via the simultaneous binding of a dimeric ligand by two SNIPR-expressing cells in trans. Center: BFP reporter expression histograms depicting a T cell titration assay wherein the TGFp ligand concentration remains constant while the primary T cells are diluted within the same volume. Measurement obtained 48 h after ligand addition. Right, Activation of an orthoLigand-responsive SNIPR driving a BFP reporter circuit in Jurkat T cells via introduction of orthoLigand Cl-activeAttorney Docket No. 048536-729001WO at 500 nM while titrating down the number of receiver cells (n = 3 technical replicates). FIG. 4B: Bright-field (top) and fluorescence (bottom) imaging of primary TGFp SNIPR NLS-mCitrine circuit human T cells in the presence or absence of recombinant TGF i 24 h after ligand exposure. FIG. 4C: Confocal maximum intensity projection images of Jurkat T cells containing a TGFp SNIPR — BFP circuit entrapped in microwells after 24 h of exposure to recombinant TGFpi. FIG. 4D: Left, Schematic of an alternative force-mediated soluble SNIPR activation mechanism wherein mechanical stress is applied via T cell motion while the receptor binds to a putatively soluble ligand which has been immobilized on the cell culture vessel via adsorption. Right, Representative histograms showing TGF SNIPR T cell activation in the presence or absence of 50 ng / mL TGFpi in a conventional polystyrene vs. low-binding culture vessel after 24 hours of ligand exposure. FIG. 4E: BFP reporter expression of TGF SNIPR T cells after addition of 100 ng mF1TGFp and the indicated concentration of chloroquine or vehicle control for 24 h (n = 2 technical replicates). Statistics were computed via two-way ANOVA with Sidak’s multiple comparisons test. FIG. 4F: BFP reporter expression of TGF SNIPR T cells in the presence of 100 ng ml-1TGFp and the indicated concentration of DMSO after 24 h of incubation (n = 2 technical replicates). Vertical dashed lines indicate the final DMSO vehicle concentrations for DAPT, GI254023X, and Chloroquine in FIG. 3B (left). The chloroquine and vehicle concentrations used to assay the synthetic receptors were 4-fold lower than that in the endogenous receptor panel.

[0033] FIGS. 5A-5F summarize the results of experiments performed for the construction of bioorthogonal orthoSNIPR signaling systems and illustrate the expanded landscape of targetable antigens for engineered T cell therapies with soluble SNIPR to CAR circuits. FIG. 5A: Left, design of a series of synthetic ligands with a range of geometries and valencies. Right, activation of an orthoLigand-responsive SNIPR driving a BFP reporter circuit in Jurkat T cells through the introduction of orthoLigands that vary in their geometry and valency (n = 6 technical replicates). FIG. 5B: Illustration demonstrating private and promiscuous signaling channels were created using a set of five heterodimer pairs (left). These pairs were computationally designed to have exclusive binding partners or accommodate promiscuous binding of other heterodimer components. Right, activation of five orthoSNIPRs driving a BFP reporter circuit in Jurkat T cells through the introduction of orthoLigands fused to a C6 scaffolding backbone. Ligands were added at 500 nM concentration (data are mean of n = 3 technical replicates). FIG. 5C: Left,Attorney Docket No. 048536-729001WO schematic of a modulation strategy for orthoLigand-responsive SNIPRs through the introduction of orthoTuners generating a pseudo-C2 structure by homodimerization. Right, BFP reporter activation in Jurkat T cells after the addition of C2 orthoLigand and the orthoTuner dimerization disrupter (n = 3 technical replicates). Statistics calculated using two-way ANOVA with Sidak’s multiple comparisons test. **P = 0.0077; ****P < 0.0001. FIG. 5D: Enhancing orthoLigands by forced lysosomal shuttling. Left, schematic of EndoTag-mediated soluble SNIPR activation. Right, activation of orthoSNIPR4 and orthoSNIPR5 in primary human T cells using recombinant orthoLigand-EndoTag 4 and 5 (respectively) 24 h after ligand exposure (n = 3 technical replicates). FIG. 5E: Activation of three different orthoSNIPRs in primary human T cells using their cognate and mismatched EndoTagged orthoLigands (200 nM, 24 h ligand exposure, representative of n = 3 technical replicates). FIG. 5F: Left, schematic of autonomous cell-to-cell communication through orthoLigand secretion. Right, activation of an orthoLigand-responsive SNIPR driving a BFP reporter circuit in Jurkat T cells through the introduction of orthoLigands secreted from sender HeLa cells (data are mean ± s.e.m. of n = 3 technical replicates). Statistics computed using one-way ANOVA with Dunnett’s multiple comparisons test.

[0034] FIGS. 6A-6B summarize the results of experiments illustrating orthoSNIPR activation tuning. FIG. 6A: Individual activation analysis of the 5 x 5 receptor ligand screen shown in FIG. 5C. n = 3 technical replicates. Error bars represent SEM. FIG. 6B: Activation of Jurkat T cells expressing orthoSNIPR and a corresponding variant bearing the hmSNIPR hinge cysteine substitution using orthoLigand C 1 -active at 200 nM (n = 3 technical replicates; error bars represent SEM). Statistics were computed using Welch’s unpaired Z-test.

[0035] FIGS. 7A-7G summarizes the results of experiments performed for in vivo evaluation of engineered cell-to-cell communication via soluble ligands. FIG. 7A: Schematic of in vitro activation experiment. Cocultured cancer cell lines secrete endogenous soluble factors that activate the cognate SNIPR, driving expression of the downstream BFP reporter. FIG. 7B: Left, in vitro activation of primary human T cells bearing TGF[3 SNIPR in coculture with the respective cancer cell line (n = 3 technical replicates; mean ± s.d ). Right, TGF01 secretion of cancer cell lines, measured by ELISA ( / / = 2 technical replicates). Error bars represent s.d. FIG. 7C: Left, in vitro activation of primary human T cells bearing VEGF SNIPR in coculture with the respective cancer cell line (n = 3 technical replicates; mean ± s.d.). Right, VEGF secretion of cancer cell lines measured by ELISA (n = 2 technical replicates). FIG. 7D: In vitro activation ofAttorney Docket No. 048536-729001WO primary human T cells bearing VEGF SNIPR in transwell cell culture with the respective cancer cell line (n = 3 technical replicates; mean ± s.d.). FIG. 7E: Left, schematic of ligand blocking coculture experiment. BFP reporter activation of primary human TGFP (middle) or VEGF (right) SNIPR T cells in coculture with A549 target cells, or with the addition of recombinant ligand. Activation was performed with and without a simultaneously administered blocking antibody and measured 24 h after treatment. Data are mean of n = 3 technical replicates ± s.d. Statistics computed using Welch’s unpaired two-tailed / -test. FIG. 7F: Relative killing efficacy of TGFp SNIPR^CAR or constitutive HER2 CAR T cells targeting A549RFP'NLScells, measured by Incucyte live-cell imaging. hmSNIPR, hinge mutant SNIPR. FIG. 7G: Incucyte imaging of VEGF SNIPR^CAR or constitutive HER2 CAR T cells targeting A549RFP'NLScells.

[0036] FIGS. 8A-8I summarize the results of experiments demonstrating that soluble antigen sensors can improve CAR-T therapy. FIG. 8A: Quantification of TGFp and VEGF isoform secretion from cancer cell lines using Multiplexing Laser Bead assay. FIG. 8B: Schematic of Incucyte cytotoxicity assay; RFP -labelled tumour cell lines induce CAR expression in soluble SNIPR —> CAR T cells by secreting the cognate SNIPR ligand. FIG. 8C: Induction of a Flag- tagged Her2 CAR by TGF or VEGF SNIPR T cells using titrated recombinant ligand (n = 3 technical replicates). FIG. 8D: Incucyte imaging of CD8+T cells bearing a panel of TGF SNIPR^CAR circuits cocultured with A375RFP'NLScells. FIG. 8E: Quantification of Her2 expression on A375RFP'NLSand early passage A375 cells sourced from ATCC. FIG. 8F: Incucyte cytotoxicity analysis of TGFp SNIPR —> Her2 CAR circuit variants, constitutive Her2 CAR, and untransduced T cells. FIG. 8G: Incucyte cytotoxicity assay of VEGF SNIPR — Her2 CAR, constitutive CAR, and untransduced T cells. FIG. 8H: Comparison of engineered CD19 expression on A549 and M28 target cells. FIG. 81: Incucyte viability analysis of A549 (left) and M28 (right) cells ectopically expressing CD 19 in coculture with TGFP or VEGF SNIPR^CD19 CAR, constitutive CD19 CAR or untransduced T cells.

[0037] FIGS. 9A-9C summarizes the results of experiments performed to evaluate in vivo antitumor efficacy of soluble SNIPR in the context of CAR T cells. FIG. 9A: Schematic of in vivo CAR efficacy and toxicity study. The cross-reactive CAR has previously been shown to induce toxicity, most prominently displayed as a weight-loss phenotype, owing to on-target and off- tumour activity targeting mainly lung tissue. NSG mice were implanted subcutaneously with A549 lung carcinoma or A375 melanoma cells. After 7 days, mice were adoptively transferredAttorney Docket No. 048536-729001WG(i.v.; intravenously) with primary human CD3+T cells bearing soluble SNIPRs driving a mousehuman cross-reactive HERZ CAR, constitutively expressed HER2 CAR or untransduced T cells. Stim., stimulated. FIG. 9B: Tumour volume (top), weight change (bottom), and survival (right) of A549 xenografted mice. Tumour volume statistics analysed using one-way ANOVA with Dunnett’s correction comparing the area under the curve versus the untransduced control group over the course of the experiment. Weight loss statistics computed by one-way ANOVA with Dunnett’s correction comparing mouse weights versus constitutive CAR on the day of peak weight loss (indicated by an arrow). Survival statistics calculated using pairwise log-rank test with Bonferroni correction factor comparing SNIPR^CAR T cells with constitutive CAR and untransduced control groups. ACT, adoptive cell transfer. FIG. 9C: Activity of SNIPR circuits driving thetrDARPin HER2 CAR in A375 melanoma xenografted mice. Data presented analogously to FIG. 9B. Tumour volume statistics analysed using one-way ANOVA with Dunnett’s correction comparing the area under the curve versus the untransduced control group over the course of the experiment. Weight loss statistics computed by one-way ANOVA with Dunnett’s correction comparing mouse weights versus constitutive CAR on the day of peak weight loss (indicated by an arrow). Survival statistics calculated using pairwise log-rank test with Bonferroni correction factor comparing SNIPR^CAR T cells with constitutive CAR and untransduced control groups. NS, not significant.

[0038] FIGS. 10A-10G summarizes the results of experiments performed to evaluate in vivo performance of SNIPR-CAR circuits. FIG. 10A: Tumour volume (left) and Kaplan-Meyer survival plots (right) of TGFp SNIPR, hinge-mutant SNIPR, or constitutive 4D5 anti-Her2 CAR T cells versus untransduced control T cells. Tumour volume statistics compare cumulative area under curve versus untransduced control using one-way ANOVA with Dunnett’s correction. Survival statistics versus untransduced control were calculated using pairwise Log-Rank test with Bonferroni correction factor. FIG. 10B: Activation of TGFp (left) and VEGF (right) SNIPR BFP T cells using recombinant human and mouse ligands (n = 3 technical replicates). FIG.10C: Tumour growth (top), weight change (bottom), and survival (right) of NSG mice bearing A375 xenografts treated with untransduced, constitutive, or SNIPR Her2 DARPin CAR T cells. Statistics are presented analogously to those shown in FIGS. 9C-9D. FIG. 10D: Schematic of CD8 hinge sequence variation in the original 4D5 CAR, original DARPin CAR, and matched truncated DARPin CAR. FIG. 10F: Incucyte comparison of cytotoxic potential of Her2 DARPinAttorney Docket No. 048536-729001WOCARs in original CD8 Hinge or truncated CD8 Hinge architectures and bearing CD28 vs. 4-1 BB costimulatory domains. FIG. 10F: Weight loss phenotype of tumour-free NSG mice injected with constitutivetrDARPin Her2 CAR T cells bearing 4-1BB (top) or CD28 (bottom) costimulatory domains. FIG. 10G: In vivo evaluation of a constitutive or SNIPR-gated mouse / human cross-reactive Mesothelin CAR in an NSG xenograft model. Tumour volume (top), weight change (bottom), and overall mouse survival (right) are shown. Statistics are presented analogously to those shown in FIG. 9C.DETAILED DESCRIPTION OF THE DISCLOSURE

[0039] The present disclosure generally relates to, inter alia, novel chimeric polypeptides, e.g., synthetic intramembrane proteolysis receptors (SNIPRs) containing an extracellular ligandbinding domain (ECD) having a binding affinity for an engineered ligand, e.g., an engineered bioorthogonal ligand. In some embodiments of the disclosure, the ECD of chimeric polypeptides and SNIPRs disclosed herein includes a first monomer polypeptide of a designed a / p heterodimer (LHD) and the engineered ligand includes a second monomer polypeptide of the designed LHD heterodimer. In some embodiments, the SNIPRs of the disclosure are bioorthogonal ligand-responsive SNIPRs that incorporate a designed LHD heterodimer comprising two biorthogonal and modular sets of interacting proteins (e.g., two monomer polypeptides), which are configured such that they are folded and soluble when alone but rapidly and specifically associate with one another to form the designed LHD heterodimer when mixed. In the bioorthogonal ligand-responsive SNIPRs of the disclosure, the first monomer polypeptide of the designed LHD heterodimer in incorporated in the ECD of the SNIPRs and the second monomer polypeptide of the designed LHD heterodimer is incorporated in the engineered bioorthogonal ligand. In some embodiments, further provided are (i) recombinant nucleic acids encoding such chimeric polypeptides and / or SNIPRs, (ii) recombinant cells that have been engineered to express a chimeric polypeptide or a SNIPR as disclosed herein. Also provided are pharmaceutical compositions including a recombinant nucleic acid and / or a recombinant cell as disclosed herein. The disclosure also provides methods for modulating an activity of a cell and / or for the treatment of various health conditions.

[0040] The fundamental basis of biochemical signal transduction lies in the ability of cells to produce, sense, and react to small diffusible molecules. This capability allows them to coordinateAttorney Docket No. 048536-729001WG intricate functions and respond to environmental stimuli beyond their immediate vicinity. For instance, morphogens play a critical role in shaping the three-dimensional pattern of an embryo during development (Schwank G & Basler K Cold Spring Harb Per sped Biol 2, 2010), while cytokines are involved in shaping cell state changes and recruiting a diverse array of immune cells to the site of disease (Berraondo P et al. Br J Cancer 120, 6-15, 2019). The ability to mimic these natural systems and interface with soluble factors in synthetic biological systems would enable engineered cells to integrate signals from distant sources and activate therapeutic programs. Furthermore, artificial signaling molecules could provide privileged communication channels between cells to specifically engage engineered cells after administration to patients. Without being bound to any particular theory, it is believed that using these bioorthogonal channels of communication, engineered cells could promote large-scale cellular coordination, facilitating complex multicellular behavior and delivering synergistic therapeutic benefits.

[0041] Despite this potential, progress toward developing sensitive, robust, and modular biosensors for soluble factors has been limited. Chimeric antigen receptor (CAR)-T cells capable of sensing TGF-0 and other soluble factors have been demonstrated (Chang ZL et al., Nat Protoc 1-18, 2020); however, they can only induce a native T cell transcriptional response and a subset of T cell functions such as cytokine secretion (Chang ZL et al., Nature Chemical Biology 14, 317-324, 2018). Immune cell activities that drive inflammation and cytotoxicity are not always desirable, especially in scenarios where the cell is simply a smart delivery agent for therapeutic molecules. Additionally, strategies employing endogenous stimulation-responsive promoters to drive payload transgene expression are often hampered by the relatively weak activity of such promoters in primary immune cells (Guo T. et al., ACS Synth. Biol., 2022, doi:10.1021 / acssynbio. lc00236 and Uchibori R. et al., Moi ' Ther Oncolytics 12, 16-25, 2018). The MESA receptor platform couples modular ligand sensing to custom transcriptional output, but achieving high sensitivity and dynamic range in therapeutic cell types remains a challenge, and reliance on viral components and a multi-chain architecture complicate translation (Schwarz KA et al., Nat Chem Biol 13, 202-209, 2017). TanGo (Kroeze WK et al., Nat Struct Mol Biol 22, 362-369, 2015) and ChaCha (Kipniss NH et al., Nat Commun 8, 1-10, 2017) are GPCR- fused protease architectures that offer an alternative approach but still require multiple components, which can constrain the use of therapeutic delivery vehicles like lentiviral or adeno- associated viral vectors. Moreover, these GPCR-based designs lack the flexibility in ligandAttorney Docket No. 048536-729001WO selection that is inherent to receptors using modular binding domains (e.g., scFvs). The OCAR platform also relies on co-delivery of two receptor chains to allow for ligand-induced dimerization and activation (Mahameed M et al., Nat Commun 13, 7350, 2022). A compact, single-chain receptor capable of modularly sensing soluble factors would overcome the limitations of current systems and unlock the potential for engineered receptors to coordinate therapeutic genetic programs in clinically-relevant cell types.

[0042] Ideally, receptors used for soluble factor detection must operate with high fidelity, generating strong signals in the ON state while minimizing basal signaling in the OFF state. These receptors should also be compact to enable efficient delivery to relevant immune cell types and minimize potential issues related to subunit stoichiometry. The synthetic Notch Receptor (synNotch) represents a prototypical engineered receptor, employing cleavage by endogenous y- secretase to release its transcription factor upon binding a cell surface ligand and demonstrating robust activation in therapeutically relevant cell types including CAR-T cells (Morsut L et al., Cell 164, 780-791 , 2016 and Roybal KT et al, Cell 167, 419-432. el 6, 2016). Recently, a new receptor with a Notch-based architecture, the SyNthetic Intramembrane Proteolysis Receptor (SNIPR), was developed. SNIPRs provide a compact and readily tunable scaffold for custom signal transduction along the Notch cleavage paradigm (Zhu I. et al., Cell 185, 1431-1443. el6, 2022). The ability of SNIPRs to respond to ligand binding robustly and selectively despite the omission of the Notch LNR regulatory domains, widely thought to be the key determinants of Notch activation, implies that SNIPRs may employ an alternate signaling pathway that bypasses the mechanosensing filter which precludes Notch and synNotch from detecting soluble ligands. Applicant set out to investigate whether the SNIPR system could be adapted to sense soluble ligands, which could enable a wide variety of applications (see, e.g., FIG. 1A).

[0043] As will be discussed more thoroughly herein, Applicant has developed a receptor architecture called synthetic intramembrane proteolysis receptor (SNIPR), that has the added ability to be activated by soluble ligands, both natural and synthetic, with remarkably low baseline activity and high fold activation, through an endocytic, pH-dependent cleavage mechanism. In particular, Applicant has demonstrated the therapeutic capabilities of the receptor platform by localizing the activity of CAR T cells to solid tumors where soluble disease- associated factors are expressed, bypassing the major hurdle of on-target off-tumor toxicity in bystander organs. Applicant further applied the SNIPR platform to engineer fully syntheticAttorney Docket No. 048536-729001WO signaling networks between cells orthogonal to natural signaling pathways, expanding the scope of synthetic biology. The design framework described herein enables cellular communication and environmental interactions, extending the capabilities of synthetic cellular networking in clinical and research contexts.

[0044] The experimental data described herein confirm that SNIPRs can utilize an alternative signaling pathway, allowing for robust and selective responses to soluble ligand binding. SNIPRs possess the unique capability to interact with a range of physiological or synthetic ligands, whether they are tethered or soluble. This versatile feature makes them an asset to the current array of cell-engineering tools, with potential applications in cancer therapy, and engineering development. Here, Applicant has shown that T cells engineered with SNIPR technology can effectively react to soluble factors and drive therapeutic payload production in solid tumor animal models. Moreover, Applicant also shows that SNIPRs can serve as a fundamental building block for constructing modular bioorthogonal communication systems in cells that can operate independently of natural signaling pathways.

[0045] The SNIPR architecture satisfies the demands of high-performance soluble factor sensing. SNIPRs bearing ligand binding domains targeting cancer-associated factors show robust activation upon titration of recombinant ligands and are capable of driving potent therapeutic responses at the site of disease, mitigating the toxicity potential of cell therapies. The ability of SNIPRs to recognize engineered orthogonal ligands expands their potential capabilities beyond natural cues and unidirectional signaling. Immune cells bearing orthoSNIPRs could coordinate to establish signaling feedback loops to more precisely pinpoint tumor coordinates within the body, and different immune cell types bearing their own receptors and payloads could interact to activate specific programs at the optimal times during treatment stages. Customization of orthoSNIPR signaling using privileged or promiscuous signaling channels enables fine tuning of engineered cell behavior, while the capability to sense and secrete endogenous signaling factors provides an interface with the local host immune environment.

[0046] The ability to increase soluble SNIPR activity with a cysteine substitution in the hinge domain suggests that additional mechanisms are available to fine-tune the response profile of these receptors. Additionally, the high response of the VEGF SNIPR T cells to A549 cell, which appear to secrete relatively little VEGF, suggests that the receptor may trigger in response to a combination of ligand states, perhaps fully soluble plus membrane-bound ligand using theAttorney Docket No. 048536-729001WO conventional activation pathway. Unraveling this behavior may provide further avenues to shape SNIPR T cell responses to the surrounding microenvironment.

[0047] Overall, soluble SNIPRs provide a versatile tool for therapeutic bioengineering and other disciplines in biology. The customizability of these receptors could enable them to sense morphogen gradients established by proteins such as NGF and the BMP family during embryonic development, or report on the local immune state in the context of cancer, autoimmunity, and infectious disease. These receptors are also suitable for use in developing complex high-order biocomputational circuits owing to their programmability via synthetic ligands and compatibility with modular payloads. Their compact genetic footprint may permit the development of multi -receptor circuits that integrate various soluble or cell surface inputs for precise localization of biological activity34. SNIPRs are an expanding platform for precise control of cells for therapeutic purposes and basic biology applications.Attorney Docket No. 048536-729001WODEFINITIONS

[0048] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0049] The singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A,” “B,” “A or B,” and “A and B.”

[0050] The terms “cell,” “cell culture,” “cell line” refer not only to the particular subject cell, cell culture, or cell line but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g, deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the originally cell, cell culture, or cell line.

[0051] The term “construct” refers to a recombinant molecule, e.g., a recombinant nucleic acid or a recombinant polypeptide, including one or more isolated sequences (e.g., nucleic acid sequence or amino acid sequences) from heterologous sources. For example, nucleic acid constructs can be chimeric nucleic acid molecules in which two or more nucleic acid sequences of different origin are assembled (e.g., operably linked) into a single nucleic acid molecule. Similarly, polypeptide constructs can be chimeric polypeptide molecules in which two or more amino acid sequences of different origin are assembled (e.g., operably linked) into a single polypeptide molecule.

[0052] As used herein, the term “chimeric antigen receptor” (CAR) refers to a polypeptideAttorney Docket No. 048536-729001WO construct comprising at least an extracellular antigen-binding domain, a TMD and a cytoplasmic signaling domain (also referred to as “an intracellular signaling domain” or ICD). In some cases, the cytoplasmic signaling domain includes a functional signaling domain derived from a stimulatory molecule. In some embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. Optionally, the ICD can further include one or more functional signaling domains derived from at least one costimulatory molecule.

[0053] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein, and refer to both RNA and DNA molecules, including nucleic acid molecules comprising cDNA, genomic DNA, synthetic DNA, and DNA or RNA molecules containing nucleic acid analogs. A nucleic acid molecule can be double-stranded or single-stranded (e.g., a sense strand or an antisense strand). A nucleic acid molecule may contain unconventional or modified nucleotides. The terms "polynucleotide sequence" and "nucleic acid sequence" as used herein interchangeably refer to the sequence of a polynucleotide molecule.

[0054] The term “operably linked”, as used herein, denotes a physical or functional linkage between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion. It should be understood that, operably linked elements may be contiguous or non-contiguous.

[0055] As used herein, the terms “orthogonal” and “bioorthogonal” in reference to a receptor or ligand refers to those receptors and ligands that are so dissimilar from those which are naturally occurring in nature (e.g., those are not found or are rarely represented) in living cells. In some embodiments, a bioorthogonal receptor or ligand refers to those that are not found in vivo, e.g., in living cells in nature.

[0056] The term “percent identity” as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be “substantially identical.” ThisAttorney Docket No. 048536-729001WG definition also refers to, or may be applied to, the complement of a sequence. This definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. Such modifications can occur naturally or synthetically. In some embodiments, sequence identity can be calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol 215:403, 1990). In some embodiments, sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof. Additional methodologies that can suitably be utilized to determine structural similarity or identity amino acid sequences include those relying on position-specific structure-scoring matrix (P3SM) that incorporates structure-prediction scores from Rosetta, as well as those based on a length-normalized edit distance as described previously in, e.g., Setcliff et al., Cell Host & Microbe 23(6), May 2018.

[0057] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human subjects) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. The subject can also be an individual who is diagnosed with a risk of the condition of interest at the time of diagnosis or later. The term "non-human animals" includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non- mammals, such as non-human primates, e.g., sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0058] The term "vector" is used herein to refer to a nucleic acid molecule or sequence capable of transferring or transporting another nucleic acid molecule. For example, a vector can be used as a gene delivery vehicle to transfer a gene into a cell. The transferred nucleic acid molecule is generally linked to, e.g., inserted into, the vector nucleic acid molecule. Generally, a vector is capable of replication when associated with the proper control elements. The term "vector" includes cloning vectors and expression vectors, as well as viral vectors and integrating vectors. An "expression vector" is a vector that includes a regulatory region, thereby capable ofAttorney Docket No. 048536-729001WO expressing DNA sequences and fragments in vitro and / or in vivo. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, e.g., replication defective retroviruses and lentiviruses. In some embodiments, a vector is a gene delivery vector.

[0059] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising,” particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0060] Headings, e.g., (a), (b), (i) etc., are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0061] As will be understood by one having ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a groupAttorney Docket No. 048536-729001WO having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0062] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.

[0063] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combinations was individually and explicitly disclosed herein.COMPOSITIONS OF THE DISCLOSURE

[0064] As described in greater detail below, one aspect of the present disclosure relates to novel chimeric polypeptides and synthetic intramembrane proteolysis receptors (SNIPRs) that include an extracellular ligand-binding domain (ECD) having a binding affinity for a bioorthogonal ligand. In some embodiments, further provided are (i) recombinant nucleic acids encoding such chimeric polypeptides and / or SNIPRs, (ii) recombinant cells that have been engineered to express a chimeric polypeptide or a SNIPR as disclosed herein. Also provided are pharmaceutical compositions including a recombinant nucleic acid and / or a recombinant cell as disclosed herein.Attorney Docket No. 048536-729001WOChimeric Polypeptides and Synthetic Jntramembrane Proteolysis Receptors (SNIPRs)

[0065] In one aspect, some embodiments disclosed herein relate to novel chimeric polypeptides and SNIPRs (e.g., orthoSNIPRs). The chimeric polypeptides and SNIPRs of the disclosure generally contain an extracellular ligand-binding domain (ECD) having a binding affinity for an engineered ligand, e.g., an engineered bioorthogonal ligand. In some embodiments of the disclosure, the ECD of chimeric polypeptides and SNIPRs disclosed herein includes a first monomer polypeptide of a designed a / 0 heterodimer (LHD) and the engineered ligand includes a second monomer polypeptide of the designed LHD heterodimer.

[0066] Implementations of embodiments of the chimeric polypeptides (e.g., SNIPRs) of the disclosure can include one or more of the following features.Extracellular ligand-binding domains (ECD)

[0067] In some embodiments, an extracellular ligand-binding domain (ECD) of the chimeric polypeptides or SNIPRs (e.g., orthoSNIPRs) disclosed herein includes a first monomer polypeptide of a designed a / p heterodimer (LHD) capable of selectively binding to an engineered ligand, wherein the engineered ligand comprises a second monomer polypeptide of the designed LHD heterodimer.

[0068] In some embodiments, the first and the second monomer polypeptides are capable of (e.g., configured for) non-covalently interacting with one another to form the designed LHD heterodimer. In some embodiments, the SNIPRs of the disclosure are bioorthogonal ligand- responsive SNIPRs that incorporate a designed LHD heterodimer comprising two biorthogonal and modular sets of interacting proteins (e.g., first and second monomer polypeptides) which are configured such that they are folded and soluble when alone but rapidly and specifically associate with one another to form the designed LHD heterodimer when mixed. In some embodiments, the ECD does not substantially bind a naturally occurring ligand. In some embodiments, the engineered ligand is a bioorthogonal ligand. One of ordinary skill in the art will understand that the term “bioorthogonal” when used in referencing a chemical compound, e.g., a ligand, means the ligand is so dissimilar from that which is naturally occurring in nature (e.g., ligand not found or are rarely represented) in living cells. In some embodiments, the bioorthogonal ligand of the disclosure is not found in vivo, e.g., in living cells in nature. In some embodiments, the engineered ligand is a soluble ligand. In some embodiments, the secondAttorney Docket No. 048536-729001WO monomer polypeptide of the designed LHD heterodimer is fused to one or more designed oligomeric scaffolds. In some embodiments, second monomer polypeptide of the designed LHD heterodimer is capable of binding to the first monomer polypeptide of the designed LHD heterodimer with specific geometry, affinity, and / or valency as determined by EC50 and Emax, respectively. One skilled in the art will appreciate that LHD heterodimers with specific geometry, affinity, and / or valency binding of the first monomer polypeptide to the second monomer polypeptide can be designed using publicly available computational design tools such as RFdiffusion, which is an open source method for structure generation, with or without conditional information, such as a motif, a target, etc. (see, e.g., Watson JL et al. Nature 620, 1089-1100, 2023, which is incorporated herein by reference). De novo design of LHD heterodimers with specific geometry, affinity, and / or valency binding of the first monomer polypeptide to the second monomer polypeptide can also be achieved using Rosetta Commons (available at https: / / rosettacommons.org / software / ). Additional information in this regard can be found in, for example, Sahtoe DD et al., Science, 2022, which is expressly incorporated herein by reference.

[0069] Exemplary designed LHD heterodimers in accordance with various embodiments of the disclosure are provided in Table 1, as well as the respective first and second monomer polypeptides.TABLE 1 : Exemplary designed LHD heterodimers.

[0070] Additional polypeptides suitable for use as the first and / or the second monomer polypeptides to form the designed LHD heterodimer as disclosed herein include those described in PCT application PCT / US2019 / 059654, which is expressly incorporated herein by reference.

[0071] In some embodiments, the first monomer polypeptide of a designed LHD heterodimerAttorney Docket No. 048536-729001WO includes an amino acid sequence selected from SEQ ID NOS: 1-5, and the second monomer polypeptide of the designed LHD heterodimer includes an amino acid sequence selected from SEQ ID NOS: 6-10. In some embodiments, the first and second monomer polypeptides of the designed LHD heterodimer include an amino acid sequence of SEQ ID NO: 1 and 6, respectively. In some embodiments, the first and second monomer polypeptides of the designed LHD heterodimer include an amino acid sequence of SEQ ID NO: 1 and 6, respectively. In some embodiments, the first and second monomer polypeptides of the designed LHD heterodimer include an amino acid sequence of SEQ ID NO: 2 and 7, respectively. In some embodiments, the first and second monomer polypeptides of the designed LHD heterodimer include an amino acid sequence of SEQ ID NO: 3 and 8, respectively. In some embodiments, the first and second monomer polypeptides of the designed LHD heterodimer include an amino acid sequence of SEQ ID NO: 4 and 9, respectively. In some embodiments, the first and second monomer polypeptides of the designed LHD heterodimer include an amino acid sequence of SEQ ID NO: 5 and 10, respectively.Linking polypeptide / juxtamembrane domain (JMD)

[0072] As described in greater detail below, the extracellular domains located N-terminally to the transmembrane domain (TMD) of the chimeric polypeptides or SNIPRs (e.g., orthoSNIPRs) of the disclosure include a linking polypeptide sequence disposed between the extracellular ligand-binding domain (ECD) and the transmembrane domain (TMD). In some embodiments, the length and amino acid composition of the linking polypeptide sequence can be optimized to vary the orientation and / or proximity of ECD and TMD relative to one another to achieve a desired activity of the chimeric polypeptides and SNIPRs as disclosed herein. In some embodiments, the length and amino acid composition of the linking polypeptide sequence can be varied as a “tuning” tool to achieve a tuning effect that would enhance or reduce the biological activity of the disclosed chimeric polypeptides and receptors. Additional information regarding the relative glycine content, length, amino acid composition, and the flexibility / stiffness of glycerin-serine linking polypeptide can be determined by any methodologies known in the art as suitable for such purposes, for example as determined by Forster resonance energy transfer (FRET) efficiencies as described in Rosmalen M. et aL, Biochemistry (2017), 56:6565-74.

[0073] In some embodiments, an single-chain peptide including about two to 100 amino acid residues (aa) e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acidAttorney Docket No. 048536-729001WG residues) is used as a linking polypeptide in the disclosed chimeric polypeptides. Tn some embodiments, the linking polypeptide sequence has a length ranging from about 5 to about 50, about 10 to about 60, about 20 to about 70, about 30 to about 80, about 40 to about 90, about 50 to about 100, about 60 to about 80, about 70 to about 100, about 30 to about 60, about 20 to about 80, about 30 to about 90 amino acid residues. In some embodiments, the linking polypeptide sequence has a length ranging from about 1 to about 10, about 5 to about 15, about 10 to about 20, about 15 to about 25, about 20 to about 40, about 30 to about 50, about 40 to about 60, about 50 to about 70 amino acid residues. In some embodiments, the linking polypeptide sequence has a length ranging from about 40 to about 70, about 50 to about 80, about 60 to 8 about 0, about 70 to about 90, or about 80 to about 100 amino acid residues. In some embodiments, the linking polypeptide sequence has a length ranging from about 1 to about 10, about 5 to about 15, about 10 to about 20, about 15 to about 25 amino acid residues.

[0074] In some embodiments, the linking polypeptide sequence has a length ranging from about 1 to about 40 amino acid residues. In some embodiments, the linking polypeptide sequence has a length ranging from 1 to about 10, about 5 to about 20, about 10 to about 30, about 15 to about 40, about 10 to about 40, about 15 to about 40, about 20 to about 40, about 25 to about 40, about 30 to about 40, about 5 to about 0, about 15 to about 30 amino acid residues. In some embodiments, the linking polypeptide sequence has a length ranging from about 5 to about 40, about 10 to about 35, about 15 to about 35, about 20 to about 35, about 5 to about 20, about 5 to about 25, about 5 to about 30, about 5 to about 35 amino acid residues.

[0075] In certain embodiments, the linking polypeptide contains only glycine and / or serine residues (e.g., glycine-serine linking polypeptide). Examples of such linking polypeptides include: Gly, Ser; Gly Gly Ser; Ser Gly Gly; Gly Ser Gly; Gly Gly Gly Ser; Ser Gly Gly Gly; Ser Gly Ser Gly; Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly; Gly Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly Gly; Gly Gly Gly Gly Gly Gly Ser; Ser Gly Gly Gly Gly Gly Gly; (Gly Gly Gly Gly Ser)n, wherein n is an integer of one or more; and (Ser Gly Gly Gly Gly)n, wherein n is an integer of one or more. In some embodiments, the linking polypeptide sequence includes at least one glycine residue. In some embodiments, the linking polypeptide sequence includes at least one serine residue. In some embodiments, the linking polypeptide sequences are modified such that the amino acid sequence Gly Ser Gly (GSG) (that occurs at the junction of traditional Gly / Ser linker polypeptide repeats) is not present. For example, in some embodiments, theAttorney Docket No. 048536-729001WO linking polypeptide includes an amino acid sequence selected from the group consisting of: (GGGXX)nGGGGS and GGGGS(XGGGS)n, where X is any amino acid that can be inserted into the sequence and not result in a polypeptide comprising the sequence GSG, and n is 0 to 4. In some embodiments, the sequence of a linking polypeptide is (GGGXlX2)nGGGGS and XI is P and X2 is S and n is 0 to 4. In some embodiments, the sequence of a linking polypeptide is (GGGXlX2)nGGGGS and XI is G and X2 is Q and n is 0 to 4. In some other embodiments, the sequence of a linking polypeptide is (GGGXlX2)nGGGGS and XI is G and X2 is A and n is 0 to 4. In some embodiments, the sequence of a linking polypeptide is GGGGS(XGGGS)n, and X is P and n is 0 to 4. In some embodiments, a linking polypeptide of the disclosure comprises or consists of the amino acid sequence (GGGGA^GGGGS. In some embodiments, a linking polypeptide comprises or consists of the amino acid sequence (GGGGQ)2GGGGS. In some embodiments, a linking polypeptide comprises or consists of the amino acid sequence (GGGPS)2GGGGS. In some embodiments, a linking polypeptide comprises or consists of the amino acid sequence GGGGS(PGGGS)2.

[0076] In some embodiments, a linking polypeptide the amino acid sequence (GGS)n wherein n is an integer from 1 to 50, for example, from 1 to 10, from 5 to 15, from 10 to 20, from 15 to 25, from 20 to 30, from 25 to 35, from 30 to 40, from 35 to 45, or from 40 to 50. In some embodiments, a linking polypeptide the amino acid sequence (GGS)n wherein n is an integer from 1 to 10. In some embodiments, a linking polypeptide the amino acid sequence (GGS)n wherein n is an integer from 10 to 20. In some embodiments, a linking polypeptide the amino acid sequence (GGS)n wherein n is an integer from 20 to 30. In some embodiments, a linking polypeptide the amino acid sequence (GGS)n wherein n is an integer from 30 to 40. In some embodiments, a linking polypeptide the amino acid sequence (GGS)n wherein n is an integer from 40 to 50. In some embodiments, n is 18, i.e., (GGS)is. In some embodiments, n is 15, i.e., (GGS)i5. In some embodiments, n is 12, z.e., (GGS)i2. In some embodiments, n is 9, z.e., (GGS)$>. In some embodiments, n is 6, i.e., (GGS)e. In some embodiments, n is 3, z.e., (GGS)a. In some embodiments, the sequence is selected so that it does not include a common protease cleavage site. In some embodiments, the sequence is selected so that it does not include a glycosylation site.

[0077] In some embodiments, the linking polypeptide has substantial sequence identity with a Notch juxtamembrane domain (JMD), which corresponds to a distinct extracellular domainAttorney Docket No. 048536-729001WO located between the heterodimerization domain (HD) and the transmembrane domain (TMD) of a Notch receptor. More information about Notch JMD can be found in Deatherage CL. et al, Sci. Adv., Apr 12;3(4), 2017 and Sulis ML et al., Blood, 112 (3): 733-740, August 2008. In some embodiments, the Notch receptor JMD is partly or completely devoid of the negative regulatory region (NRR) and / or the heterodimerization domain (HD). In some embodiments, the Notch JMD sequence may be the JMD sequence from Notch 1, Notch2, Notch3, or Notch4, and can be derived from a non-human homolog, such as those from Drosophila, Gallus, Danio, and the like. Four to 50 amino acid residues of the remaining Notch sequence (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, amino acid residues) can be used as a polypeptide linker. In some embodiments, the length and amino acid composition of the linker polypeptide sequence are varied to alter the orientation and / or proximity of the ECD and the TMD relative to one another to achieve a desired activity of the chimeric polypeptide, such as the signal transduction level when ligand induced or in the absence of ligand.

[0078] In some embodiments, the linking polypeptide has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a Notch JMD wherein a LIN-12-Notch repeat (LNR) and / or a HD of a Notch receptor has been deleted. In some embodiments, the linking polypeptide has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a Notch JMD wherein at least one LNR of a Notch receptor has been deleted. In some embodiments, the linking polypeptide has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a Notch JMD wherein at least two LNRs of a Notch receptor has been deleted. In some embodiments, the linking polypeptide has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a Notch JMD wherein one, or two, or all three LNRs of a Notch receptor has been deleted. In some embodiments, the linking polypeptide has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a Notch JMD which does not include a Notch NRR or HD of a Notch receptor, e.g., complete absence of the Notch extracellular subunit (NEC).Hinge linkers

[0079] As described in greater detail herein, the linking polypeptide of the chimeric polypeptides or SNIPRs (e.g., orthoSNIPRs) in accordance with some embodiments of the disclosure incorporate a hinge linker, which is an extracellular oligomerization domain to promote formation of oligomeric forms, e.g., dimeric or trimeric form of the chimericAttorney Docket No. 048536-729001WO polypeptides. It is believed, without being bound by any theory, that this design allows oligomerization / clustering of extracellular domains (ECD) and subsequently brings together intracellular domains (ICD) to activate cell signaling, e.g., T-cell signaling. In these instances, the Notch ECDs located N-terminally to the TMD include an oligomerization domain (e.g., a polypeptide hinge domain) containing one or more polypeptide motifs that promote oligomer formation of the chimeric polypeptides via intermolecular disulfide bonding. In these instances, the hinge domain generally includes a flexible oligo- or polypeptide connector region disposed between the ECD and the TMD. Thus, the polypeptide hinge domain provides flexibility between the ECD and TMD and also provides sites for intermolecular disulfide bonding between two or more chimeric polypeptide monomers to form an oligomeric complex. In some embodiments, the hinge domain includes motifs that promote dimer formation of the chimeric polypeptides disclosed herein. In some embodiments, the hinge domain includes motifs that promote trimer formation of the chimeric polypeptides disclosed herein e.g., a hinge domain derived from 0X40).

[0080] Hinge polypeptide sequences suitable for the compositions and methods of the disclosure can be naturally-occurring hinge polypeptide sequences (e.g., those from naturally- occurring immunoglobulins). Alternatively, a hinge polypeptide sequence can be a synthetic sequence that corresponds to a naturally-occurring hinge polypeptide sequence, or can be an entirely synthetic hinge sequence, or can be engineered, designed, or modified to provide desired and / or improved properties, e.g., modulating transcription. Suitable hinge polypeptide sequences include, but are not limited to, those derived from IgA, IgD, and IgG subclasses, such as IgGl hinge domain, IgG2 hinge domain, IgG3 hinge domain, and IgG4 hinge domain, or a functional variant thereof. In some embodiments, the hinge polypeptide sequence contains one or more CXXC motifs. In some embodiments, the hinge polypeptide sequence contains one or more CPPC motifs. Additional information in this regard can be found in, for example, a recent review by Vidarsson G. et al., Frontiers Immunol. (October 20, 2014) 5:520, which is hereby incorporated by reference in its entirety.

[0081] Accordingly, in some embodiments, the hinge domain of the chimeric Notch receptors disclosed herein includes a hinge polypeptide sequence derived from an IgGl hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgG2 hinge domain or a functional variant thereof. In someAttorney Docket No. 048536-729001WO embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgG3 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgG4 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgA hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgD hinge domain or a functional variant thereof.

[0082] Additional hinge polypeptide sequences suitable for the compositions and methods disclosed herein include, but are not limited to, hinge polypeptide sequences derived from a CD8a hinge domain, a CD28 hinge domain, a CD 152 hinge domain, a PD-1 hinge domain, a CTLA4 hinge domain, an 0X40 hinge domain, an FcyRIIIoc hinge domain, and functional variants thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from a CD8oc hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from a CD28 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an 0X40 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgG4 hinge domain or a functional variant thereof.

[0083] The length and / or amino acid composition of the hinge domain are selected to confer flexibility and the capacity for oligomerization. One skilled in the art will readily appreciate that the length and amino acid composition of the hinge polypeptide sequence can be optimized to vary the orientation and / or proximity of the ECD and the TMD relative to one another, as well as of the chimeric polypeptide monomers to one another, to achieve a desired activity of the chimeric polypeptide of the disclosure. In some embodiments, a single-chain peptide including about one to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as a hinge domain. In some embodiments, the hinge domain includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the hinge domain includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the hinge domain includes about 40 to 70, about 50Attorney Docket No. 048536-729001WO to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. Tn some embodiments, the hinge domain includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues.

[0084] In some embodiments, the linking polypeptide includes a hinge domain capable of promoting oligomer formation of the chimeric polypeptide via intermolecular disulfide bonding. In some embodiments, the hinge domain is derived from a CD8a hinge domain, a CD28 hinge domain, a PD-1 hinge domain, a CTLA4 hinge domain, an 0X40 hinge domain, an IgGl hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, and an IgG4 hinge domain, or a functional variant of any thereof. In some embodiments, the hinge domain includes one or more mutations at cysteine residues that form the intermolecular disulfide bonds.

[0085] In some embodiments, the linking polypeptide is derived from the Robol JMD, which contains a fibronectin repeat (Fn) domain, with a short polypeptide sequence between the Fn repeats and the TMD. The Fn Notch linking polypeptide does not contain a Notch negative regulatory region (NRR), or the Notch HD domain. The Fn linking polypeptide can contain 1, 2, 3, 4, or 5 Fn repeats. In some embodiments, the chimeric polypeptides comprises a Fn linking polypeptide having about 1 to about 5 Fn repeats, about 1 to about 3 Fn repeats, or about 2 to about 3 Fn repeats. The short polypeptide sequence between the Fn repeats and the TMD can be from about 2 to about 30 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, amino acid residues). In some embodiments, the short polypeptide sequence can be between about 5 and about 20 amino acids, of any sequence. In some embodiments, the short polypeptide sequence can be between about 5 and about 20 naturally- occurring amino acids, of any sequence. In some embodiments, the short polypeptide sequence can be between about 5 and about 20 amino acids, of any sequence but having no more than one proline. In some embodiments, the short polypeptide sequence can be between about 5 and about 20 amino acids, and about 50% or more of the amino acids are glycine. In some embodiments, the short polypeptide sequence can be between about 5 and about 20 amino acids, where the amino acids are selected from glycine, serine, threonine, and alanine. In some embodiments, the length and amino acid composition of the Fn linking polypeptide sequence can be varied to alter the orientation and / or proximity of the ECD and the TMD relative to one another to achieve a desired activity of the chimeric polypeptide of the disclosure.

[0086] In some embodiments of the present disclosure, the chimeric polypeptides of theAttorney Docket No. 048536-729001WO present disclosure do not include a LIN-12-Notch repeat (LNR) and / or a heterodimerization domain (HD) of a Notch receptor. In some embodiments of the present disclosure, the chimeric polypeptides of the disclosure do not include a LIN-12-Notch repeat (LNR) of a Notch receptor. In some embodiments of the present disclosure, the chimeric polypeptides of the disclosure do not include a heterodimerization domain (HD) of a Notch receptor. In some embodiments of the present disclosure, the chimeric polypeptides of the present disclosure are completely devoid of the negative regulatory region (NRR) of a Notch receptor, i.e.., both the LIN-12-Notch repeat (LNR) and heterodimerization domain (HD) of the Notch receptor are completely absent.Transmembrane domains

[0087] As described above, the chimeric polypeptides of the disclosure include a TMD comprising one or more ligand-inducible proteolytic cleavage sites. In some embodiments, the ligand-inducible proteolytic cleavage site is a y-secretase cleavage site. Examples of proteolytic cleavage sites includes those identified in a Notch receptor (e.g., S2 or S3). Additional proteolytic cleavage sites suitable for the compositions and methods disclosed herein include, but are not limited to, a metalloproteinase cleavage site for a MMP selected from collagenase- 1, -2, and -3 (MMP-1, - 8, and -13), gelatinase A and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and -11), matrilysin (MMP-7), and membrane metalloproteinases (MT1-MMP and MT2-MMP). For example, the cleavage sequence of MMP-9 is Pro-X-X-Hy (wherein, X represents an arbitrary residue; Hy, a hydrophobic residue such as Leu, He, Vai, Phe, Trp, Tyr, Vai, Met, and Pro, e.g., Pro-X-X-Hy-(Ser / Thr), e.g., Pro-Leu / Gln-Gly-Met-Thr-Ser, or Pro- Leu / Gln-Gly-Met-Thr. Another example of a suitable protease cleavage site is a plasminogen activator cleavage site, e.g., a urokinase plasminogen activator (uPA) or a tissue plasminogen activator (tPA) cleavage site. Another example of a suitable protease cleavage site is a prolactin cleavage site. Specific examples of cleavage sequences of uPA and tPA include sequences comprising Val-Gly-Arg. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a tobacco etch virus (TEV) protease cleavage site, e.g., Glu- Asn-Leu-Tyr-Thr-Gln-Ser, where the protease cleaves between the glutamine and the serine. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is an enterokinase cleavage site, e.g., Asp-Asp-Asp-Asp-Lys, where cleavage occurs after the lysine residue. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a thrombin cleavage site, e.g., Leu- Vai -Pro- Arg. AdditionalAttorney Docket No. 048536-729001WG suitable linkers comprising protease cleavage sites include sequences cleavable by the following proteases: a PreScission™ protease (a fusion protein comprising human rhinovirus 3C protease and glutathione-S-transferase), a thrombin, cathepsin B, Epstein-Barr virus proteas, MMP-3 (stromelysin), MMP-7 (matrilysin), MMP-9; thermolysin-like MMP, matrix metalloproteinase 2 (MMP-2), cathepsin L; cathepsin D, matrix metalloproteinase 1 (MMP-1), urokinase-type plasminogen activator, membrane type 1 matrixmetalloprotemase (MT-MMP), stromelysin 3 (or MMP-11), thermolysin, fibroblast collagenase and stromelysin- 1, matrix metalloproteinase 13 (collagenase-3), tissue-type plasminogen activator(tPA), human prostate-specific antigen, kallikrein (hK3), neutrophil elastase, and calpain (calcium activated neutral protease). Proteases that are not native to the host cell in which the receptor is expressed (for example, TEV) can be used as a further regulatory mechanism, in which activation of the receptor is reduced until the protease is expressed or otherwise provided. Additionally, a protease may be tumor-associated or disease-associated (expressed to a significantly higher degree than in normal tissue), and serve as an independent regulatory mechanism. For example, some matrix metalloproteases are highly expressed in certain cancer types.

[0088] Generally, the TMD suitable for the chimeric polypeptides disclosed herein can be any transmembrane domain of a Type 1 transmembrane receptor including at least one y-secretase cleavage site. Detailed description of the structure and function of the y-secretase complex as well as its substrate proteins, including amyloid precursor protein (APP) and Notch, can, for example, be found in a recent review by Zhang et aL, Frontiers Cell Neurosci (2014). Nonlimiting suitable TMDs from Type 1 transmembrane receptors include those from CLSTN1, CLSTN2, APLP1, APLP2, LRP8, APP, BTC, TGBR3, SPN, CD44, CSF1R, CXCL16, CX3CL1, DCC, DLL1, DSG2, DAG1, CDH1, EPCAM, EPHA4, EPHB2, EFNB1, EFNB2, ErbB4, GHR, HLA-A, and IFNAR2, wherein the TMD includes at least one y-secretase cleavage site. Additional TMDs suitable for the compositions and methods described herein include, but are not limited to, transmembrane domains from Type 1 transmembrane receptors IL1R1, IL1R2, IL6R, INSR, ERN1, ERN2, JAG2, KCNE1, KCNE2, KCNE3, KCNE4, KL, CHL1, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGER, ROBO1, SORCS3, SORCS1, SORL1, SDC1, SDC2, SPN, TYR, TYRP1, DCT, VASN, FLT1, CDH5, PKHD1, NECTIN1, PCDHGC3, NRG1, LRP1B, CDH2, NRG2, PTPRK, SCN2B, Nradd, and PTPRM. In some embodiments, the TMD of the chimeric polypeptides or Notch receptors of the disclosure is aAttorney Docket No. 048536-729001WOTMD derived from the TMD of a member of the calsyntenin family, such as, alcadein alpha and alcadein gamma. In some embodiments, the TMD of the chimeric polypeptides or Notch receptors of the disclosure is a TMD known for Notch receptors. In some embodiments, the TMD of the chimeric polypeptides or Notch receptors of the disclosure is a TMD derived from a different Notch receptor. For example, in a Mini Notch based on human Notchl, the Notchl TMD can be substituted with a Notch2 TMD, Notch3 TMD, Notch4 TMD, or a Notch TMD from a non-human animal such as Dcmio rerio, Drosophila melanogaster, Xenopus laevis, or Gallus gallus.Stop-transfer sequences (STS)

[0089] In some embodiments, the chimeric polypeptides and SNIPRs of the present disclosure further include a stop-transfer-sequence (STS) which comprises a charged, hydrophilic domain located between the TMD and the ICD. Without being bound to any particular theory, this domain disposed between the TMD and the ICD prevents the ICD from entering the plasma membrane. In some embodiments, a single-chain peptide comprising about 1 to about 40 amino acid residues (e. , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) in which most of the residues have charged side chains under physiological conditions can be used as a STS. In short STS embodiments (e.g., less than about 6 amino acids), about 5 or 6 of the amino acids will have charged side chains. In some embodiments, the STS includes about 1 to 15, about 5 to 20, about 8 to 25, about 10 to 30, about 12 to 35, about 14 to 40, about 5 to 40, about 10 to 35, about 15 to 30, about 20 to 25, about 20 to 40, about 10 to 30, about 4 to 20, or about 5 to 25 amino acid residues. In some embodiments, the STS includes about 4 to 10, about 5 to 12, about 6 to 14, about 7 to 18, about 8 to 20, about 9 to 22, about 10 to 24, or about 11 to 26 amino acid residues. In some embodiments, the STS includes about 4 to 10 residues, such as, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

[0090] In some embodiments, the STS includes a sequence having at least about 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to the STS domain of a Type 1 receptor. In some embodiments, the STS includes an amino acid sequence having at least 90% sequence identity to the STS domain of a Type 1 receptor.

[0091] In some embodiments, the STS comprises a sequence comprising only Lys (K) or Arg (R) in the first 4 residues. In some embodiments, the STS comprises one, two, three, four, five,Attorney Docket No. 048536-729001WO or more basic residues. In some embodiments, the STS comprises five, four, three, two, one, or zero aromatic residues or residues with hydrophobic and / or bulky side chains.Intracellular Domain (TCP)

[0092] As described above, the chimeric polypeptides of the disclosure includes a transcriptional regulator. The transcriptional regulator of the disclosure is a biochemical element that acts to promote or inhibit the transcription of a promoter-driven DNA sequence.Transcriptional regulators suitable for the compositions and methods of the disclosure can be naturally-occurring transcriptional regulators or can be engineered, designed, or modified so as to provide desired and / or improved properties, e.g., modulating transcription. In some embodiments, the transcriptional regulator directly regulates differentiation of the cell. In some embodiments, the transcriptional regulator indirectly modulates differentiation of the cell by modulating the expression of a second transcription factor. It will be understood by one having ordinary skill in the art that a transcriptional regulator can be a transcriptional activator or a transcriptional repressor. In some embodiments, the transcriptional regulator is a transcriptional repressor. In some embodiments, the transcriptional regulator is a transcriptional activator. In some embodiments, the transcriptional regulator can further include a nuclear localization signal.

[0093] In some embodiments, the transcriptional regulator includes a DNA-binding domain and an effector domain. Exemplary effector domains suitable for the compositions and methods of the disclosure include transcription activating domains, transcription repressor domains, and epigenetic effector domains. In instances where the effector domain of the transcriptional regulator includes a transcription activating domain, examples of suitable transcription activating domains include Herpes Simplex Virus Protein 16 (HSV VP 16) activation domain, an activation domain consisting of four tandem copies of VP16 (VP64), a p65 activation domain ofNFKB; an Epstein-Barr virus R transactivator activation domain (Rta), a tripartite activator consisting of VP64, and Rta activation domains (VPR), and a histone acetyltransferase core domain of the human ElA-associated protein p300 (p300 HAT core activation domain).

[0094] In instances where the effector domain of the transcriptional regulator includes a transcription repressor domain, examples of suitable transcription repressor domains include a Kruppel associated box repression domain (KRAB), a Repressor Element Silencing Transcription Factor repression domain (REST), a WRPW motif of the hairy-related basic helixloop-helix repressor proteins repression domain (WRPW), a DNA (cytosine-5)-Attorney Docket No. 048536-729001WO methyltransferase 3B repression domain (DNMT3B), and an HP1 alpha chromoshadow repression domain. In some embodiments, the effector domain of the transcriptional regulator includes an epigenetic effector domain. Exemplary epigenetic effector domains include a DNA methyltransferase DNMT (DNMT1, DNMT3), HAT1, GCN5, PCAF, MLL, SET, DOTI, SUV39H, G9a, KAT2A / B, EZH1 / 2, TET1 / 2, a SIRT family protein effector domain, a histone deacetylase, LSD1, and a KDM family protein effector domain. In some embodiments, the effector domain is from a human or humanized polypeptide.

[0095] Examples of suitable DNA-binding domain (DBD) of the transcriptional regulator include a GAL4-DBD, a TetR-DBD, a zinc finger (ZF) DBD, and a zinc-finger homeodomain (ZFHD) DBD. In some embodiments, the intracellular domain of the chimeric polypeptides of the disclosure further includes a nuclear transport signal sequence or a membrane localization signal such as a CD8A signal.

[0096] In some embodiments, the transcriptional regulator is selected from Gal4-VP16, Gal4- VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, and HAP 1 -VP 16. In some embodiments, the transcriptional regulator is Gal4-VP64.

[0097] One skilled in the art will appreciate that the complete amino acid sequence of a chimeric polypeptide SNIPR (e.g., orthoSNIPR) of the disclosure can be used to construct a back-translated gene. For example, a DNA oligomer containing a nucleotide sequence coding for a given chimeric polypeptide SNIPR can be synthesized. For example, several small oligonucleotides coding for portions of the desired chimeric polypeptide or SNIPR can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0098] In addition to generating desired chimeric polypeptides SNIPR (e.g, orthoSNIPR) via expression of nucleic acid molecules that have been engineered by recombinant molecular biological techniques, a subject chimeric polypeptide SNIPR (c.g, orthoSNIPR) in accordance with the present disclosure can be chemically synthesized. Chemically synthesized polypeptides are routinely generated by those of skill in the art.

[0099] Once assembled (by synthesis, recombinant methodologies, site-directed mutagenesis or other suitable techniques), the nucleic acid sequences encoding a chimeric polypeptide or SNIPR (e.g, orthoSNIPR) as disclosed herein can be inserted into an expression vector and operably linked to an expression control sequence appropriate for expression of the chimericAttorney Docket No. 048536-729001WO polypeptide SNIPR (e.g., orthoSNIPR) in the desired transformed host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is known in the art, in order to obtain high expression levels of a transfected gene in a host, take should be taken to ensure that the gene is operably linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.Nucleic Acid Molecules

[0100] In one aspect, some embodiments disclosed herein relate to nucleic acid molecules that include nucleotide sequences encoding the chimeric polypeptides provided herein. In some embodiments, the recombinant nucleic acids of the disclosure can be configured as, e.g., incorporated into, expression cassettes or vectors containing these nucleic acid molecules operably linked to heterologous nucleic acid sequences such as, for example, regulatory sequences which facilitate (e.g., allow) in vivo expression of the receptor in a host cell.

[0101] In some embodiments, provided herein is a recombinant nucleic acid molecule including a nucleotide sequence that encodes a chimeric polypeptide including, from N-terminus to C-terminus: (a) an extracellular ligand-binding domain (ECD) including a first monomer polypeptide of a designed o / p heterodimer (LHD) capable of selectively binding to an engineered ligand, wherein the engineered ligand includes a second monomer polypeptide of the designed LHD heterodimer; (b) a linking polypeptide; (c) a transmembrane domain (TMD) from a Type 1 transmembrane receptor including one or more ligand-inducible proteolytic cleavage sites; and (d) an intracellular domain (ICD) including a transcriptional regulator, wherein binding of the engineered ligand to the extracellular binding domain induces cleavage at the one or more ligand-inducible proteolytic cleavage sites.

[0102] In some embodiments, the recombinant nucleic acid of the disclosure further includes a response element, wherein the response element includes: (a) a cognate target sequence to which the DBD of the transcriptional regulator binds; (b) an engineered responsive promoter operably linked to the cognate target sequence; and (c) a polynucleotide of interest. In principle, there are no particular limitations with regard to suitable polynucleotides of interest. Exemplary polynucleotides of interest includes those encoding a regulatory RNA, a regulatory protein, a therapeutic protein, or a reporter molecule. In some embodiments, the polynucleotide of interest encodes a reporter molecule. Suitable reporter molecules include, but are not limited to,Attorney Docket No. 048536-729001WO fluorescent proteins, herpes simplex virus type 1 (HSV-1) thymidine kinase (TK), and gas vesicle proteins. In some embodiments, the reporter protein is a fluorescent reporter protein. In some embodiments, the fluorescent reporter protein is green fluorescent protein (GFP) gene, blue fluorescent protein (BFP) gene, yellow fluorescent protein (YFP) gene, luciferase gene, or mCherry gene. In some embodiments, the reporter protein includes an eGFP polypeptide.

[0103] In some embodiments, the polynucleotide of interest encodes a therapeutic protein. In some embodiments, the therapeutic protein is a recombinant antigen-specific receptor. In some embodiments, the recombinant antigen-specific receptor is an engineered T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0104] In some embodiments, the antigen-specific receptor is a T-cell receptor (TCR). A TCR generally includes two polypeptides (e.g., polypeptide chains), such as an a-chain of a TCR, a P- chain of a TCR, a y-chain of a TCR, a 6-chain of a TCR, or a combination thereof. Such polypeptide chains of TCRs are known in the art. The antigen-specific TCR can include any amino acid sequence, provided that the TCR can specifically bind to and / or immunologically recognize an antigen, such as a cancer antigen or epitope thereof. In some embodiments, the TCR is an endogenous TCR, e.g., a TCR that is endogenous or native to (naturally-occurring) the T cell. In such a case, the T cell expressing the endogenous TCR can be a T cell that was isolated from a mammal which is known to express the particular cancer antigen. For example, in some embodiments, the T cell is a primary T cell isolated from a mammal having a cancer. In some embodiments, the T cell is a TIL or a T cell isolated from a human cancer patient.

[0105] In some embodiments, the immune cells include and / or express a chimeric antigen receptor (CAR). Generally, a CAR includes an antigen binding domain, e.g., a single-chain variable fragment (scFv) of an antibody, fused to a transmembrane domain and an intracellular domain. In this case, the antigenic specificity of a CAR can be encoded by a scFv which specifically binds to the antigen, or an epitope thereof. CARs, and methods of making them, are known in the art.

[0106] In some embodiments, the polynucleotide of interest encodes a regulatory RNA. Exemplary regulatory RNAs suitable for the compositions and methods of the disclosure include the regulatory RNA is siRNA, shRNA, and miRNA. In some embodiments, the polynucleotide of interest encodes a protein, a regulatory RNA, or an antisense oligonucleotide.

[0107] Nucleic acid molecules of the present disclosure can be nucleic acid molecules of anyAttorney Docket No. 048536-729001WO length, including nucleic acid molecules that are generally between about 5 Kb and about 50 Kb, for example between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.

[0108] In some embodiments, the recombinant nucleic acid molecule of the disclosure is operably linked to a heterologous nucleic acid sequence, such as, for example a regulatory sequence (e.g., promoter sequence) or a sequence encoding signal peptide. In some embodiments, the recombinant nucleic acid molecule is further configured as (e.g., incorporated into) an expression cassette or a vector.

[0109] Accordingly, some embodiments disclosed herein relate to vectors or expression cassettes including a recombinant nucleic acid molecule as disclosed herein. As used herein, the term “expression cassette” refers to a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and / or translation of the coding sequences in a host cell, in vivo and / or ex vivo. The expression cassette may be inserted into a vector for targeting to a desired host cell and / or into a subject. As such, the term expression cassette may be used to refer to an expression construct. Also provided herein are vectors, plasmids or viruses containing one or more of the nucleic acid molecules encoding any of the chimeric polypeptides or SNIPRs e.g., orthoSNIPRs) disclosed herein. The nucleic acid molecules described above can be contained within a vector or expression cassette that is capable of directing their expression in, for example, a cell that has been transformed / transduced with the vector or expression cassette. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available, or readily prepared by a skilled artisan. Additional vectors can also be found in, for example, Ausubel, F. M., et al. (2014, supra) and Sambrook etal. (2012, supra). In some embodiments, the vector is viral vector. Viral vectors that can be used in the disclosure include, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), lentivirus, and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.). In some embodiments, the vector is a lentiviral vector, an adeno virus vector, an adeno-associated virus vector, a baculovirus, or a retroviral vector. In some embodiments, the vector is a lentiviral vector.Attorney Docket No. 048536-729001WO

[0110] It should be understood that not all vectors and expression control sequences will function equally well to express the nucleic acid sequences described herein. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation. For example, in selecting a vector, the host must be considered because the vector must replicate in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. For example, vectors that can be used include those that allow the DNA encoding the chimeric polypeptides or SNIPRs (e.g., orthoSNIPRs) of the present disclosure to be amplified in copy number. Such amplifiable vectors are known in the art.

[0111] Accordingly, in some embodiments, the chimeric polypeptides or SNIPRs (e.g., orthoSNIPRs) the present disclosure can be expressed from vectors, generally expression vectors. The vectors are useful for autonomous replication in a host cell or may be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome (e.g., non-episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses) are also included.

[0112] Exemplary recombinant expression vectors can include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, operably linked to the nucleic acid sequence to be expressed.

[0113] DNA vector can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in, for example, Sambrook el al. (2012) Molecular Cloning: A Laboratory ManuaX (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and other standard molecular biology laboratory manuals.

[0114] The nucleic acid sequences encoding the chimeric polypeptides or SNIPRs (e.g., orthoSNIPRs) of the present disclosure can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell.Attorney Docket No. 048536-729001WOMethods for codon optimization are known in the art. Codon usages within the coding sequence of the chimeric polypeptides or SNIPRs (e.g., orthoSNIPRs) disclosed herein can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a particular host cell.

[0115] Vectors suitable for use include T7-based vectors for use in bacteria, the pMSXND expression vector for use in mammalian cells, and baculovirus-derived vectors for use in insect cells. In some embodiments nucleic acid inserts, which encode the subject chimeric polypeptide or SNIPR (e.g., orthoSNIPR) in such vectors, can be operably linked to a promoter, which is selected based on, for example, the cell type in which expression is sought.

[0116] In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the actual nucleic acid sequence encoding the subject chimeric polypeptide or SNIPR (e.g., orthoSNIPR), particularly as regards potential secondary structures. Hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the nucleic acid sequences of this disclosure, their secretion characteristics, their ability to fold the polypeptides correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the nucleic acid sequences.

[0117] Within these parameters one of skill in the art may select various vector / expression control sequence / host combinations that will express the desired DNA sequences on fermentation or in large scale animal cell culture, for example, using CHO cells or COS 7 cells.

[0118] The choice of expression control sequence and expression vector, in some embodiments, will depend upon the choice of host. A wide variety of expression host / vector combinations can be employed. Non-limiting examples of useful expression vectors for eukaryotic hosts, include, for example, vectors with expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Non-limiting examples of useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including col El, pCRI, pER32z, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as M13 and filamentous single stranded DNA phages. Non-limitingAttorney Docket No. 048536-729001WO examples of useful expression vectors for yeast cells include the 2p plasmid and derivatives thereof. Non-limiting examples of useful vectors for insect cells include pVL 941 and pFastBac™ 1.

[0119] Skilled artisans will readily appreciate numerous promoters and other regulatory elements which can be used to direct expression of nucleic acids. For example, a cytomegalovirus or metallothionein promoter can be used in mammalian cells, a T7 promoter can be used in bacteria, and a polyhedrin promoter can be used in insect cells. Also, in the case of higher eukaryotes, tissue-specific and cell type-specific promoters are widely available. These promoters are so named for their ability to direct expression of a nucleic acid molecule in a given tissue or cell type within the body.

[0120] As described in greater detail below, prokaryotic or eukaryotic cells that contain and express a nucleic acid molecule that encodes a subject chimeric polypeptide or SNIPR disclosed herein are also features of the disclosure. A cell of the disclosure is a transfected cell, e.g., a cell into which a nucleic acid molecule, for example a nucleic acid molecule encoding a chimeric polypeptide or SNIPR, has been introduced by means of recombinant DNA techniques. The progeny of such a cell are also considered within the scope of the disclosure.

[0121] An exemplary method of constructing a DNA sequence encoding the chimeric polypeptides or SNIPRs of the disclosure is by chemical synthesis. This includes direct synthesis of a peptide by chemical means of the protein sequence encoding for a chimeric polypeptide or SNIPR exhibiting the properties described. Alternatively, a gene which encodes the desired chimeric polypeptide or SNIPR can be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired chimeric polypeptide or SNIPR, and suitably selecting those codons that are favored in the host cell in which the recombinant chimeric polypeptides or SNIPR will be produced. In this regard, it is well recognized in the art that the genetic code is degenerate-that an amino acid may be coded for by more than one codon. Accordingly, it will be appreciated by those skilled in the art that for a given DNA sequence encoding a particular chimeric polypeptide or SNIPR, there will be many DNA degenerate sequences that will code for that chimeric polypeptide or SNIPR. For example, it will be appreciated that in addition to the DNA sequences for chimeric polypeptides and SNIPRs in the specification, there will be many degenerate DNA sequences that code for the chimeric polypeptides and SNIPRs disclosed herein. These degenerate DNA sequences areAttorney Docket No. 048536-729001WO considered within the scope of this disclosure. Therefore, “degenerate variants thereof’ in the context of this disclosure means all DNA sequences that code for and thereby enable expression of a particular chimeric polypeptide or SNIPR.

[0122] The nucleic acid sequence encoding the subject chimeric polypeptide or SNIPR, whether prepared by site directed mutagenesis, chemical synthesis or other methods, can also include DNA sequences that encode a signal sequence. Such signal sequence, if present, should be one recognized by the cell chosen for expression of the chimeric polypeptide or SNIPR. It can be prokaryotic, eukaryotic or a combination of the two. In general, the inclusion of a signal sequence depends on whether it is desired to secrete the chimeric polypeptide or SNIPR as disclosed herein from the recombinant cells in which it is made. If the chosen cells are prokaryotic, it generally is preferred that the DNA sequence does not encode a signal sequence. If the chosen cells are eukaryotic, it generally is preferred that a signal sequence be included.

[0123] The nucleic acid molecules provided herein can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide. These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded (e.g., either a sense or an antisense strand).

[0124] The nucleic acid molecules of the disclosure are not limited to sequences that encode the chimeric polypeptides; some or all of the non-coding sequences that lie upstream or downstream from a coding sequence (e.g., the coding sequence of a chimeric polypeptide or SNIPR) can also be included. Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or by performance of the polymerase chain reaction (PCR). In the event the nucleic acid molecule is a ribonucleic acid (RNA), molecules can be produced, for example, by in vitro transcription.

[0125] Exemplary recombinant nucleic acid molecules of the present disclosure can include fragments not found as such in the natural state. Thus, this disclosure encompasses recombinant molecules, such as those in which a nucleic acid sequence (for example, a sequence encoding a chimeric polypeptide or SNIPR) is incorporated into a vector (e.g., a plasmid or viral vector) orAttorney Docket No. 048536-729001WO into the genome of a heterologous cell (or the genome of a homologous cell, at a position other than the natural chromosomal location).RECOMBINANT CE LS AND CULTURES

[0126] As discussed above, the recombinant nucleic acid molecule of the present disclosure can be introduced into a host cell, such as a human T cell or cancer cell, to produce a recombinant cell containing the nucleic acid molecule. Accordingly, some embodiments of the disclosure relate to recombinant cells that contain (a) a recombinant nucleic acid as disclosed herein, and / or (b) a chimeric polypeptide as disclosed herein. Some embodiments of the disclosure relate to methods for making a recombinant cell (e.g., an engineered cell), including (a) providing a host cell capable of protein expression; and transducing the provided host cell with a recombinant nucleic acid of the disclosure to produce a recombinant cell. Introduction of the nucleic acid molecules of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome- mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0127] Accordingly, in some embodiments, the recombinant nucleic acid molecules of the disclosure can be introduced into a host cell by viral or non-viral delivery vehicles known in the art to produce a recombinant cell. For example, the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for a stable or transient expression. Accordingly, in some embodiments of the disclosure, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be completed using classical random genomic recombination techniques or with more precise genome editing techniques such as using zinc-finger proteins (ZNF), guide RNA directed CRISPR / Cas9, DNA- guided endonuclease genome editing NgAgo (Natronobacterhim gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nucleases).

[0128] The nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle, or can be delivered by viral or non-viral delivery means and methods known in the art, such asAttorney Docket No. 048536-729001WO electroporation. For example, introduction of nucleic acids into cells may be achieved by viral transduction. In a non-limiting example, baculoviral virus or adeno-associated virus (AAV) can be engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.

[0129] Lentiviral-derived vector systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as genedelivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profde; and (vii) a relatively easy system for vector manipulation and production.

[0130] In some embodiments, the recombinant cells of the disclosure further include an engineered response element comprising a cognate target sequence to which the DBD of the transcriptional regulator binds. In some embodiments, the engineered response element includes i) a cognate target sequence to which the DBD of the transcriptional regulator binds, ii) a promoter sequence, wherein the cognate target sequence is operably linked to the 5' end of the promoter sequence, and iii) a polynucleotide of interest operably linked to the promoter sequence, wherein binding of the transcriptional regulator to the cognate target sequence modulates transcription initiation of a polynucleotide of interest.

[0131] In some embodiments, the chimeric polypeptide and the engineered response element are encoded by the same nucleic acid molecule. In some embodiments, the chimeric polypeptide and the engineered response element are encoded by two separate nucleic acid molecules, e.g., the chimeric polypeptide is encoded by a first nucleic acid molecule and the engineered response element is encoded by a second nucleic acid molecule. In some embodiments, the engineered response element is present in a nucleic acid vector, plasmid, DNA mini circle, mini chromosome, or in a host chromosome.

[0132] In various embodiments of the disclosure, host cells can be genetically engineered (e.g.,Attorney Docket No. 048536-729001WO transduced or transformed or transfected) with, for example, a vector construct of the present application that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the polypeptides of interest. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.

[0133] In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is a prokaryotic cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a mouse cell. In some embodiments, the animal cell is a human cell. In some embodiments, the cell is a non-human primate cell. In some embodiments, the mammalian cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some embodiments, the recombinant cell is an immune system cell, e.g., a B cell, a monocyte, a NK cell, a natural killer T (NKT) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell (TH), a cytotoxic T cell (TCTL), a memory T cell, a gamma delta (yS) T cell, another T cell, a hematopoietic stem cell, or a hematopoietic stem cell progenitor.

[0134] In some embodiments, the immune system cell is a lymphocyte. In some embodiments, the lymphocyte is a T lymphocyte. In some embodiments, the lymphocyte is a T lymphocyte progenitor. In some embodiments, the T lymphocyte is a CD4+ T cell or a CD8+ T cell. In some embodiments, the T lymphocyte is a CD8+ T cytotoxic lymphocyte cell. Non-limiting examples of CD8+ T cytotoxic lymphocyte cell suitable for the compositions and methods disclosed herein include naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, effector CD8+ T cells, CD8+ stem memory T cells, and bulk CD8+ T cells. In some embodiments, the T lymphocyte is a CD4+ T helper lymphocyte cell. Suitable CD4+ T helper lymphocyte cells include, but are not limited to, naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, effector CD4+ T cells, CD4+ stem memory T cells, and bulk CD4+ T cells.

[0135] As discussed above, some embodiments of the disclosure relate to various methods for making a recombinant cell, including (a) providing a host cell capable of protein expression; andAttorney Docket No. 048536-729001WO transducing the provided host cell with a recombinant nucleic acid of the disclosure to produce a recombinant cell. Non-limiting exemplary embodiments of the disclosed methods for making a recombinant cell can further include one or more of the following features. In some embodiments, the host cell is obtained by leukapheresis performed on a sample obtained from a subject, and the cell is transduced ex vivo. In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle. In some embodiments, the methods further include isolating and / or purifying the produced cells. Accordingly, the recombinant cells produced by the methods disclosed herein are also within the scope of the disclosure.

[0136] Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. For example, DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, nucleoporation, hydrodynamic shock, and infection. In some embodiments, the nucleic acid molecule is introduced into a host cell by a transduction procedure, electroporation procedure, or a biolistic procedure. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.

[0137] In a related aspect, some embodiments of the disclosure relate to a cell culture including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any one of suitable culture media for the cell cultures described herein. In some embodiments, the recombinant cell expresses a chimeric polypeptide or SNIPR (e.g., orthoSNIPR) as described herein. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.PHARMACEUTICAL COMPOSITIONS

[0138] As discussed above, the chimeric polypeptides, SNIPRs (e.g, orthoSNIPRs), recombinant nucleic acids, recombinant cells, and / or cell cultures of the disclosure can beAttorney Docket No. 048536-729001WO incorporated into compositions, including pharmaceutical compositions. Such compositions generally include the chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, and / or cell cultures as described herein and a pharmaceutically acceptable carrier. Accordingly, in one aspect, some embodiments of the disclosure relate to pharmaceutical compositions for treating, preventing, ameliorating, reducing, or delaying the onset of one or more health conditions, for example a proliferative disease (e. , cancer). In some embodiments, the pharmaceutical composition includes at least one chimeric polypeptide, SNIPR (e.g., orthoSNIPR), nucleic acid, recombinant cell, and / or cell culture as disclosed herein, in an admixture with a pharmaceutically acceptable carrier. Some embodiments of the disclosure relate to a pharmaceutical composition including a pharmaceutically acceptable carrier and one or more of the following: (a) a chimeric polypeptide (for example, a SNIPR, e.g., an orthoSNIPR) of the disclosure; (b) a recombinant nucleic acid molecule of the disclosure; and (c) a recombinant cell of the disclosure. In some embodiments, the composition includes a recombinant nucleic acid of the disclosure and a pharmaceutically acceptable carrier. In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle. In some embodiments, the composition includes a recombinant cell of the disclosure and a pharmaceutically acceptable carrier.

[0139] In certain embodiments, the pharmaceutical compositions in accordance with some embodiments disclosed herein include cell cultures that can be washed, treated, combined, supplemented, or otherwise altered prior to administration to a subject in need thereof. Furthermore, administration can be at varied doses, time intervals or in multiple administrations.

[0140] The pharmaceutical compositions provided herein can be in any form that allows for the composition to be administered to a subject. In some specific embodiments, the pharmaceutical compositions are suitable for human administration. As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The carrier can be a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, including injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumAttorney Docket No. 048536-729001WO chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E.W. Martin. In some embodiments, the pharmaceutical composition is sterilely formulated for administration into a subject. In some embodiments, the subject is a human. One of ordinary skilled in the art will appreciate that the formulation should suit the intended mode of administration.

[0141] In some embodiments, the pharmaceutical compositions of the present disclosure are formulated to be suitable for the intended route of administration to a subject. For example, the pharmaceutical composition may be formulated to be suitable for one or more of the following administration routes: intranasal administration, transdermal administration, intrathecal administration, intraperitoneal administration, intramuscular administration, intratracheal administration, intranodal administration, intratumoral administration, intraarticular administration, intravenous administration, subcutaneous administration, intravaginal administration, intraocular administration, rectal administration, and oral administration. In some particular embodiments of the disclosure, the composition is formulated intramuscular administration.

[0142] For example, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™. (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringeability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. Prevention of microbial contamination can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be generally to includeAttorney Docket No. 048536-729001WO isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and / or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0143] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.METHODS OF THE DISCLOSURE

[0144] As discussed in greater detail below, administration of any one of the therapeutic compositions described herein, e.g., chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, can be used for modulating an activity of a cell, or in the prevention and / or treatment of relevant health conditions, such as health disorders and proliferative diseases (e.g., cancer). In some embodiments, the chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions as described herein can be incorporated into therapeutic agents for use in methods of treating a subject or individual who has, who is suspected of having, or who may be at high risk for developing one or more health conditions, such as health disorders and proliferative diseases (e.g., cancers). In some embodiments, the subject is a patient under the care of a physician. In some embodiments, the proliferative is a cancer. In some embodiments, the cancer is a pediatric cancer. In some embodiments, the cancer is an adult malignancy.

[0145] Accordingly, in one aspect, some embodiments of the disclosure relate to methods for modulating (e.g., stimulating or inhibiting) an activity of a target cell in a subject, the methods include administering to the subject an effective amount of a composition comprising one or more of the chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions as disclosed herein, wherein the administered composition modulates an activity of the target cell.

[0146] In some embodiments, an activity of the target cell may be inhibited if its proliferationAttorney Docket No. 048536-729001WO is reduced, if its pathologic or pathogenic behavior is reduced, if it is destroyed or killed, and the like. Inhibition includes a reduction of the measured quantity of at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, an activity of the target cell may be stimulated if its proliferation is increased or if its pathologic or pathogenic behavior is increased. Stimulation includes an increase of the measured quantity of at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the methods include administering to the subject an effective number of the recombinant cells as disclosed herein, wherein the recombinant cells modulate an activity of the target cell in the subject. In some embodiments, the modulation of the activity the target cell results in the death of the target cell.

[0147] Generally, the target cell of the disclosed methods can be any cell and can be, for example a leukemia cell, an acute myeloma leukemia cell, an anaplastic lymphoma cell, an astrocytoma cell, a B-cell cancer cell, a breast cancer cell, a colon cancer cell, an ependymoma cell, an esophageal cancer cell, a glioblastoma cell, a glioma cell, a leiomyosarcoma cell, a liposarcoma cell, a liver cancer cell, a lung cancer cell, a mantle cell lymphoma cell, a melanoma cell (e.g., A375 cell), a neuroblastoma cell, a non-small cell lung cancer cell, an oligodendroglioma cell, an ovarian cancer cell, a pancreatic cancer cell, a peripheral T cell lymphoma cell, a renal cancer cell, a sarcoma cell, a stomach cancer cell, a carcinoma cell, a mesothelioma cell, or a sarcoma cell. In some embodiments, the target cell is a pathogenic cell. In some embodiments, the target cell is a cancer cell. In some embodiments, the modulation of the activity the target cell results in the death of the target cell.

[0148] Some embodiments of the disclosure relate to methods for modulating an activity of a cell, the methods including: (a) providing a recombinant cell as disclosed herein; and (b) contacting the recombinant cell with the engineered ligand, wherein binding of the engineered ligand to the extracellular ligand-binding domain results in cleavage of a ligand-inducible proteolytic cleavage site and release of the intracellular domain, wherein the release of the intracellular domain results in modulation of an activity of the recombinant cell. One skilled in the art upon reading the present disclosure will appreciate that the disclosed methods can beAttorney Docket No. 048536-729001WO carried out in vivo, ex vivo, or in vitro.

[0149] In some embodiments, the release of the intracellular domain results in binding of the transcriptional regulator of the released intracellular domain to a cognate target sequence, which results in modulation of the expression initiation of a polynucleotide of interest, which in turn results in modulation of an activity of the recombinant cell.

[0150] Activities of a cell that can be modulated using a method of the present disclosure include, but are not limited to, expression of a selected gene, proliferation, apoptosis, non- apoptotic death, differentiation, dedifferentiation, migration, secretion of a molecule, cellular adhesion, and cytolytic activity. In some embodiments, the released transcriptional regulator modulates expression of an gene product. In some embodiments, the released transcriptional regulator modulates expression of an endogenous gene product. In some embodiments, the transcriptional regulator modulates expression of a heterologous gene product. A heterologous gene product is one that is not normally produced by the cell. For example, the cell can be genetically modified with a nucleic acid comprising a nucleotide sequence encoding the heterologous gene product.

[0151] In some embodiments, the gene product is a secreted gene product. In some embodiments, the gene product is a cell surface gene product. In some cases, the gene product is an intracellular gene product. In some embodiments, the released transcriptional regulator simultaneously modulates expression of two or more gene products in the cell.

[0152] Gene products whose expression can be modulated include, but are not limited to, a chemokine, a chemokine receptor, a chimeric antigen receptor, a cytokine, a cytokine receptor, a differentiation factor, a growth factor, a growth factor receptor, a hormone, a metabolic enzyme, a pathogen-derived protein, a proliferation inducer, a receptor, an RNA guided nuclease, a sitespecific nuclease, a T cell receptor, a toxin, a toxin derived protein, a transcriptional regulator, a transcriptional activator, a transcriptional repressor, a translational regulator, a translational activator, a translational repressor, an activating immuno-receptor, an antibody, an apoptosis inhibitor, an apoptosis inducer, an engineered T cell receptor, an immuno-activator, an immunoinhibitor, an immune cell receptor, and an inhibiting immuno-receptor. In some embodiments, the released transcriptional regulator modulates differentiation of the cell, and wherein the cell is an immune cell, a stem cell, a progenitor cell, or a precursor cell.

[0153] Some embodiments of the disclosure relate to methods for the prevention and / orAttorney Docket No. 048536-729001WO treatment of a health condition in a subject in need thereof, the method comprising administering to the subject an effective amount of a therapeutic agent comprising one or more of the chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions as disclosed herein, wherein the administered composition confers a prevention and / or treatment of the health condition in the subject.

[0154] Administration of therapeutic agents described herein, e.g, chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, can be used in the stimulation of an immune response. In some embodiments, chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions as described herein are administered to a subject or individual after induction of remission of cancer with chemotherapy, or after autologous or allogeneic hematopoietic stem cell transplantation.

[0155] An effective amount of the therapeutic agents described herein, e.g., chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, is determined based on the intended goal, for example tumor regression. For example, where existing cancer is being treated, the amount of a therapeutic agent disclosed herein to be administered may be greater than where administration of the therapeutic agent is for prevention of cancer. One of ordinary skill in the art would be able to determine the amount of a therapeutic agent to be administered and the frequency of administration in view of this disclosure. The quantity to be administered, both according to number of treatments and dose, also depends on the subject or individual to be treated, the state of the subject or individual, and the protection desired. Precise amounts of the therapeutic agent also depend on the judgment of the practitioner and are peculiar to each subject. Frequency of administration could range from 1-2 days, to 2-6 hours, to 6-10 hours, to 1-2 weeks or longer depending on the judgment of the practitioner.

[0156] Longer intervals between administration and lower amounts of therapeutic agents may be employed where the goal is prevention. For instance, amounts of therapeutic agents administered per dose may be 50% of the dose administered in treatment of active disease, and administration may be at weekly intervals. One of ordinary skill in the art, in light of this disclosure, would be able to determine an effective amount of therapeutic agents and frequency of administration. This determination would, in part, be dependent on the particular clinicalAttorney Docket No. 048536-729001WO circumstances that are present (e.g., type of cancer, severity of cancer).

[0157] In certain embodiments, it may be desirable to provide a continuous supply of the therapeutic agents to the subject to be treated, e.g., a patient. In some embodiments, continuous perfusion of the region of interest (such as a tumor) may be suitable. The time period for perfusion would be selected by the clinician for the particular subject and situation, but times could range from about 1-2 hours, to 2-6 hours, to about 6-10 hours, to about 10-24 hours, to about 1-2 days, to about 1-2 weeks or longer. Generally, the dose of the therapeutic agent via continuous perfusion will be equivalent to that given by single or multiple injections, adjusted for the period of time over which the doses are administered.

[0158] One of ordinary skill in the art would be familiar with techniques for administering therapeutic agents to a subject or individual. Furthermore, one of ordinary skill in the art would be familiar with techniques and pharmaceutical reagents necessary for preparation of these therapeutic agents prior to administration to a subject or individual.

[0159] In certain embodiments of the present disclosure, the therapeutic agents will be an aqueous composition that includes the chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions as described herein. Aqueous compositions of the present disclosure contain an effective amount of a therapeutic agent disclosed herein in a pharmaceutically acceptable carrier or aqueous medium. Thus, the “pharmaceutical preparation” or “pharmaceutical composition” of the disclosure can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the recombinant cells disclosed herein, its use in the manufacture of the pharmaceutical compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. For human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA Center for Biologies.

[0160] One of ordinary skill in the art would appreciate that biological materials should be extensively dialyzed to remove undesired small molecular weight molecules and / or lyophilized for more ready formulation into a desired vehicle, where appropriate. The therapeutic agents described herein, e.g., chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleicAttorney Docket No. 048536-729001WO acids, recombinant cells, cell cultures, and / or pharmaceutical compositions, will then generally be formulated for administration by any known route, such as parenteral administration. Determination of the amount of therapeutic agents to be administered will be made by one of skill in the art, and will in part be dependent on the extent and severity of cancer, and whether the recombinant cells are being administered for treatment of existing cancer or prevention of cancer. The preparation of the therapeutic agents containing the chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions of the disclosure will be known to those of skill in the art in light of the present disclosure.

[0161] Upon formulation, therapeutic agents will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The therapeutic agents can be administered in a variety of dosage forms, such as the type of injectable solutions described above. For parenteral administration, the therapeutic agents disclosed herein should be suitably buffered. As discussed in greater detail below, the therapeutic agents as described herein may be administered with other therapeutic agents that are part of the therapeutic regiment of the subject or individual, such as other immunotherapy or chemotherapy.

[0162] The chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be used to cure established tumors, inhibit tumor growth or metastasis of cancer in the treated subject as compared to the tumor growth or metastasis in subjects who have not been administered one of the therapeutic compositions disclosed herein. In some embodiments, the chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be used to stimulate proliferation and / or killing capacity of CAR T-cells in the treated subject as compared to subjects who have not been administered one of the therapeutic compositions disclosed herein.Administration of recombinant cells to a subject

[0163] In some embodiments, the methods of the disclosure involve administering an effective amount or number of the recombinant cells provided herein to a subject in need thereof. This administering step can be accomplished using any method of implantation delivery in the art. For example, the recombinant cells can be infused directly in the subject’s bloodstream or otherwise administered to the subject.Attorney Docket No. 048536-729001WO

[0164] In some embodiments, the methods disclosed herein include administering, which term is used interchangeably with the terms “introducing,” implanting,” and “transplanting,” recombinant cells into a subject or individual, by a method or route that results in at least partial localization of the introduced cells at a desired site such that a desired effect(s) is / are produced. The recombinant cells or their differentiated progeny can be administered by any appropriate route that results in delivery to a desired location in the subject or individual where at least a portion of the administered cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of the subject or individual, i.e., long-term engraftment.

[0165] When provided prophylactically, the recombinant cells described herein can be administered to a subject in advance of any symptom of a disease or condition to be treated. Accordingly, in some embodiments the prophylactic administration of a recombinant cell population prevents the occurrence of symptoms of the disease or condition.

[0166] When provided therapeutically in some embodiments, recombinant cells are provided at (or after) the onset of a symptom or indication of a disease or condition, e.g., upon the onset of disease or condition.

[0167] For use in the various embodiments described herein, an effective amount of recombinant cells as disclosed herein, can be at least 102cells, at least 5 * 102cells, at least 103cells, at least 5 x io3cells, at least 104cells, at least 5 x io4cells, at least 105cells, at least 2 x 1 C cells, at least 3 x 105cells, at least 4 x 105cells, at least 5 x 1 C cells, at least 6 x 105cells, at least 7 x io5cells, at least 8 x io5cells, at least 9 x io5cells, at least 1x106cells, at least 2 x 106cells, at least 3 x io6cells, at least 4 x IQ6cells, at least 5 x io6cells, at least 6 x io6cells, at least 7 x io6cells, at least 8 x 106cells, at least 9 x io6cells, or multiples thereof. The recombinant cells can be derived from one or more donors or can be obtained from an autologous source. In some embodiments, the recombinant cells are expanded in culture prior to administration to a subject in need thereof.

[0168] In some embodiments, the delivery of a recombinant cell composition (e.g., a composition including a plurality of recombinant cells according to any of the cells described herein) into a subject by a method or route results in at least partial localization of the cell composition at a desired site. A composition including recombinant cells can be administered byAttorney Docket No. 048536-729001WO any appropriate route that results in effective treatment in the subject, e.g., administration results in delivery to a desired location in the subject where at least a portion of the composition delivered, e.g., at least 1 x 104cells, is delivered to the desired site for a period of time. Modes of administration include injection, infusion, and instillation. “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. For the delivery of cells, delivery by injection or infusion is a standard mode of administration.

[0169] In some embodiments, the recombinant cells are administered systemically, e.g., via infusion or injection. For example, a population of recombinant cells are administered other than directly into a target site, tissue, or organ, such that it enters, the subject’s circulatory system and, thus, is subject to metabolism and other similar biological processes.

[0170] The efficacy of a treatment including any of the compositions provided herein for the treatment of a disease or condition can be determined by a skilled clinician. However, one skilled in the art will appreciate that a treatment is considered effective if any one or all of the signs or symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of a subject to worsen as assessed by decreased hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in a subject or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0171] Measurement of the degree of efficacy is based on parameters selected with regard to the disease being treated and the symptoms experienced. In general, a parameter is selected that is known or accepted as correlating with the degree or severity of the disease, such as a parameter accepted or used in the medical community. For example, in the treatment of a solid cancer, suitable parameters can include reduction in the number and / or size of metastases, number of months of progression-free survival, overall survival, stage or grade of the disease, the rate of disease progression, the reduction in diagnostic biomarkers (for example withoutAttorney Docket No. 048536-729001WO limitation, a reduction in circulating tumor DNA or RNA, a reduction in circulating cell-free tumor DNA or RNA, and the like), and combinations thereof. It will be understood that the effective dose and the degree of efficacy will generally be determined with relation to a single subject and / or a group or population of subjects. Therapeutic methods of the disclosure reduce symptoms and / or disease severity and / or disease biomarkers by at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100%.

[0172] As discussed above, a therapeutically effective amount or number includes an amount of a therapeutic composition or number of recombinant cells that is sufficient to promote a particular beneficial effect when administered to a subject, such as one who has, is suspected of having, or is at risk for a disease. In some embodiments, an effective amount or number includes an amount or number sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount or number can be determined by one of ordinary skill in the art using routine experimentation.Additional therapies

[0173] As discussed supra, any one of the chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be administered to a subject in need thereof as a sole therapy (e.g, monotherapy). In addition or alternatively, in some embodiments of the disclosure, the chimeric polypeptides, SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, cell cultures, and / or pharmaceutical compositions described herein can be administered to a subject in combination with one or more additional therapeutic agents, e.g., at least one, two, three, four, or five additional therapies. Suitable therapies to be administered in combination with the compositions of the disclosure include, but are not limited to chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery. Other suitable therapies include chemotherapeutics, anti-cancer agents, and anti-cancer therapies.

[0174] Administration “in combination with” one or more additional therapeutic includes simultaneous (e.g., concurrent) and consecutive administration in any order. In some embodiments, the one or more additional therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery.Attorney Docket No. 048536-729001WO

[0175] Accordingly, in some embodiments, the methods of the disclosure include administration of a composition disclosed herein to a subject individually as a sole therapy (e. ., monotherapy). In some embodiments, a composition of the disclosure is administered to a subject as a first therapy in combination with a second therapy, such as an anti -cancer agent, a chemotherapeutic, or an anti-cancer therapy. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in rotation. In some embodiments, the first therapy is administered at the same time as the second therapy. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.KITS

[0176] Also provided herein are various kits for the practice of a method described herein. In particular, some embodiments of the disclosure provide kits for modulating an activity of a target cell, e.g., a cell in a subject. Some embodiments of the disclosure provide kits for the prevention of a health condition in a subject in need thereof. Some other embodiments relate to kits for methods of treating a health condition in a subject in need thereof. For example, provided herein, in some embodiments, are kits that include one or more of the chimeric polypeptides, such as soluble SNIPRs (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, and / or pharmaceutical compositions as provided and described herein, as well as written instructions for making and / or using the same.

[0177] In some embodiments, the kits of the disclosure further include one or more means useful for the administration of any one of the provided chimeric polypeptides (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to a subject or individual. For example, in some embodiments, the kits of the disclosure further include one or more syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer any one of the provided chimeric polypeptides (e.g., orthoSNIPRs), recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to a subject orAttorney Docket No. 048536-729001WO individual. In some embodiments, a kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the other kit components for a desired purpose, e.g., for preventing and / or treating a health condition in a subject in need thereof.

[0178] Any of the above-described kits can further include one or more additional reagents, where such additional reagents can be one or more of the following: dilution buffers; reconstitution solutions, wash buffers, control reagents, control expression vectors, negative control polypeptides, positive control polypeptides, reagents suitable for in vitro or ex vivo production of the chimeric polypeptides, e.g., orthoSNIPRs.

[0179] In some embodiments, the components of a kit can be in separate containers. In some other embodiments, the components of a kit can be combined in a single container.

[0180] In some embodiments, a kit can further include instructions for using the components of the kit to practice the methods disclosed herein. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kit as a package insert, in the labeling of the container of the kit or components thereof (e.g., associated with the packaging or sub-packaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0181] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0182] No admission is made that any reference cited herein constitutes prior art. TheAttorney Docket No. 048536-729001WG discussion of the references states what their authors assert, and the inventors reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0183] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.EXAMPLES

[0184] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

[0185] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). C rrent Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press;Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry . New York, NY:Attorney Docket No. 048536-729001WGWiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and / or parameters unless otherwise noted.EXAMPLE 1General Experimental ProceduresPlasmid Assembly

[0186] Plasmids were derived from previously reported constructs, with receptor vectors originating in pHR PGK (AddGene 79120) and reporter constructs cloned into pHR_Gal4UAS_PGK_mCherry (AddGene 79124). All constructs reported in this work were generated via NEBuilder HiFi DNA Assembly (NEB E2621). A summary of the constructs described herein is shown in Table 2 below.TABLE 2:Attorney Docket No. 048536-729001WOAttorney Docket No. 048536-729001WOAttorney Docket No. 048536-729001WO

[0187] Sequences are listed in Tables 3A-3D.TABLE 3A: ReceptorsAttorney Docket No. 048536-729001WOTABLE 3B: RespondersAtorney Docket No. 048536-729001WOTABLE 3C: Constitutive CARsTABLE 3D: Ligand expressionAttorney Docket No. 048536-729001WO C6-79-101A-mcherrry

[0188] The origins of the components described in this disclosure are listed in Table 4 below.TABLE 4:Attorney Docket No. 048536-729001WOCell culture and lentivira! production

[0189] Lenti-X 293T cells (Clontech 632180) were cultured in DMEM supplemented with 10% FBS (Millipore Sigma), lx Sodium Pyruvate (Millipore Sigma), and 50 U / mL penicillinstreptomycin (MP Biochemicals). A375 cells expressing nuclear-localized RFP were a generous gift of the Marson lab at UCSF. A549 cells (CCLZR013) were obtained from the UCSF Cell and Genome Engineering Core. A375 and A549 cell lines were grown in 293T media. Caco-2 cells were obtained from the ATCC (HTB-37) and grown in DMEM (Gibco 11965092) supplemented with 20% FBS and 50 U / mL penicillin-streptomycin. M28 cells were obtained from the Gerwin laboratory at the National Cancer Institute and grown in RPMI 1640 (Gibco) with 10% FBS, 50 U / mL penicillin-streptomycin, and lx GlutaMAX (Gibco). Cells obtained from ATCC or the UCSF Cell and Genome Engineering Core were authenticated by the supplier. All cell lines were tested for mycoplasma (ATCC 30-1012K). For viral production, 1E6 Lenti-X 293T cells were seeded in a 6 well vessel at lE6 / well in 2.5 mL of 293T media. One day after seeding, cells were transfected with a packaging mix consisting of 1.5 pg of the transgene expression vector, 1.34 pg of pCMVdR8.91, and 0.17 pg of pMD2.G, using TransIT-Lenti Transfection Reagent (Minis) (3 pl of the reagent per 1 pg of total DNA). Two days after transfection, viral supernatants were harvested via centrifugation and used immediately for transduction.

[0190] For Jurkat experiments, HEK293T cells were initially cultured in DMEM (Gibco) supplemented with 10% FCII (Gibco), and 50 U / mL penicillin-streptomycin (Gibco), then with “induction” DMEM media supplemented with 10% FCII, 10 mM sodium butyrate, and 50 U / mL penicillin-streptomycin, and later with “viral” DMEM media supplemented with 10% FCII, 0.1 mM HEPES, lx GlutaMax, lx MEM non-essential amino acids and 50 U / mL penicillin- streptomycin. Jurkat T Cells were cultured in RPMI 1640 supplemented with lx GlutaMax (Gibco), 10% FCII, and 50 U / mL penicillin-streptomycin. For lentivirus production, 293T cells were seeded at 400k cells / mL in either a 12-well vessel or T75 plate and grown to 90% confluency. Then, 22 ug of expression vector was mixed with 22 ug of pCMV and 4.5 ug of pVSV-G (for 12-well transfections these ug quantities of DNA were divided by 4). This DNA mixture was then packaged using PEImax (Fisher Scientific) and lx OptiMem (Gibco) and added to the media of the 293T cells. Then, 12-17 hours later the media was removed from theseAttorney Docket No. 048536-729001WO cells and replaced with “induction” DMEM media containing 10 mM sodium pyruvate. After incubating for 6-8 hours, the “induction” media was replaced with “viral” media. Two days later, viral supernatants were harvested using LentiX Concentrator (Takara), concentrated by centrifugation, and used immediately for transduction.Primary human T cell culture

[0191] T cells were isolated from anonymized donor blood post-apheresis (Vitalant) in bulk CD3+format via positive selection (Stem Cell 17851). For Incucyte experiments, CD8+populations were isolated (Stem Cell 15063). Use of donor material was approved by the UCSF Institutional Review Board. Upon isolation, T cells were frozen in liquid nitrogen in RPMI-1640 (Thermo Fisher 11875093) supplemented with 20% human AB serum (Valley Biomedical Inc., HP1022) and 10% DMSO. For experiments, T cells were thawed at 37°C, washed, and cultured in human T cell media consisting of X- VIVO 15 (Lonza #04-418Q) with 5% Human AB Serum, 10 mM N-acetyl L-Cysteine (Sigma-Aldrich #A9165) neutralized with IM NaOH (Sigma- Aldrich S2770) and supplemented with 30 units / mL of IL-2 (NCI BRB Preclinical Repository). One day post-thaw, T cells were stimulated with washed Dynabeads (Thermo Fisher 11132D) using a 1 :3 cell Lead ratio. On the following day, untitered lentiviral supernatant was added at a 1 : 1 total volume ratio. After 24 hours of infection, cells were gently pelleted (400xg for 5 minutes) and depleted media was exchanged for fresh complete media. After three days of subsequent expansion, Dynabeads were removed via magnetic separation and cells were sorted (Beckton Dickinson FACSARIA II) for populations expressing both epitope-tagged receptors and constitutive fluorophore-expressing reporter circuits (where applicable). Experiments typically commenced 4 to 7 days post-sort. All experiments were performed in complete human T cell media aside from Incucyte assays, which were performed in RPMI / 10% FBS.Flow cytometry and sorting

[0192] For sorting, T cells were pelleted and resuspended in 100 pL of PBS with 2% FBS and antibody at a 1 : 100 ratio. After 20-30 minutes of staining at room temperature, cells were pelleted and resuspended in PBS with 2% FBS and kept on ice until sorting. For flow cytometry measuring a fluorescent reporter gene, cells were directly analyzed and gated on scatter (FSC-A vs. SSC-A), single cells (FSC-H vs. FSC-A), and the constitutive co-fluorophore expressed by the reporter construct (see, e.g., FIG. 2A). For cytometry experiments involvingAttorney Docket No. 048536-729001WG immunohistochemistry, cells were washed once in PBS prior to staining for 20-30 minutes at room temperature, and then washed 3x prior to analysis. Antibodies used in the present disclosure are listed in Table 5 below.TABLE 5: AntibodiesIn vitro SNIPR activation

[0193] Recombinant soluble factors were gently resuspended in their respective manufacturer- recommended reconstitution buffers and frozen at -80°C as single use aliquots. After thawing at room temperature, proteins were diluted in T cell media to the appropriate concentration and either added directly to the T cells in 96 well plates at a 1 : 100 dilution, or pre-diluted in media to 2x final concentration and added in a 1 : 1 ratio. Cells were mixed briefly and incubated at 37°C until reporter expression was assayed via flow cytometry (BD FACSymphony X50 SORP or LSR II SORP). Data shown represents 72 hours of ligand exposure unless otherwise indicated. Representative Gating is shown in FIG. 2B. Recombinant soluble factors employed in experiments are listed in Table 6 and inhibitors are shown in Table 7.TABLE 6: Soluble factorsAttorney Docket No. 048536-729001WOTABLE 7: Inhibitors

[0194] Data collected for Natural Ligands heat map was at 100 pM while Synthetic Ligands experiment was performed at 25 pM.

[0195] Where unspecified, TGF-P refers to human TGF-P 1 and VEGF refers to human VEGFa. Activation of latent human TGF-P 1 was performed by incubation at 80 °C for 5 minutes as described by Brown et al35. For the TRE3G promoter, doxycycline hyclate (Abeam abl41091) was added as the ligand analogue reagent. For chemical inhibitor experiments, DAPT, GI 254023X and Chloroquine were used at a working concentration of 10 pM, 10 pM and 25-100 pM, respectively. The blocking antibodies, pan TGF-P blocker (R&D MAB1835) and Bevacizumab (Selleck, Cat. No. A2006) were used at a concentration of 0.17 pg / mL and 1.4 pg / mL, respectively. Chemical inhibitors and blocking antibodies were added immediately prior to ligands.

[0196] For Jurkat experiments, Jurkat T Cells were harvested and resuspended at 4xl06cells / mL in fresh RPMI media. De-novo designed OrthoLigands were diluted to the appropriate concentrations in lx PBS and were added to freshly washed cells at 1 / 10 dilution (20 pL into 200 pL) in 96 well plates. Assay plates were incubated at 37 °C for 17-24 hours and then analyzed using flow cytometry. The OrthoLigands and inhibitors used in these experiments are also listed Table 6 and Table 7, respectively.Microwell fabrication

[0197] Microwells were prepared as previously described (Engl W. et al., Nat Cell Biol 16,Attorney Docket No. 048536-729001WG584-591, 2014). In brief, a master SU8 mold with periodic arrays of microwells (24 pm x 40 pm diameter x depth, 10 pm well-to-well spacing) was fabricated on a silicon wafer via photolithography. The master mold was then passivated via Ch-plasma treatment followed by trichloro(lH,lH,2H,2H-perfluorooctyl)silane (Sigma- Aldrich) vapor deposition under vacuum. Polydimethylsiloxane (PDMS) was cast onto the wafer and cured to create a negative of the master mold. After carefully removing PDMS blocks from the wafer, a circular PDMS stamp (4 mm in diameter) was cut and placed on the coverslip (No. 1.5) of a glass-bottom dish (MatTek). A drop of pre-polymer solution (BIO 133, My Polymers) was added to one side of the PDMS stamp, facilitating gap-filling between the substrate and the stamp by capillarity. After curing with UV exposure, the PDMS stamp was removed carefully. The final microwell array pattern was treated with Pluronic acid F127 (Sigma-Aldrich) overnight to block nonspecific binding of soluble ligands to the microwells.Protein expression and purification

[0198] OrthoSNIPR ligands were expressed in E. coli BL21 (NEB). Briefly, the DNA fragments encoding the design sequences were assembled into pET-29 vectors via Gibson assembly and further transformed into BL21 strain with heat-shock. Protein expression was induced by the autoinduction system and proteins were purified with immobilized metal affinity chromatography (IMAC). The elutions were further purified by FPLC SEC using Superdex 75 10 / 300 GL or Superdex 10 / 300200 columns (GE Healthcare). Protein concentrations were determined by NanoDrop (Thermo Scientific) and normalized by extinction coefficients. Proteins were diluted to the appropriate concentrations in lx PBS and applied to cell culture media using 1 / 100 dilutions.Confocal microscopy

[0199] HeLa cells were engineered to express the SNIPR receptor with a cleavable GFP domain in place of a transcription factor. These cells were seeded in 18 well glass bottom p- Slides (Ibidi, Cat. No. 81817) at a density of 15k / well. mCherry fused SNIPR ligands were incubated with the cultured cells for 15 min, 3 h, 6 h or 24 h, Lysotracker (Thermo Fisher Scientific, Cat. No. L7526) was added 30 min before imaging. Cells were washed 3 times in PBS and immediately proceeded to imaging. Confocal laser scanning microscopy was performed on a Nikon AIR HD25 system equipped with a LU-N4 laser unit (Lasers used: 488 nm, 561 nm, 640Attorney Docket No. 048536-729001WO nm). Data was acquired using a 20*, NA 0.75, WD 1 .00 mm air objective (Plan Apochromat Lambda) in combination with 1 multi-alkaline (EM 650 LP) and 2 GaAsP detectors (DM 560 LP EM 524 / 42 (503-545) and DM 652 EM 600 / 45 (578-623)). Acquisition was controlled via NIS Elements software and data was analyzed via Fiji and custom-written Python Scripts.

[0200] Jurkat T cells expressing mCherry-fused TGF-P SNIPRs were seeded into microwells. Fluorescence labeling of recombinant TGF-P 1 was performed by the following procedures. Recombinant TGF-pi (10 pg) was dissolved in HEPES buffer (pH8, 100 mM) and then mixed with 5 equivalent of Alexa Fluor 647 NHS ester (Thermo Fisher A37573) (20 mg / ml in DMSO). After 1 hour reaction, excess dyes were removed by passing the solution through spin desalting column (Zeba microspin, Thermo Fisher). To induce SNIPR activation, Alexa Fluor 647 labeled TGF-pi was added to the cells entrapped in microwells to a final concentration of 100 ng / ml. Cells were stained with Lysotracker (Thermo Fisher L7526) for 30 minutes immediately prior to each imaging session at 6 hours and 24 hours. Live cell imaging was performed using a 60* Plan-Apo oil objective (N.A. 1 .42) on an Olympus Fluoview 3000 laser scanning confocal microscope. Z-stack images were acquired and data was analyzed in Fiji.In vitro cell-cell communication assays

[0201] For in vitro detection of cell-produced TGF-P and VEGFu, target cells were seeded in a flat bottom 96 well culture vessel at 20k / well in each cell type’s native media. After 24 hours of adherence and growth, the growth media was aspirated and replaced with 40k / well T cells in human T cell media. BFP reporter activation was determined via flow cytometry after 48 hours of coculture. Similarly, the in vitro co-culture in transwell cell system required 50k / well of target cells in the transwell membrane (Corning, Cat. No. 3388). After 24 hours of incubation, T cell were seeded at lOOk / well T cells at the bottom of the well, and cells were incubated for 72 hours before BFP reporter activation was determined via flow cytometry. For the production of TGF- pi and VEGF by ELISA, target cells were seeded at lOOk / well in a flat bottom 96 well culture vessel and incubated for 72 hours. After incubation, a hTGF-pi ELISA (Thermo Fisher Scientific BMS249-4,) and hVEGF ELISA kit (Thermo Fisher Scientific KHG0111) were performed following the manufacturer’s protocol. Following this same methodology, target cells were seeded and supernatant was collected for a Luminex TGFp 3-plex Discovery Assay® Multi Species Array (TGFpi-3) and a VEGF-A,B,C Assay® performed by Eve Technologies. To generate OrthoLigand sender cells, HeLa cells were transiently transfected via electroporationAttorney Docket No. 048536-729001WO with a plasmid encoding an orthoLigand that was preceded by a modified serum albumin secretion tag. The cells were then grown for 48 hours to allow for protein expression before collecting the media. To eliminate any potential cell contamination, the media was centrifuged at 500 xg. To assess the functionality of the orthoLigand, the conditioned media from the sender cells was added to Jurkat cells, which served as the receiver cells. The cells were incubated for 24 hours before being analyzed for BFP expression. BFP signal was indicative of successful orthoLigand-mediated signaling between the sender and receiver cells.Incucyte Imaging

[0202] For in vitro cell live cell imaging killing assays, A375, A549, or M28 target cells bearing nuclear-localized mKate2 were seeded in flat bottom 96 well plates in their native media. After 24 hours, media was aspirated and immediately replaced with CD8+T cells in RPMI1640 (Gibco) + 10% FBS + 50 U / mL penicillin / streptomycin + 30 U / mL IL-2 so as to maintain an expected 2:1 effector:target ratio at the start of the experiment. Plates were imaged using the Incucyte S3 Live-Cell Analysis System (Essen Bioscience) with 3 or 4 images collected per well and an imaging period of 4 - 6 hours for at least 7 days of coculture. Cells were counted via automated segmentation of the fluorescent target cell nuclei using the Incucyte software.In vivo xenograft models

[0203] All animal work was conducted under approval from the UCSF Institutional Animal Care and Use Committee (protocol # AN177022-03C). All experiments employed NOD.Cg- Prkdc' ‘cl2rf ">!‘'Vj! / SzJ (NSG) (RRID:IMSR_JAX:005557) mice of age 8 - 12 weeks at the onset of experimentation. For all tumor models, dissociated cancer cells in 0.1 mL serum-free DMEM were implanted subcutaneously in the flank. T cells were administered retroorbitally in 0.1 mL PBS 7 days post-tumor injection (5 days post-sort). Experiments were performed with 1E6 tumor cells and 6E6 T cells per mouse, except for in FIG. 9C which employed 1.5E6 tumor cells and 9E6 T cells. Tumor size was monitored via caliper, and for experiments employing mouse / human cross-reactive CARs, mice were weighed to a precision of 0.1 g daily for one week post T cell injection and then at all subsequent tumor measurement time points. Mice were euthanized upon tumor measurement along any axis of > 20 mm or upon reaching a tumor volume of > 2000 mm3, where volume = Vi * largest axis * (smallest axis)2, on weight loss of 15% below initial weight at tumor injection as specified in Applicant’s I ACUC -approvedAttorney Docket No. 048536-729001WO protocol, or when displaying humane euthanasia criteria for other reasons including impaired mobility, observable behavioral distress (labored respiration, hunched appearance), or tumor ulceration covering >50% of tumor surface area. Sample size not pre-determined via statistical methods. Randomization and blinding were not performed.Statistics and Reproducibility

[0204] Data points represent individual technical replicates unless otherwise stated. Technical replicates were performed as distinct samples. For tumor curve comparison, Area Under Curve measurements were compared; for mice which were euthanized prior to the end of the study, the final tumor volume measurement was prorated through the final time point. Comparison of mouse weight loss was performed on the day of peak weight loss for each individual experiment. Individual biological donors used are provided in Table 2.Software

[0205] Chart plotting and statistical analysis were performed in GraphPad Prism 10. Flow cytometry and sorting were performed using BD FACSDiva, and post-hoc gating and analysis were conducted using FlowJo 10.8.0. Imaging colocalization analysis was performed with a custom-written python script reporting the Pearson correlation coefficient.EXAMPLE 2QrthoSNIPRs enable sensing of natural and engineered soluble factors

[0206] This Example describes experiments performed to demonstrate that the orthoSNIPRs as disclosed herein can serve as a versatile receptor architecture that can be adapted for bioorthogonal cell signaling in T cells for detecting cancer-related and inflammatory soluble signaling molecules.

[0207] As reported previously, SNIPRs remain inactive in the absence of ligands but exhibit a robust transcriptional response when membrane-bound ligands are encountered, despite lacking the LNR domains thought to be critical for regulating Notch activation. The experiments described in this Example were performed to investigate as to whether SNIPRs may activate through an alternative pathway to the conventional force-induced LNR stretching model (Sloas, DC et al., Nat Biotechnol 1-9 (2023) doi: 10.1038 / s41587-022-01638-y), enabling the detection of soluble factors that are inaccessible to Notch family receptors. In particular, to assess this potential, the inventors initially designed SNIPRs with extracellular domains (ECDs)Attorney Docket No. 048536-729001WO incorporating scFvs derived from a set of antibodies against the broadly tumor-associated cytokines transforming growth factor 0 (TGF-0) (Chang ZL et al., Nature Chemical Biology 14, 317-324, 2018) and vascular endothelial growth factor a (VEGF) (Liang WC et al., Journal of Biological Chemistry 281, 951-961, 2006). TGF-0 is a critical tumor signaling molecule that is intrinsically tumorigenic, suppressive against tumor-infiltrating immune cells, and broadly expressed across a variety of solid cancers (Neuzillet C. et al., Pharmacology & Therapeutics 147, 22-31, 2015). Likewise, VEGF is a pleiotropic tumor-associated signaling molecule that can modulate the immune environment but is best known for its role in stimulating angiogenesis within the tumor (Liang WC et al. , Journal of Biological Chemistry 281, 951-961 (2006). The inventors found that primary human CD3+ T cells bearing TGF-0 SNTPR driving a BFP reporter circuit signaled upon addition of recombinant activated TGF-0 (FIG. IB). Similarly, it was observed that a VEGF SNIPR activated upon titration of recombinant VEGF (FIG. 1C). However, equivalent synNotch receptors failed to signal upon addition of the cognate ligands. The inventors demonstrated the tunability of the SNIPR by varying both the scFv identity and orientation (FIG. 2A) and the potency of the receptor’s transactivation domain (FIG. 2B). It was also found that receptor activity can be further improved by replacing the cysteine residue in the hinge domain with a serine residue (FIG. 2C). Both classes of SNIPR strongly activated in response to their respective ligands across multiple human T cell donors (FIG. 2D) and were specific to their designated ligand (FIG. 2E). The TGF-0 SNIPR is specific to the active versus the latent form of TGF-0 (FIG. 2F), suggesting its potential for tumor microenvironment detection (Metelli A. et al., Cancer Research 76, 7106-7117, 2016), and was cross-reactive across human TGF-0 isoforms, albeit with a preference for TGF-01 (FIG. 2G). To expand the range of possible soluble targets in disease environments, additional functional SNIPRs capable of detecting the stromal signaling factor fibroblast growth factor 2 (FGF-2) as well as interferon- Y (IFN-y), a key inflammatory cytokine, were also developed (FIG. ID). Collectively, these findings indicate that SNIPRs can serve as a versatile receptor architecture for detecting cancer- related and inflammatory soluble signaling molecules.

[0208] The inventors next investigated whether the SNIPR system could be adapted for bioorthogonal cell signaling in T cells. The inventors built SNIPRs incorporating one subunit of a computationally designed “LHD” heterodimer (Sahtoe DD et al., Science 375, eabj7662, 2022) as the extracellular domain, and evaluated signaling in response to the soluble heterodimericAttorney Docket No. 048536-729001WO partner (FIG. IE). Addition of the cognate subunit resulted in strong transcriptional response. In contrast, no signaling was observed when the LHD heterodimer was incorporated into synNotch system. This orthoSNIPR system is designed as bioorthogonal since the LHD subunits do not interact with naturally occurring proteins, and hence enables the creation of "private" channels of communication between engineered cells for more precise control over coordinated cellular activity.EXAMPLE 3OrthoSNIPRs access a distinct activation mechanism when sensing soluble ligands

[0209] This Example describes experiments performed to investigate the ability of the orthoSNIPRs as disclosed herein to detect soluble ligands.

[0210] The ability of SNIPRs to detect soluble ligands is a novel behavior for Notch-like receptors. Understanding the mechanism of soluble factor-regulated SN1PR activation has implications for receptor engineering and optimization while potentially elucidating new possibilities for Notch signaling beyond what has been observed in nature. Notch and synNotch signaling is typically considered to be restricted to surface-bound ligands (Gordon WR et al., Journal of Cell Science 121, 3109-3119, 2008), and previous literature has explicitly demonstrated the inability of a soluble ligand to activate synNotch in contrast to a membrane- tethered analogue (Morsut L et al., Cell 164, 780-791, 2016 and Toda S. et al., Science 370, 327-331, 2020). The homodimeric nature of TGF-P and VEGF suggested a model of mechanical cross-activation in trans, but SNIPR activation is insensitive to T cell dilution (FIGS. 4A-4B). The ability to induce BFP reporter expression in single cells entrapped in microwells underscores that soluble SNIPR activation does not rely on contact between adjacent cells (FIG. 4C). An alternative putative model proposed that ligands may apply force on the receptor by adsorbing to the culture plate, but using low-binding plastic vessels did not abrogate signaling (FIG. 4D).

[0211] Beyond the canonical mechanical model of force-mediated Notch signaling at the plasma membrane, evidence for the role of receptor endocytosis in Notch activation has emerged (Steinbuck, MP et al., The Journal of Immunology 200, 997-1007, 2018), and the localization of y-secretase activity to acidic compartments implies that pH may also play a role in some contexts (Maesako M. et al., J. Neurosci. 42, 145-154, 2022). To probe the behavior of SNIPRs as they enter the activation pathway, experiments were designed to perturb each potential step usingAttorney Docket No. 048536-729001WO small molecule inhibitors (FIG. 3A). All Notch-based receptors including soluble SNIPRs and CD19-responsive synNotch are sensitive to y-secretase inhibition via DAPT, implying a common final trigger for transcription factor release, while doxycycline-mediated induction from the control pTRE promoter was insensitive to this perturbation (FIG. 3B). Surprisingly, it was found that inhibition of ADAM protease, which is responsible for the initial cleavage event upon Notch ligand binding and LNR conformational change, hindered the activation of both the CD 19- responsive synNotch and SNIPR, but did not affect the soluble SNIPRs disclosed herein. In contrast, blocking endosomal acidification via chloroquine selectively inhibited the soluble SNIPRs disclosed herein, suggesting a model in which soluble factor binding triggers an endocytic cascade culminating in proteolysis within the endosome.

[0212] Additional experiments further demonstrated that orthoSNIPRs recapitulated the above result in conjunction with a panel of synthetic ligands. Inhibition of the TGF-P SNIPR by chloroquine was dose-dependent (FIG. 4E) while the effect of the DMSO vehicle was minimal at the assayed concentrations (FIG. 4F). In accordance with the receptor inhibition assay, confocal imaging confirmed that the TGF-P SNIPR colocalizes with a fluorescently labeled TGF-P 1 ligand upon exposure, and both receptor and ligand subsequently redistribute to internal compartments (FIG. 3C). Additionally, fluorescently labeled TGF-P SNIPR (FIG. 3D) and orthoLigand (FIG. 3E) colocalize with LysoTracker, an endocytic marker, upon coincubation.

[0213] Given that the experimental data described herein suggests that SNIPR activation occurs via the endosomal axis, additional experiments were performed to investigate the mechanism by which ligand binding could stimulate receptor internalization. A potential mechanism is based on the observation that the natural ligand-responsive SNIPRs all bind to dimeric ligands. Receptor oligomerization may induce endocytosis via an avidity-like mechanism in which multiplexed low affinity binding motifs against the native endocytic machinery cooperatively drive internalization of the clustered complex (Zhao C. et al., Biophysical Journal 117, 646-658, 2019). Without being bound to any particular theory, it is believed that ligand-mediated SNIPR dimerization may be responsible for receptor internalization. When the chemically-inducible DmrA (FKBP) homodimerization domain (Rollins CT et al., Proc Natl Acad Sci USA 97, 7096-7101, 2000) was inserted into the TGF-P SNIPR scaffold (FIG. 3F), the receptor demonstrated strong ligand-independent activation upon addition of the API 903 homodimerizer. Ligand-induced receptor multimerization may serve asAttorney Docket No. 048536-729001WO the proximal trigger for the endocytic cascade culminating in endosomal transcription factor release and gene expression.EXAMPLE 4Tunability of orthoSNIPR signaling

[0214] This Example describes experiments performed to investigate the tunability of orthoSNIPR signaling.

[0215] In these experiments, the inventors generated ligands with the same binding domain but differing valency and geometry by fusing them to a series of designed oligomeric scaffolds (Edman, N. I. et al. Preprint at doi.org / 10.1101 / 2023.03.14.532666, 2023) (FIG. 5A). These ligands generate a range of signaling strengths as reflected in both ECso and Emax, with higher valency ligands generally having increased sensitivity, cooperativity, and maximal activation up to a 40-fold increase over the baseline level. To enable the creation of bioorthogonal communication networks, a series of orthoSNIPR receptor-ligand pairs using five different synthetic heterodimer pairs was generated. As described above, one subunit of each heterodimer was used as the extracellular domain of the SNIPR, and the second subunit was fused to a designed hexameric scaffold conferring robust signaling. To maximize the versatility of the signaling networks that could be generated from these synthetic receptors, the inventors selected three heterodimer pairs that previous characterization with purified proteins showed are completely orthogonal, and two heterodimers that cross interact; the former enable insulated communication pathways, the latter combinatorial modulation as observed with BMP signaling (Antebi YE et al., Cell 170, 1184-1196. e24, 2017) (FIGS. 5B and 6A). As predicted from the biochemical properties of the binding domains, three of the receptors are completely orthogonal and respond only to their ligands, while the other two recapitulate the more relaxed specificity of the parent designs. Such cross-reactive components enable the design of complex networks with fewer parts than would be possible with fully orthogonal receptors, allowing for encoding more information in therapeutic cells by payload-limited gene delivery vectors.

[0216] Thus, the experimental results described herein indicates that the orthoSNIPR system can be readily expanded beyond one receptor to form the basis of a complex communication network that relies on private and promiscuous signals to process and respond to their environments and execute user-defined functions in therapy, development, or homeostasis.Attorney Docket No. 048536-729001WG

[0217] The orthoSNIPR platform offers several avenues for signal tuning in addition to modulating ligand valency. Similar to the TGF-P and VEGF SNIPRs, orthoSNIPR signaling output increases upon mutation of the hinge cysteine to a serine residue (FIG. 6B). A more dynamic approach employs engineered signaling pathways that are conditional on the presence of additional factors, which could have considerable therapeutic utility. For example, if CAR-T cells detect a signal from other engineered cells indicating they are in the wrong environment, or if patients experience CRS symptoms, off- or damper-switches could mitigate toxicity. To enable such conditional signaling, the inventors designed a weakly homomeric ligand that strongly induces receptor signaling unless another engineered factor, termed “orthoTuner,” is present in the environment. OrthoTuner has higher affinity for the subunits of the homodimeric ligand than they have for each other, and hence forms heterodimers that contain only one receptorinteracting LHD module (FIG. 5C). Addition of orthoTuner inhibits signaling in a dosedependent manner. This conditionality enables modulation of the strength of orthoSNIPR signaling by external inputs or communication between engineered cells.

[0218] Deciphering the mechanism of soluble SNIPR activation suggested an approach to enhancing the signal strength of orthogonal output by promoting its endocytosis. Tethering orthoLigands to binders against lysosomal shuttling proteins (EndoTags) promotes SNIPR internalization and activation of its payload (see companion manuscript). EndoTagged orthoLigands efficiently activate their cognate SNIPRs in primary T cells (FIGS. 5D-5E). The inventors extended their work on the orthoSNIPR system to demonstrate autonomous cell signaling with cell-derived rather than exogenous ligands. To this end, the inventors engineered a “sender cell” that secretes the ligand. When the “receiver” Jurkat T cells expressing the cognate receptor were cultured in the presence of the media conditioned by the sender cells, the inventors observed signaling in response to the secreted ligand (FIG. 5F). The inventors chose to isolate the sender cells from the receiver cell to avoid cell membrane-based trans-activation and guarantee that any response is due to the soluble ligand. Sender cells secreting isolated orthoLigand activated SNIPR-bearing receiver cells, and secreted EndoTagged orthoLigand augmented the response. The experimental results described herein demonstrated that with the orthoSNIPR communication system, cells can autonomously communicate and share information in a channel that is completely private and biorthogonal.Attorney Docket No. 048536-729001WOEXAMPLE 5Expanding the landscape of targetable antigens for engineered T cell therapies with soluble SNIPR to CAR circuits

[0219] This Example describes experiments performed to demonstrate that SNIPRs could detect physiological production of endogenous soluble factors rather than overexpressed or supplemented recombinant ligands.

[0220] In these experiments, SNIPR — BFP circuit T cells in co-culture with a panel of potential target human tumor cell lines were assayed (FIG. 7A). The activation of BFP expression in TGF SNIPR-T cells generally scales with the production of TGF-pi (FIG. 7B) across tumor cell lines, as determined by ELISA. The disproportionate response to Caco-2 can be reconciled through Luminex analysis, revealing co-production of TGF-P2 alongside the primary TGF- 1 isoform (FIG. 8A). Activation of VEGF SNIPR T-cells in direct co-culture with target cells shows a less clear correlation to the measured VEGFa concentration, with A549 cells stimulating disproportionate BFP expression (FIG. 7C). Luminex analysis revealed that VEGFa is the sole variant of VEGF produced (FIG. 8A), ruling out cross-homolog reactivity as the cause. Interestingly, preventing direct cell-cell contact using a transwell cell culture system reconciles the BFP expression pattern with the measured ligand secretion (FIG. 7D), indicating heightened activity of A549 in a co-culture setting. Nonetheless, the specificity of activation was confirmed via inhibition of SNIPR activation via TGF and VEGF-blocking antibodies (FIG.7E), which effectively blunted BFP expression including in co-culture with A549. The heightened activation potential of A549 cell line may be due to surface display of VEGF or other undetermined mechanisms that rely on cell contact.

[0221] Soluble antigen sensors could improve the safety and efficacy of CAR-T therapy by selectively restricting CAR expression within the tumor, imparting asynchronous IF — THEN combinatorial logic and enhancing the specificity of potentially promiscuous CARs. A receptor intended for therapeutic use must strike a balance between low basal signaling to avoid off-target toxicity and robust activation to drive a sufficient level of CAR transgene expression for effective cancer cell killing. This is particularly challenging given the lower sensitivity of CARs compared to natural TCR counterparts (Salter Al et al., Sci. Signal. 14, 2021). The inventors sought to construct circuits in which soluble SNIPR-primed activation drives expression of a CAR capable of addressing solid tumor antigens (FIG. 8B). It was then verified that, similarly toAttorney Docket No. 048536-729001WGBFP, the CAR payload is expressed by SNIPR CAR T cells in a dose-dependent manner (FIG. 8C). To determine the optimal CAR payload for targeting A375 melanoma cells, which have previously been implicated in TGF-mediated CAR-T suppression (Roth TL et al., Cell 181, 728-744. e21, 2020), the inventors constructed TGF-P SNIPR —> CAR circuits bearing a panel of CARs against potential endogenous A375 antigens and assayed their in vitro killing efficacy using a live cell imaging assay (FIG. 8D). Of the variants tested, a Her2-specific CAR was the most efficacious and was therefore selected for subsequent experiments. While this result was surprising in light of literature suggesting a dearth of Her2 expression in A375 cells (Milenic, DE et al., mAbs 2, 550-564, 2010), the presence of this antigen was confirmed in RFP-expressing target cells used for live cell killing assays as well in the unmodified cell line (FIG. 8E). It was then shown that both TGF-P and VEGF-responsive SNIPR circuits driving the Her2 CAR exhibit robust killing of both A549 lung adenocarcinoma cells (FIGS. 7F-7G) and A375 cells (FIGS. 8F-8G). Consistent with previous work, SNIPR — CAR circuits displayed slower killing kinetics than constitutively expressed CAR, owing to the temporal delay in CAR expression subsequent to primary ligand encounter (Zhu I. et al., Cell 185, 1431-1443. el6, 2022). To verify the target specificity of these circuits, A549 and M28 cells overexpressing matched levels of CD 19 were constructed (FIG. 8H). As predicted by reporter gene induction in co-culture, the TGF-P SNIPR shows specificity for A549, while the VEGF SNIPR demonstrates preferential A549 targeting (FIG. 81).

[0222] Next, additional experiments were performed to evaluate the in vivo performance of SNIPR — CAR circuits. Mice bearing subcutaneously embedded A375 melanoma tumors underwent adoptive T cell transfer 7 days post tumor seeding. TGF-P SNIPR —> Her2 CAR T cells significantly slowed tumor growth relative to untransduced control T cells, while SNIPRs bearing the activating hinge mutation eliminated the tumors with efficacy similar to that of the constitutive CAR (FIG. 10A). To assess the ability of soluble SNIPRs to improve the therapeutic window of CARs prone to off-tumor toxicity, the human-specific 4D5 Her2 CAR was replaced with a recently described mouse / human cross-reactive DARPin-based variant (Hammill JA et al., J Immunother Cancer 3, 55, 2015). Previous work has shown that constitutive DARPin CAR-T cells administered to NSG mice results in toxicity and weight loss due to on-target / off-tumor toxicity against low levels of Her2 expression in lung tissue (Hammill JA et al., Molecular Therapy - Oncolytics 17, 278-292, 2020), consistent with the fatal pulmonary toxicity reportedAttorney Docket No. 048536-729001WG in an early clinical trial of human Her2-targeting CARs (Morgan RA et al., Mol Ther 18, 843- 851, 2010). SNIPR CAR circuits are expected to enhance the safety of CARs with such promiscuous activity by confining their expression to the tumor (FIG. 9A). The ability of TGF-P and VEGF SNTPRs to activate in response to both human and mouse target ligands (FIG. 10B) further enhances the clinical relevance of this model, although TGF-P and VEGF expression in immunocompromised NSG mice may not recapitulate that of syngeneic models.

[0223] The performance of the SNIPR DARPin CAR circuits was first evaluated in an A549 adenocarcinoma xenograft model. Consistent with the previously published data, mice injected with constitutive DARPin CAR-T cells experienced rapid weight loss to the humane euthanasia threshold (FIG. 9B). In contrast, mice receiving the SNIPR CAR circuits maintained a healthy weight and were able to robustly control tumor growth, emphasizing the potential of this approach for generating potent cell-based therapies with improved safety profiles. The inventors then sought to validate the above-described circuits in an A375 melanoma xenograft model. Despite retarding tumor growth, the SNIPR circuit T cells failed to clear the tumors, suggesting that A375 xenografts are more resistant to T cell cytotoxicity (FIG. 10C). In vitro cytotoxicity analysis revealed that the DARPin CAR, constructed as reported with an extended CD8 hinge domain, is less potent than the original 4D5-based version, but it was found that a modified construct bearing a truncated hinge (DARPintrCAR) matching that in the 4D5 CAR partially restored efficacy (FIGS. 10D-10E). Unexpectedly, it was also found that this truncation reduced in vivo toxicity of the constitutive CAR-T cells, necessitating the installation of the CD28 costimulatory domain and injection of a higher dose to recapitulate weight loss (FIG. 10F). Then, the performance of the SNIPR —trCARs was evaluated in the A375 xenograft model, increasing the tumor dosage proportionally to the T cell amount. The TGF-P and VEGF SNIPR —>trCAR circuits were well -tolerated and highly efficacious, while constitutive DARPintrCAR-T cells rapidly induced weight loss within several days of infusion, albeit not to the euthanasia threshold, and then failed to control tumor outgrowth (FIG. 9B). To test an orthogonal toxicity model, the inventors also evaluated their constructs using a cross- reactive anti-Mesothelin nanobody -based CAR payload (Brown PD et al., Growth Factors 3, 35-43, 1990) using A375 tumor cells ectopically expressing Mesothelin. Similarly to the cross- reactive Her2 CARs, the SNIPRs significantly increased the therapeutic window of this potent and highly toxic CAR (FIG. 10G).Attorney Docket No. 048536-729001WO

[0224] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.REFERENCES1. Schwank, G. & Basler, K. Regulation of Organ Growth by Morphogen Gradients. Cold Spring Harb Perspect Biol 2, a001669 (2010).2. Berraondo, P. et al. Cytokines in clinical cancer immunotherapy. Br J Cancer 120, 6-15 (2019).3. Chang, Z. L., Hou, A. J. & Chen, Y. Y. Engineering primary T cells with chimeric antigen receptors for rewired responses to soluble ligands. Nat Protoc 1-18 (2020) doi : 10.1038 / s41596-020-0294-8.4. Chang, Z. L. el al. Rewiring T-cell responses to soluble factors with chimeric antigen receptors. Nature Chemical Biology 14, 317-324 (2018).5. Guo, T., Ma, D. & Lu, T. K. Sense-and-Respond Payload Delivery Using a Novel Antigen- Inducible Promoter Improves Suboptimal CAR-T Activation. ACS Synth. Biol. (2022) doi : 10.1021 / acssynbio.1 c00236.6. Uchibori, R. et al. Functional Analysis of an Inducible Promoter Driven by Activation Signals from a Chimeric Antigen Receptor. Mol Ther Oncolytics 12, 16-25 (2018).7. Schwarz, K. A., Daringer, N. M., Dolberg, T. B. & Leonard, J. N. Rewiring human cellular input-output using modular extracellular sensors. Nat Chem Biol 13, 202-209 (2017).8. Kroeze, W. K. et al. PRESTO-Tango as an open-source resource for interrogation of the druggable human GPCRome. Nat Struct Mol Biol 22, 362-369 (2015).9. Kipniss, N. H. et al. Engineering cell sensing and responses using a GPCR-coupled CRISPR- Cas system. Nat Commun 8, 1-10 (2017).10. Mahameed, M., Wang, P., Xue, S. & Fussenegger, M. Engineering receptors in the secretory pathway for orthogonal signalling control. Nat Commun 13, 7350 (2022).11. Morsut, L. et al. Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors. Cell 164, 780-791 (2016).Attorney Docket No. 048536-729001WO Roybal, K. T. et al. Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Cell 167, 419-432. el6 (2016). Zhu, I. et al. Modular design of synthetic receptors for programmed gene regulation in cell therapies. Cell 185, 1431-1443.el6 (2022). Sloas, D. C., Tran, J. C., Marzilli, A. M. & Ngo, J. T. Tension-tuned receptors for synthetic mechanotransduction and intercellular force detection. Nat Biotechnol 1-9 (2023) doi : 10.1038 / s41587-022-01638-y . Liang, W.-C. et al. Cross-species Vascular Endothelial Growth Factor (VEGF)-b locking Antibodies Completely Inhibit the Growth of Human Tumor Xenografts and Measure the Contribution of Stromal VEGF *. Journal of Biological Chemistry 281, 951-961 (2006). Neuzillet, C. et al. Targeting the TGF pathway for cancer therapy. Pharmacology & Therapeutics 147, 22-31 (2015). Metelli, A. etal. Surface Expression of TGFp Docking Receptor GARP Promotes Oncogenesis and Immune Tolerance in Breast Cancer. Cancer Research 76, 7106-7117 (2016). Sahtoe, D. D. et al. Reconfigurable asymmetric protein assemblies through implicit negative design. Science 375, eabj7662 (2022). Gordon, W. R., Arnett, K. L. & Blacklow, S. C. The molecular logic of Notch signaling - a structural and biochemical perspective. Journal of Cell Science 121, 3109-3119 (2008). Toda, S. et al. Engineering synthetic morphogen systems that can program multicellular patterning. Science 370, 327-331 (2020). Steinbuck, M. P., Arakcheeva, K. & Winandy, S. Novel TCR-Mediated Mechanisms of Notch Activation and Signaling. The Journal of Immunology 200, 997-1007 (2018). Maesako, M., Houser, M. C. Q., Turchyna, Y., Wolfe, M. S. & Berezovska, O. Presenilin / y- Secretase Activity Is Located in Acidic Compartments of Live Neurons. J. Neurosci. 42, 145— 154 (2022). Zhao, C. etal. Receptor Heterodimerization Modulates Endocytosis through Collaborative and Competitive Mechanisms. Biophysical Journal 117, 646-658 (2019). Rollins, C. T. et al. A ligand-reversible dimerization system for controlling protein-protein interactions. Proc Natl Acad Sci USA 97, 7096-7101 (2000). Edman, N. I. et al. Modulation of FGF pathway signaling and vascular differentiation using designed oligomeric assemblies. 2023.03.14.532666 Preprint atAttorney Docket No. 048536-729001WO https: / / doi.org / 10.1101 / 2023.03.14.532666 (2023). Antebi, Y. E. et al. Combinatorial Signal Perception in the BMP Pathway. Cell 170, 1184- 1196. e24 (2017). Salter, A. I. etal. Comparative analysis of TCR and CAR signaling informs CAR designs with superior antigen sensitivity and in vivo function. Sci. Signal. 14, (2021). Roth, T. L. et al. Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies. Cell 181, 728-744. e21 (2020). Milenic, D. E. et al. Targeting HER2. mAbs 2, 550-564 (2010). Hammill, J. A. et al. Designed ankyrin repeat proteins are effective targeting elements for chimeric antigen receptors. JImmunother Cancer 3, 55 (2015). Hammill, J. A. et al. A Cross-Reactive Small Protein Binding Domain Provides a Model to Study Off-Tumor CAR-T Cell Toxicity. Molecular Therapy - Oncolytics 17, 278-292 (2020). Morgan, R. A. et al. Case Report of a Serious Adverse Event Following the Administration of T Cells Transduced With a Chimeric Antigen Receptor Recognizing ERBB2. Mol Ther 18, 843-851 (2010). Prantner, A. M. et al. Anti-Mesothelin Nanobodies for Both Conventional and Nanoparticle- Based Biomedical Applications. Journal of Biomedical Nanotechnology 11, 1201-1212 (2015). Williams, J. Z. et al. Precise T cell recognition programs designed by transcriptionally linking multiple receptors. Science 370, 1099-1104 (2020). Brown, P. D., Wakefield, L. M , Levinson, A. D. & Sporn, M. B. Physicochemical Activation of Recombinant Latent Transforming Growth Factor-beta’s 1, 2, and 3. Growth Factors 3, 35- 43 (1990). Engl, W ., Arasi, B., Yap, L. L., Thiery, J. P. & Viasnoff, V. Actin dynamics modulate mechanosensitive immobilization of E-cadherin at adherens junctions. Nat Cell Biol 16, 584- 591 (2014). Teng, F. et al. Programmable synthetic receptors: the next-generation of cell and gene therapies. Sig Transduct Target Ther 9, 1-25 (2024).

Claims

Attorney Docket No. 048536-729001WOCLAIMSWHAT IS CLAIMED IS:

1. A chimeric polypeptide comprising, from N-terminus to C-terminus: a) an extracellular ligand-binding domain (ECD) comprising a first monomer polypeptide of a designed a / p heterodimer (LHD) capable of selectively binding to an engineered ligand, wherein the engineered ligand comprises a second monomer polypeptide of the designed LHD heterodimer; b) a linking polypeptide; c) a transmembrane domain (TMD) from a Type 1 transmembrane receptor comprising one or more ligand-inducible proteolytic cleavage sites; and d) an intracellular domain (ICD) comprising a transcriptional regulator, wherein binding of the engineered ligand to the extracellular binding domain induces cleavage at the one or more ligand-inducible proteolytic cleavage sites.

2. The chimeric polypeptide construct of claim 1, wherein the first and the second monomers are capable of non-covalently interact to form the designed LHD heterodimer.

3. The chimeric polypeptide of any one of claims 1-2, wherein the ECD does not substantially bind a naturally occurring ligand.

4. The chimeric polypeptide of any one of claims 1-3, wherein the engineered ligand is a bioorthogonal ligand.

5. The chimeric polypeptide of any one of claims 1-4, wherein the engineered ligand is a soluble ligand.

6. The chimeric polypeptide of any one of claims 1-5, wherein the second monomer polypeptide of the designed LHD heterodimer is fused to one or more designed oligomeric scaffolds.

7. The chimeric polypeptide of any one of claims 1-6, wherein the second monomer polypeptide of the designed LHD heterodimer is capable of binding to the first monomerAttorney Docket No. 048536-729001WO polypeptide of the designed LHD heterodimer with specific geometry, affinity, and / or valency as determined by EC50 and Emax, respectively.

8. The chimeric polypeptide of any one of claims 1-7, wherein the first monomer polypeptide of a designed LHD heterodimer comprises an amino acid sequence selected from SEQ ID NOS: 1-5, and the second monomer polypeptide of the designed LHD heterodimer comprises an amino acid sequence selected from SEQ ID NOS: 6-10.

9. The chimeric polypeptide of any one of claims 1-8, wherein the linking polypeptide comprises:(i) a polypeptide hinge domain;(ii) at least about 80% sequence identity to a Notch juxtamembrane domain (IMD);(ii) at least about 80% sequence identity to a Notch IMD wherein the LIN-12-Notch repeat (LNR) and / or a heterodimerization domain (HD) of a Notch receptor has been deleted;(iv) at least about 80% sequence identity to a ROBO1 JMD including at least one fibronectin repeat; or(v) a polypeptide having about 2 to about 40 amino acids.

10. The chimeric polypeptide of any one of claims 1-9, wherein the chimeric polypeptide does not comprise a LIN-12-Notch repeat (LNR) and / or a heterodimerization domain (HD) of a Notch receptor.

11. The chimeric polypeptide of any one of claims 1-10, further comprising a stop-transfer- sequence (STS) in between the transmembrane domain and the intracellular domain.

12. The chimeric polypeptide of claims 1-11, wherein the linking polypeptide comprises a hinge domain capable of promoting oligomer formation of the chimeric polypeptide via intermolecular disulfide bonding.

13. The chimeric polypeptide of claim 12, wherein the hinge domain is derived from a CD8a hinge domain, a CD28 hinge domain, a PD-1 hinge domain, a CTLA4 hinge domain, an 0X40 hinge domain, an IgGl hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, and an IgG4 hinge domain, or a functional variant of any thereof.Attorney Docket No. 048536-729001WO14. The chimeric polypeptide of any one of claims 12-13, wherein the hinge domain comprises one or more mutations at cysteine residues that form the intermolecular disulfide bonds.

15. The chimeric polypeptide of any one of claims 1-14, wherein the ligand-inducible proteolytic cleavage site is a y-secretase cleavage site.

16. The chimeric polypeptide of any one of claims 1-15, wherein the transcriptional regulator comprises a DNA-binding domain and an effector domain, wherein the effector domain selected from the group consisting of a transcription activating domain, a transcription repressor domain, or an epigenetic effector domain.

17. The chimeric polypeptide of claim 16, wherein the effector domain comprises a transcription activating domain selected from the group consisting of Herpes Simplex Virus Protein 16 (HSV VP 16) activation domain; an activation domain consisting of four tandem copies of VP16 (VP64); a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator activation domain (Rta); a tripartite activator consisting of VP64, and Rta activation domains (VPR); and a histone acetyltransferase core domain of the human ElA-associated protein p300 (p300 HAT core activation domain).

18. The chimeric polypeptide of claim 16, wherein the effector domain comprises a transcription repressor domain selected from the group consisting of a Kruppel associated box repression domain (KRAB); a Repressor Element Silencing Transcription Factor repression domain (REST); a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins repression domain (WRPW); a DNA (cytosine-5)-methyltransferase 3B repression domain (DNMT3B); and an HP1 alpha chromoshadow repression domain.

19. The chimeric polypeptide of claim 16, wherein the effector domain comprises an epigenetic effector domain selected from the group consisting of a DNA methyltransferase DNMT (DNMT1, DNMT3), HAT1, GCN5, PCAF, MLL, SET, DOTI, SUV39H, G9a, KAT2A / B, EZH1 / 2, TET1 / 2, a SIRT family protein effector domain, a histone deacetylase, LSD1, and a KDM family protein effector domain.

20. The chimeric polypeptide of any one of claims 16-19, wherein the effector domain comprises a domain from a human or humanized polypeptide.Attorney Docket No. 048536-729001WO21 . The chimeric polypeptide of claim 16, wherein the DNA-binding domain (DBD) comprises a GAL4-DBD, a TetR-DBD, a zinc finger (ZF) DBD, or a zinc-finger homeodomain (ZFHD) DBD.

22. The chimeric polypeptide of any one of claims 1-21, wherein the intracellular domain further comprises a nuclear transport signal sequence.

23. A recombinant nucleic acid comprising a nucleotide sequence that encodes a chimeric polypeptide according to any one of claims 1-22.

24. The recombinant nucleic acid of claim 23, wherein the nucleotide sequence is incorporated into an expression cassette or an expression vector.

25. The recombinant nucleic acid of claim 24, wherein the expression vector is a viral vector.

26. The recombinant nucleic acid of claim 25, wherein the viral vector is a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, or a retroviral vector.

27. The recombinant nucleic acid of any one of claims 23-26, wherein the recombinant nucleic acid further comprises a response element, wherein the response element comprises:(a) a cognate target sequence to which the DBD of the transcriptional regulator binds;(b) an engineered responsive promoter operably linked to the cognate target sequence; and(c) a polynucleotide of interest.

28. The recombinant nucleic acid of claim 27, wherein the polynucleotide of interest encodes a regulatory RNA, a regulatory protein, a therapeutic protein, or a reporter molecule.

29. The recombinant nucleic acid of claim 28, wherein the reporter molecule is a fluorescent protein, a herpes simplex virus type 1 (HSV-1) thymidine kinase (TK), or a gas vesicle protein.

30. The recombinant nucleic acid of claim 28, wherein the therapeutic protein is a recombinant antigen-specific receptor.Attorney Docket No. 048536-729001WO31 . The recombinant nucleic acid of claim 30, wherein the recombinant antigen-specific receptor is an engineered T cell receptor (TCR) or a chimeric antigen receptor (CAR).

32. The recombinant nucleic acid of claim 28, wherein the regulatory RNA is a siRNA, shRNA, or miRNA.

33. The recombinant cell of claim 28, wherein the polynucleotide of interest encodes a protein, a regulatory RNA, or an antisense oligonucleotide.

34. A recombinant cell comprising:(a) a chimeric polypeptide according to any one of claims 1-22; and / or(b) a recombinant nucleic acid according to any one of claims 23-33.

35. The recombinant cell of claim 34, wherein the recombinant cell is a eukaryotic cell.

36. The recombinant cell of claim 35, wherein the eukaryotic cell is a mammalian cell.

37. The recombinant cell of claim 36, wherein the mammalian cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell.

38. The recombinant cell of claim 37, wherein the immune cell is a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, a cytotoxic T cell, or other T cell.

39. The recombinant cell of any one of claims 34-37, further comprising an engineered response element comprising i) a cognate target sequence to which the DBD of the transcriptional regulator binds, ii) a promoter sequence, wherein the cognate target sequence is operably linked to the 5' end of the promoter sequence, and iii) a polynucleotide of interest operably linked to the promoter sequence, wherein binding of the transcriptional regulator to the cognate target sequence modulates transcription initiation of a polynucleotide of interest.

40. The recombinant cell of claim 39, wherein the chimeric polypeptide is encoded by a first nucleic acid molecule and the engineered response element is encoded by a second nucleic acid molecule.Attorney Docket No. 048536-729001WO41 . The recombinant cell of claim 39, wherein the engineered response element is present in a nucleic acid vector, plasmid, DNA minicircle, minichromosome, or host chromosome.

42. The recombinant cell of any one of claims 37-41, wherein the immune cell is a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, a cytotoxic T cell, or other T cell.

43. A cell culture comprising a recombinant cell according to any one of claims 34-42, and a culture medium.

44. A method for making an engineered cell, comprising:(a) providing a cell capable of protein expression; and(b) transducing the cell with the recombinant nucleic acid of any one of claims 23-33.

45. The method of claim 44, wherein the recombinant nucleic acid is a recombinant nucleic acid of any one of claims 23-32, and wherein the method further comprises: c. transducing the cell with a second recombinant nucleic acid that encodes a response element, wherein the response element comprises: i. a cognate target sequence to which the DBD of the transcriptional regulator binds; ii. an engineered responsive promoter operably linked to the cognate target sequence; and iii. a polynucleotide of interest.

46. The method of claim 44, wherein the recombinant nucleic acid is a recombinant nucleic acid of any one of claims 27-32.

47. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and: a) a chimeric polypeptide according to any one of claims 1-22; b) a recombinant nucleic acid according to any one of claims 23-33; and / or c) a recombinant cell according to any one of claims 34-42.

48. The pharmaceutical composition of claim 47, wherein the composition comprises a recombinant nucleic acid according to any one of claims 23-33, and a pharmaceutically acceptable carrier.Attorney Docket No. 048536-729001WO49. The pharmaceutical composition of claim 48, wherein the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle.

50. The pharmaceutical composition of claim 47, wherein the composition comprises a recombinant cell according to any one of claims 34-42, and a pharmaceutically acceptable carrier.

51. A method for modulating an activity of a target cell in a subject, the method comprising administering to the subject an effective amount of a composition comprising a chimeric polypeptide according to any one of claims 1-22; a recombinant nucleic acid according to any one of claims 23-33; and / or an effective number of the recombinant cells according to any one of claims 34-42, wherein the administered polypeptide construct, recombinant nucleic acid and / or recombinant cells confers a modulation of an activity of the target cell in the subject.

52. The method of claim 51, wherein the target cell is a pathogenic cell.

53. The method of claim 52, wherein the target cell is a cancer cell.

54. The method of any one of claims 51-53, wherein modulation of the activity the target cell results in the death of the target cell.

55. A method for modulating an activity of a cell, the method comprising: a) providing a recombinant cell according to any one of claims 34-42; and b) contacting the recombinant cell with the engineered ligand, wherein binding of the engineered ligand to the extracellular ligand-binding domain results in cleavage of a ligandinducible proteolytic cleavage site and release of the intracellular domain, wherein the release of the intracellular domain results in modulation of an activity of the recombinant cell.

56. The method of claim 55, wherein the contacting is carried out in vivo, ex vivo, or in vitro.

57. The method of claim 55-56, wherein the release of the intracellular domain results in binding of the transcriptional regulator of the released intracellular domain to a cognate target sequence, which results in modulation of the expression initiation of a polynucleotide of interest, which results in modulation of an activity of the recombinant cell.Attorney Docket No. 048536-729001WO58. The method of any one of claims 55-57, wherein the activity of the cell to be modulated is selected from the group consisting of: expression of a selected gene, proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of a molecule, cellular adhesion, and cytolytic activity.

59. The method of any one of claims 55-58, wherein the transcriptional regulator modulates expression of an endogenous gene product.

60. The method of any one of claims 55-58, wherein the transcriptional regulator modulates expression of a heterologous gene product.

61. The method of claim 59-60, wherein the gene product is selected from the group consisting of chemokine, a chemokine receptor, a chimeric antigen receptor, a cytokine, a cytokine receptor, a differentiation factor, a growth factor, a growth factor receptor, a hormone, a metabolic enzyme, a pathogen-derived protein, a proliferation inducer, a receptor, an RNA guided nuclease, a site-specific nuclease, a T cell receptor, a toxin, a toxin derived protein, a transcriptional regulator, a transcriptional activator, a transcriptional repressor, a translational regulator, a translational activator, a translational repressor, an activating immuno-receptor, an antibody, an apoptosis inhibitor, an apoptosis inducer, an engineered T cell receptor, an immunoactivator, an immuno-inhibitor, an immune cell receptor, and an inhibiting immuno-receptor.

62. The method of any one of claims 55-61, wherein the released transcriptional regulator modulates differentiation of the cell, and wherein the cell is an immune cell, a stem cell, a progenitor cell, or a precursor cell.

63. A method for the prevention and / or treatment of a health condition in a subject in need thereof, the method comprising administering to the subject a therapeutic agent comprising an effective amount of the chimeric polypeptide according to any one of claims 1-22; the recombinant nucleic acid according to any one of claims 23-33; and / or an effective number of the recombinant cells according to any one of claims 34-42, wherein the administered polypeptide construct, the recombinant nucleic acid, and / or the recombinant cell confers a prevention and / or treatment of the health condition in the subject.Attorney Docket No. 048536-729001WO64. A kit for modulating an activity of a cell, inhibiting a target cell, or treating a health condition in a subject in need thereof, the kit comprising one or more of the following: a) a chimeric polypeptide according to any one of claims 1-22; b) a recombinant nucleic acid according to any one of claims 23-33; c) a recombinant cell according to any one of claims 34-42; and d) a pharmaceutical composition according to any one of claims 47-49; and optionally written instructions for using the kit.

65. A kit for modulating an activity of a cell, the kit comprising one or more of the following: a) a chimeric polypeptide according to any one of claims 1-22; b) a recombinant nucleic acid according to any one of claims 23-33; and c) an engineered response element comprising: i) a cognate target sequence to which the DBD of the transcriptional regulator binds; ii) an engineered responsive promoter operably linked to the cognate target sequence; and iii) a polynucleotide of interest; wherein binding of the transcriptional regulator to the cognate target sequence modulates transcription initiation of the polynucleotide of interest.

66. The use of one or more of the following for the prevention and / or treatment of a health condition: a) a chimeric polypeptide according to any one of claims 1-22; b) a recombinant nucleic acid according to any one of claims 23-33; c) a recombinant cell according to any one of claims 34-42; and d) a pharmaceutical composition according to any one of claims 47-49.

67. The use of one or more of the following for the manufacturing of a medicament for the prevention and / or treatment of a health condition: a) a chimeric polypeptide according to any one of claims 1-22; b) a recombinant nucleic acid according to any one of claims 23-33; c) a recombinant cell according to any one of claims 34-42; and d) a pharmaceutical composition according to any one of claims 47-49.

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