Binding-triggered transcriptional switches and methods of using same

Abstract

To provide binding-triggered transcriptional switch polypeptides, nucleic acids comprising nucleotide sequences encoding the binding-triggered transcriptional switch polypeptides, and host cells genetically modified with the nucleic acids.SOLUTION: Provided are chimeric Notch receptor polypeptides, nucleic acids comprising nucleotide sequences encoding the chimeric Notch receptor polypeptides, and host cells transduced and / or genetically modified with the nucleic acids. Also provided are transgenic organisms comprising a nucleic acid encoding a binding triggered transcriptional switch polypeptide and / or a chimeric Notch receptor polypeptide. The binding triggered transcriptional switch polypeptides and the chimeric Notch receptor polypeptides are useful in a variety of applications.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 120,256, filed February 24, 2015; U.S. Provisional Patent Application No. 62 / 257,153, filed November 18, 2015; and U.S. Provisional Patent Application No. 62 / 269,758, filed December 18, 2015, which applications are incorporated herein by reference in their entireties.

[0002] Statement of Federally Funded Research This invention was made with government support under grant numbers EY016546; P50 GM081879; and R01 GM055040 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Incorporation by reference of sequence listings provided as text files A sequence listing is provided herewith as a text file "UCSF-511WO_SeqList_ST25.txt," which was created on January 26, 2016 and is 649 KB in size. The contents of the text file are incorporated herein by reference in their entirety. [Background technology]

[0004] Introduction Notch receptors are transmembrane proteins that mediate cell-cell contact signaling and play a central role in the development and other aspects of cell-cell communication, e.g., communication between two contacting cells, one of which is the "receiving" cell and the other the "sending" cell. Notch receptors expressed in the receiving cell recognize their ligands (the Delta family of proteins) expressed on the sending cell. Engagement of Notch and Delta on these contacting cells results in two stages of proteolysis of the Notch receptor, which ultimately causes the release of the intracellular portion of the receptor from the membrane into the cytoplasm. This released domain alters the behavior of the receiving cell by functioning as a transcriptional regulator. Notch receptors are involved in and required for various cellular functions during development and are important for the function of numerous cell types across multiple species. Summary of the Invention

[0005] overview The present disclosure provides binding-triggered transcriptional switch polypeptides, nucleic acids comprising nucleotide sequences encoding the binding-triggered transcriptional switch polypeptides, and host cells genetically modified with the nucleic acids. The present disclosure provides transgenic organisms comprising nucleic acids encoding the binding-triggered transcriptional switch polypeptides of the present disclosure. Methods of locally regulating cellular activity using one or more binding-triggered transcriptional switch polypeptides, and localized cellular activation systems using one or more binding-triggered transcriptional switch polypeptides are provided. The binding-triggered transcriptional switch polypeptides of the present disclosure are useful in a variety of applications, which are also provided.

[0006] The present disclosure provides chimeric Notch receptor polypeptides, nucleic acids comprising nucleotide sequences encoding the chimeric Notch receptor polypeptides, and host cells genetically modified with the nucleic acids. The present disclosure provides transgenic organisms comprising nucleic acids encoding the chimeric Notch receptor polypeptides of the present disclosure. The chimeric Notch receptor polypeptides of the present disclosure are useful in a variety of applications, which are also provided.

[0007] The present disclosure provides chimeric polypeptides (also referred to herein as "chimeric Notch receptor polypeptides") comprising, covalently linked from N-terminus to C-terminus: a) an extracellular domain comprising a first member of a specific binding pair; b) a Notch receptor polypeptide having a length of between 50 amino acids and 1000 amino acids and comprising one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, wherein the first member of the specific binding pair is heterologous to the Notch receptor polypeptide, and binding of the first member of the specific binding pair to a second member of the specific binding pair induces cleavage of the Notch receptor polypeptide at the one or more ligand-inducible proteolytic cleavage sites, thereby releasing the intracellular domain. In some cases, the Notch receptor polypeptide has a length of between 300 amino acids and 400 amino acids. In some cases, the chimeric Notch receptor polypeptide comprises a linker interposed between the extracellular domain and the Notch receptor polypeptide. In some cases, the intracellular domain is a transcriptional activator. In some cases, the intracellular domain is a transcriptional repressor. In some cases, the intracellular domain is a site-specific nuclease. In some cases, the site-specific nuclease is a Cas9 polypeptide. In some cases, the intracellular domain is a recombinase. In some cases, the intracellular domain is an inhibitory immunoreceptor. In some cases, the intracellular domain is an activating immunoreceptor. In some cases, the first member of the specific binding pair comprises an antibody-based recognition scaffold. In some cases, the first member of the specific binding pair comprises an antibody. In some cases, when the first member of the specific binding pair is an antibody, the antibody specifically binds to a tumor-specific antigen, a disease-associated antigen, or an extracellular matrix component. In some cases, when the first member of the specific binding pair is an antibody, the antibody specifically binds to a cell-surface antigen, a soluble antigen, or an antigen immobilized on an insoluble substrate. In some cases, when the first member of the specific binding pair is an antibody, the antibody is a single-chain Fv.In some cases, the first member of the specific binding pair is a nanobody, a single domain antibody, a diabody, a triabody, or a minibody. In some cases, the first member of the specific binding pair is a non-antibody-based recognition scaffold. In some cases, when the first member of the specific binding pair is a non-antibody-based recognition scaffold, the non-antibody-based recognition scaffold is an avimer, a DARPin, an adnectin, an avimer, an affibody, an anticalin, or an affilin. In some cases, the first member of the specific binding pair is an antigen. In some cases, when the first member of the specific binding pair is an antigen, the antigen is an endogenous antigen. In some cases, when the first member of the specific binding pair is an antigen, the antigen is an exogenous antigen. In some cases, the first member of the specific binding pair is a ligand for a receptor. In some cases, the first member of the specific binding pair is a receptor. In some cases, the first member of the specific binding pair is a cell adhesion molecule (e.g., all or a portion of the extracellular region of a cell adhesion molecule). In some cases, the first member of the specific binding pair comprises a first dimerization domain and the second member of the specific binding pair comprises a second dimerization domain; for example, in some cases, binding of the first dimerization domain to the second dimerization domain is induced by a small molecule dimerizer, and in other cases, binding of the first dimerization domain to the second dimerization domain is induced by light. In some cases, the Notch receptor polypeptide comprises an amino acid sequence having at least 75% amino acid sequence identity to any one of the amino acid sequences depicted in Figures 2A-2G. In some cases, the Notch receptor polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to any one of the amino acid sequences depicted in Figures 2A-2G. In some cases, the Notch receptor polypeptide comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of the amino acid sequences depicted in Figures 2A-2G. In some cases, the Notch receptor polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to any one of the amino acid sequences shown in Figures 2A-2G.In some cases, a Notch receptor polypeptide comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of the amino acid sequences depicted in Figures 2A-2G. In some cases, a Notch receptor polypeptide comprises an amino acid sequence having at least 75% amino acid sequence identity to any one of the amino acid sequences depicted in Figure 3. In some cases, a Notch receptor polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to any one of the amino acid sequences depicted in Figure 3. In some cases, a Notch receptor polypeptide comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of the amino acid sequences depicted in Figure 3. In some cases, a Notch receptor polypeptide comprises an amino acid sequence having at least 95% amino acid sequence identity to any one of the amino acid sequences depicted in Figure 3. In some cases, a Notch receptor polypeptide comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of the amino acid sequences depicted in Figure 3. In some cases, a Notch receptor polypeptide comprises the following sequence: TIFF0007719836000001.tif37151. In some cases, the Notch receptor polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% amino acid sequence identity to TIFF0007719836000001.tif37151. In some cases, the one or more ligand-inducible proteolytic cleavage sites are selected from S1, S2, and S3 proteolytic cleavage sites. In some cases, the S1 proteolytic cleavage site is a furin-like protease cleavage site comprising the amino acid sequence Arg-X-(Arg / Lys)-Arg, where X is any amino acid. In some cases, the S2 proteolytic cleavage site is an ADAM-17 type protease cleavage site comprising an Ala-Val dipeptide sequence. In some cases, the S3 proteolytic cleavage site is a γ-secretase cleavage site comprising a Gly-Val dipeptide sequence.

[0008] The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a chimeric Notch receptor polypeptide as described herein. The present disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding a chimeric Notch receptor polypeptide as described herein. The present disclosure provides a host cell genetically modified with the nucleic acid or the expression vector. In some cases, the host cell is a eukaryotic cell. In some cases, the host cell is a mammalian cell. In some cases, the host cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the host cell is genetically modified with a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the intracellular domain of the chimeric polypeptide is a transcriptional activator. In some cases, the nucleotide sequence encoding the CAR is operably linked to a transcriptional control element that is activated by the intracellular domain of the chimeric polypeptide.

[0009] The present disclosure provides a method of modulating the activity of a cell expressing a chimeric Notch receptor polypeptide of the present disclosure as described herein, comprising contacting the cell with a second member of a specific binding pair, wherein binding of the first member of the specific binding pair to the second member of the specific binding pair induces cleavage of the Notch receptor polypeptide at one or more ligand-inducible proteolytic cleavage sites, thereby releasing the intracellular domain, and wherein release of the intracellular domain modulates the activity of the cell. In some cases, the contacting is carried out in vivo, ex vivo, or in vitro. In some cases, the second member of the specific binding pair is on the surface of the second cell, immobilized on an insoluble substrate, present in an extracellular matrix, present in an artificial matrix, or soluble. In some cases, release of the intracellular domain modulates cell proliferation. In some cases, release of the intracellular domain modulates apoptosis in the cell. In some cases, release of the intracellular domain induces cell death by a mechanism other than apoptosis. In some cases, release of the intracellular domain regulates gene expression in the cell through transcriptional regulation, chromatin control, translation, trafficking, or post-translational processing. In some cases, release of the intracellular domain regulates cell differentiation. In some cases, release of the intracellular domain regulates cell migration. In some cases, release of the intracellular domain regulates expression and secretion of molecules from the cell. In some cases, release of the intracellular domain regulates adhesion of the cell to a second cell or to an extracellular matrix. In some cases, release of the intracellular domain induces de novo expression of a gene product in the cell. In some cases, when release of the intracellular domain induces de novo expression of a gene product in the cell, the gene product is a transcriptional activator, a transcriptional repressor, a chimeric antigen receptor, a second chimeric Notch receptor polypeptide, a translational regulator, a cytokine, a hormone, a chemokine, or an antibody.

[0010] The present disclosure provides a method for modulating the activity of a cell expressing a chimeric Notch receptor polypeptide of the present disclosure as described herein, comprising contacting the cell with a second member of a specific binding pair, wherein binding of the first member of the specific binding pair to the second member of the specific binding pair induces cleavage of the Notch receptor polypeptide at one or more ligand-inducible proteolytic cleavage sites, thereby releasing an intracellular domain, which is a transcription factor that induces transcription of a nucleic acid encoding an effector polypeptide that modulates the activity of the cell. In some cases, the contacting is carried out in vivo, ex vivo, or in vitro. In some cases, the second member of the specific binding pair is on the surface of the second cell, immobilized on an insoluble substrate, present in an extracellular matrix, present in an artificial matrix, or soluble. In some cases, the effector polypeptide is an apoptosis inducer, apoptosis inhibitor, an activating immunoreceptor, an inhibitory immunoreceptor, a transcriptional activator, a transcriptional repressor, a cytokine, a growth factor, a hormone, a receptor, an antibody, or a site-specific nuclease.

[0011] The present disclosure provides a method of modulating an activity of a cell, the method comprising contacting the cell with a second member of a first specific binding pair, wherein the cell expresses: i) a first chimeric Notch receptor polypeptide of the present disclosure as described herein, comprising the first member of the first specific binding pair; and ii) at least a second chimeric Notch receptor polypeptide of the present disclosure as described herein, comprising a first member of a second specific binding pair, wherein the first and second specific binding pairs are different from one another, the intracellular domain of the first chimeric Notch receptor polypeptide provides a first effector function; and the intracellular domain of the second chimeric Notch receptor polypeptide provides a second effector function that is different from the first effector function, and wherein the released first and second intracellular domains modulate an activity of the cell. In some cases, the contacting is performed in vivo. In some cases, the contacting is performed ex vivo. In some cases, the contacting is performed in vitro.

[0012] The present disclosure provides a method of activating a T cell, the method comprising contacting a T cell as described herein, wherein the T cell has been genetically modified with i) a chimeric Notch receptor polypeptide of the present disclosure; and ii) one or more nucleic acids comprising a nucleotide sequence encoding a CAR, with an immobilized antigen, wherein the extracellular domain of the chimeric Notch receptor polypeptide comprises an antibody specific for a first antigen, and wherein said contacting results in release of a transcriptional activator and production of a CAR in the cell, wherein the CAR provides for activation of the T cell after binding of a second antigen.

[0013] The present disclosure provides methods for modulating the activity of a cell, the method comprising contacting the cell with a second member of a first specific binding pair, wherein the cell expresses: i) a first chimeric Notch receptor polypeptide of the present disclosure, comprising the first member of the first specific binding pair; and ii) at least a second chimeric Notch receptor polypeptide of the present disclosure, comprising the first member of the second specific binding pair, wherein the first and second specific binding pairs are different from each other, and the nucleotide sequence encoding the second chimeric Notch receptor is operably linked to a transcriptional control element that is activated or repressed by the intracellular domain of the first chimeric Notch receptor polypeptide. In some cases, the contacting is performed in vivo. In some cases, the contacting is performed ex vivo. In some cases, the contacting is performed in vitro.

[0014] The present disclosure provides a method of activating a T cell, the method comprising contacting a T cell as described herein (wherein the T cell has been genetically modified with i) a chimeric Notch receptor polypeptide of the present disclosure; and ii) one or more nucleic acids comprising a nucleotide sequence encoding a CAR, wherein the intracellular domain of the chimeric Notch receptor polypeptide is a transcriptional activator) with an immobilized antigen, wherein the extracellular domain of the chimeric polypeptide comprises an antibody specific for a first antigen, said contacting resulting in release of the transcriptional activator and production of a CAR in the cell, wherein the CAR provides for activation of the T cell after binding of a second antigen.

[0015] The present disclosure provides methods for modulating the activity of a cell, the method comprising contacting a cell with an antigen immobilized on a surface, wherein the cell expresses a chimeric Notch receptor polypeptide of the present disclosure, and a first member of a specific binding pair binds to the antigen, and the contacting results in release of an intracellular domain and modulation of the activity of the cell. In some cases, the intracellular domain is a transcription factor that modulates differentiation of the cell.

[0016] The present disclosure provides a method for locally regulating an activity of a cell, the method comprising: expressing in a cell a binding-triggered transcriptional switch comprising an extracellular domain comprising a first member of a specific binding pair, a binding-transducer, and an intracellular domain; and contacting the cell with a second member of the specific binding pair, wherein binding of the first member of the specific binding pair to the second member of the specific binding pair induces the binding-transducer to transmit a binding signal to activate the intracellular domain, thereby producing an activated intracellular domain, and the activated intracellular domain regulates an activity of the cell selected from the group consisting of expression of a gene product of the cell, proliferation of the cell, apoptosis of the cell, non-apoptotic death of the cell, differentiation of the cell, dedifferentiation of the cell, migration of the cell, secretion of a molecule from the cell, and cell adhesion of the cell.

[0017] In some cases, the activated intracellular domain regulates the expression of an endogenous gene product of the cell.

[0018] In some cases, the endogenous gene product of the cell is selected from the group consisting of a chemokine, a chemokine receptor, a cytokine, a cytokine receptor, a differentiation factor, a growth factor, a growth factor receptor, a hormone, a metabolic enzyme, a proliferation inducer, a receptor, a small molecule second messenger synthesis enzyme, a T cell receptor, a transcriptional activator, a transcriptional repressor, a transcriptional activator, a transcriptional repressor, a translational regulator, a translational activator, a translational repressor, an activating immunoreceptor, an apoptosis inhibitor, an apoptosis inducer, an immunoactivator, an immunoinhibitor, and an inhibitory immunoreceptor.

[0019] In some cases, the endogenous gene product of the cell is a secreted gene product. In some cases, the endogenous gene product of the cell is a surface-expressed gene product. In some cases, the activated intracellular domain simultaneously regulates the expression of two or more endogenous gene products of the cell. In some cases, the activated intracellular domain regulates the expression of a heterologous gene product of the cell.

[0020] In some cases, the heterologous gene product of the cell is selected from the group consisting of 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 site-specific nuclease, a small molecule second messenger synthesis enzyme, a T cell receptor, a toxin-derived protein, a transcriptional activator, a transcriptional repressor, a transcriptional activator, a transcriptional repressor, a translational regulator, a translational activator, a translational repressor, an activating immunoreceptor, an antibody, an apoptosis inhibitor, an apoptosis inducer, an engineered T cell receptor, an immunoactivator, an immunoinhibitor, an inhibitory immunoreceptor, an RNA-guided DNA-binding protein, and a second binding-triggered transcriptional switch.

[0021] In some instances, the heterologous gene product in the cell is selected from the group consisting of 806, 9E10, 3F8, 81C6, 8H9, abagovomab, abatacept, abciximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alefacept, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, AMG 102, anatumomab-mafenatox, anetumab-ravtansine, anifrolumab, anrukinzumab, apolizumab, arcitumomab, ascrinvacumab, aselizumab, atacicept, atezolizumab, atinumab, atlizumab / tocilizumab, atorlimumab, AVE1642, bapine Ozumab, Basiliximab, Bavituximab, Bectumomab, Begelomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab-mertansine, Blinatumomab, Brosozumab, BMS-93655 9, bococizumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, brontiximab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cBR96-doxorubicin immunoconjugate, CC49, CDP791, cedelizumab, certolizumab pegol, cetuximab, cG250, Ch.14.18, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, coltuximab ravtansine ravtansine, conatumumab, concizumab, CP 751871, CR6261, crenezumab, CS-1008, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, dectrekumab, demcizumab, denintuzumabmafodotin, denosumab, dellotuximab biotin, detumomab, dinutuximab, ziridabu, dorlimomab alitox, drozitumab, durigotumab, dupilumab, durvalumab, dusizizumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enabat Tuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enotikumab, Ensituximab, Epitumomab situxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etanercept, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, F19, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FB TA05, Felvizumab, Fezakinumab, Ficlatuzumab, Fizitumumab, Filibumarb, Frivumab, Fritikumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Furlanumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavirimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Golimumab Cimab, Guselkumab, HGS-ETR2, hu3S193, huA33, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Idarucizumab, IGN101, IgN311, Igovomab, IIIA4, IM-2C6, IMAB362, Imalumab, IMC-A12, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotinvedotin), infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, ipilimumab, iratumumab, isatuximab, itolizumab, ixekizumab, J591, KB004, keliximab, KW-2871, labetuzumab, lambrolizumab, lampalizumab, lebrikizumab, remaresomab, lenzilumab, lerdelimumab, lexatumumab, ribivirumab, rifastuzumab vedotin, ligelizumab, rilotomab satratetraxetan satetrazole, lintuzumab, lirilumab, roderucizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lurizumab pegol, lumiliximab, lumretuzumab, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, MEDI4736, mepolizumab, methelimumab, METMAB, milatuzumab, minletumomab, mirvetuximab soravtansinesoravtansine), mitumomab, MK-0646, MK-3475, MM-121, mogamulizumab, MORAb-003, morolimumab, motavizumab, MOv18, moxetumomab-pasudotox, MPDL33280A, muromonab CD3, nacolomab-tafenatox, namilumab, naptumomab-estafenatox Natox, narutuzumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentan, obiltoxaximab, obinutuzumab, occaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumumab, olokizumab, omalizumab Mab, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, otreltuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pancomab, panobacumab, palsatuzumab, Pascolizumab, pasotuximab, pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placuramab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, PRO 140, Kirizumab, R1507, Racotumomab, Ladletumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Linucumab, Rituximab, Lobatumumab, Lorezumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Sacituzumab govitecan, Samalizumab, Sarilumab, Satumomab pendetide, SCH900105, secukinumab, seribantumab, cetoxaximab, sevirumab, SGN-CD19A, SGN-CD33A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab Tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab-paptox, Talexuzumab, Tefibazumab, Terimomab-allitox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab, Tetulomab, TGN1412, Ticilimumab / Tremelimumab, Tigatuzumab, Tildrakizumab, TNX-650, Tocilizumab , toralizumab, tosatoxumab, tositumomab, tobetumab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tremelimumab, trevogrumab, tucotuzumab-celmoleukin, tuvilumab, ublituximab, urocuplumab, urelumab, urtoxazumab, ustekinumab The antibody is selected from the group consisting of bortuzumab, bundeltuzumab vedotin, vanticutumab, vanucizumab, bapaliximab, varlilumab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, diralimumab, and zolimomab alitox.

[0022] In some cases, the heterologous gene product of the cell is a secreted gene product. In some cases, the heterologous gene product of the cell is a surface-expressed gene product. In some cases, the activated intracellular domain simultaneously regulates the expression of two or more heterologous gene products of the cell. In some cases, the contacting is performed in vivo, ex vivo, or in vitro.

[0023] In some cases, the second member of the specific binding pair is on the surface of a second cell, immobilized on an insoluble substrate, present in an extracellular matrix, present in an artificial matrix, or is soluble. In some cases, the intracellular transcription factor directly regulates cell differentiation. In some cases, the transcription factor indirectly regulates cell differentiation by regulating the expression of a second transcription factor.

[0024] In some cases, the cell is an immune cell and the activity of the cell is differentiation of the immune cell. In some cases, the cell is an immune cell and the intracellular domain is a transcription factor that regulates differentiation of the cell and the activity of the cell is differentiation of the immune cell. In some cases, the transcription factor directly regulates differentiation of the immune cell. In some cases, the transcription factor indirectly regulates differentiation of the immune cell by regulating expression of a second transcription factor.

[0025] In some cases, the cell is a stem cell and the activity of the cell is differentiation of the stem cell. In some cases, the cell is a progenitor or precursor cell and the activity of the cell is differentiation of the progenitor or precursor cell.

[0026] In some cases, activation of the intracellular domain regulates expression of an endogenous gene of the cell through transcriptional regulation, chromatin control, translation, trafficking, or post-translational processing. In some cases, activation of the intracellular domain regulates cell adhesion to a second cell or to the extracellular matrix.

[0027] In some cases, the binding-transducer comprises a ligand-inducible proteolytic cleavage site, and binding of the first member of the specific binding pair to the second member of the specific binding pair induces cleavage of the binding-transducer at the ligand-inducible proteolytic cleavage site, thereby transducing the binding signal and activating the intracellular domain by proteolytically releasing the intracellular domain.

[0028] The present disclosure provides a method for modulating an activity of a cell, comprising contacting the cell with a second member of a first specific binding pair and a second member of a second specific binding pair, wherein the cell: i) a first binding-triggered transcriptional switch comprising an extracellular domain comprising a first member of a first specific binding pair, a binding transducer, and an intracellular domain; and ii) a second binding-triggered transcriptional switch comprising at least an extracellular domain comprising a first member of a second specific binding pair, a binding transducer, and an intracellular domain. wherein the intracellular domain of the first binding-triggered transcriptional switch provides a first effector function and the intracellular domain of the second binding-triggered transcriptional switch provides a second effector function different from the first effector function, wherein binding of the first and second members of the first and second specific binding pairs induces the binding-transducer to transduce a binding signal to activate the first and second intracellular domains.

[0029] In some cases, the effector function of the intracellular domain of the first binding-triggered transcriptional switch regulates expression of a cellular gene product.

[0030] In some cases, the cellular gene product is an endogenous gene product of the cell. In some cases, the cellular gene product is a heterologous gene product of the cell. In some cases, the cellular gene product is a cellular gene product selected from the group consisting of a chemokine, a chemokine receptor, a cytokine, a cytokine receptor, a differentiation factor, a growth factor, a growth factor receptor, a hormone, a metabolic enzyme, a proliferation inducer, a receptor, a small molecule second messenger synthesis enzyme, a T cell receptor, a transcriptional activator, a transcriptional repressor, a transcriptional activator, a transcriptional repressor, a translational regulator, a translational activator, a translational repressor, an activating immunoreceptor, an apoptosis inhibitor, an apoptosis inducer, an immunoactivator, an immunoinhibitor, and an inhibitory immunoreceptor.

[0031] In some cases, the effector function of the intracellular domain of the second binding-triggered transcriptional switch regulates expression of a cellular gene product. In some cases, the cellular gene product is an endogenous gene product of the cell. In some cases, the cellular gene product is a heterologous gene product of the cell.

[0032] In some cases, the cellular gene product is selected from the group consisting of a chemokine, a chemokine receptor, a cytokine, a cytokine receptor, a differentiation factor, a growth factor, a growth factor receptor, a hormone, a metabolic enzyme, a proliferation inducer, a receptor, a small molecule second messenger synthesis enzyme, a T cell receptor, a transcriptional activator, a transcriptional repressor, a transcriptional activator, a transcriptional repressor, a translational regulator, a translational activator, a translational repressor, an activating immunoreceptor, an apoptosis inhibitor, an apoptosis inducer, an immunoactivator, an immunoinhibitor, and an inhibitory immunoreceptor.

[0033] In some cases, at least one of the binding-transducers of the first and second binding-triggered transcriptional switches comprises a ligand-inducible proteolytic cleavage site, and binding of the first and second members of the respective specific binding pairs induces cleavage of the binding-transducer at the ligand-inducible proteolytic cleavage site, thereby transducing a binding signal and activating the respective intracellular domain by proteolytically releasing the intracellular domain.

[0034] In some cases, the binding-transducers of the first and second binding-triggered transcriptional switches both comprise a ligand-inducible proteolytic cleavage site.

[0035] In some examples, the method further includes contacting the cell with a soluble inhibitory molecule that competitively inhibits binding of the first member of the specific binding pair to the second member of the specific binding pair, thereby preventing induction of the binding transducer to transmit a binding signal to activate the intracellular domain, and contacting the cell with the soluble inhibitory molecule includes applying or administering the soluble inhibitory molecule to the first cell and / or placing the cell in the presence of a second cell that expresses the soluble inhibitory molecule. In some examples, the second cell constitutively expresses the soluble inhibitory molecule. In some examples, the second cell conditionally expresses the soluble inhibitory molecule.

[0036] The present disclosure provides a method for regulating an activity of a cell, the method comprising contacting the cell with a second member of a first specific binding pair, wherein the cell expresses i) a first binding-triggered transcriptional switch comprising an extracellular domain comprising the first member of the first specific binding pair, a binding transducer, and an intracellular domain; and ii) at least a second binding-triggered transcriptional switch comprising an extracellular domain comprising the first member of a second specific binding pair, a binding transducer, and an intracellular domain, wherein a nucleotide sequence encoding the second binding-triggered transcriptional switch is operably linked to a transcriptional control element that is activated or repressed by the intracellular domain of the first binding-triggered transcriptional switch.

[0037] In some cases, the contacting is carried out in vivo, ex vivo, or in vitro. In some cases, the second member of the first specific binding pair is on the surface of a second cell, immobilized on an insoluble substrate, present in an extracellular matrix, present in an artificial matrix, or is soluble.

[0038] In some cases, activation of the intracellular domain of the second binding-triggered transcriptional switch regulates an activity of a cell selected from the group consisting of expression of a gene product of the cell, proliferation of the cell, apoptosis of the cell, non-apoptotic death of the cell, differentiation of the cell, dedifferentiation of the cell, migration of the cell, secretion of a molecule from the cell, and cell adhesion of the cell.

[0039] In some cases, the activity of the cell is expression of a cellular gene product, in some cases, the cellular gene product is a cellular gene product selected from the group consisting of a chemokine, a chemokine receptor, a cytokine, a cytokine receptor, a differentiation factor, a growth factor, a growth factor receptor, a hormone, a metabolic enzyme, a proliferation inducer, a receptor, a small molecule second messenger synthesis enzyme, a T cell receptor, a transcriptional activator, a transcriptional repressor, a transcriptional activator, a transcriptional repressor, a translational regulator, a translational activator, a translational repressor, an activating immunoreceptor, an apoptosis inhibitor, an apoptosis inducer, an immunoactivator, an immunoinhibitor, and an inhibitory immunoreceptor.

[0040] In some cases, at least one of the binding-transducers of the first and second binding-triggered transcriptional switches comprises a ligand-inducible proteolytic cleavage site, and binding of the first and second members of the respective specific binding pairs induces cleavage of the binding-transducer at the ligand-inducible proteolytic cleavage site, thereby transducing a binding signal and activating the respective intracellular domain by proteolytically releasing the intracellular domain.

[0041] The present disclosure provides a method for tracking cell-cell contact, the method comprising: expressing, in each cell of a first plurality of cells, a binding-triggered transcriptional switch comprising an extracellular domain comprising a first member of a specific binding pair, a binding transducer, and an intracellular domain; expressing, in each cell of a second plurality of cells, a second member of the specific binding pair; and contacting the first plurality of cells with the second plurality of cells, wherein binding of the first member of the specific binding pair to the second member of the specific binding pair induces the binding transducer to transduce a binding signal of the binding-triggered transcriptional switch, thereby activating the intracellular domain, and wherein activation of the intracellular domain induces expression of a detectable reporter sufficient to track cell-cell contact in space, time, or a combination thereof.

[0042] In some cases, the first plurality of cells, the second plurality of cells, or both are neurons. In some cases, the binding-transducer comprises a ligand-inducible proteolytic cleavage site, and binding of the first member of the specific binding pair to the second member of the specific binding pair induces cleavage of the binding-transducer at the ligand-inducible proteolytic cleavage site, thereby transducing a binding signal and activating the intracellular domain by proteolytically releasing the intracellular domain.

[0043] In some cases, the binding-triggered transcriptional switch, including those described above and herein, is a SynNotch polypeptide.

[0044] The present disclosure also provides a localized cell activation system, the system comprising a cell comprising: an expressed binding-triggered transcriptional switch comprising an extracellular domain comprising a first member of a first specific binding pair, a binding transducer, and an intracellular domain; and a nucleic acid encoding a binding-triggered activating polypeptide comprising a first member of a second specific binding pair operably linked to a transcriptional control element induced by the intracellular domain of the first binding-triggered transcriptional switch, wherein upon contact with a second member of the first specific binding pair, the binding-triggered activating polypeptide is expressed, and upon contact with a second member of the second specific binding pair, the binding-triggered activating polypeptide activates the cell.

[0045] In some cases, the cell is selected from the group consisting of an immune cell, a progenitor or precursor cell, a stem cell, and a neuron. In some cases, the cell is an immune cell, the binding-triggered transcriptional switch is an antigen-triggered transcriptional switch, the binding-triggered activating polypeptide is an antigen-triggered activating polypeptide, and upon contact with a second member of the first specific binding pair, the antigen-triggered activating polypeptide is expressed, and upon contact with a second member of the second specific binding pair, the antigen-triggered activating polypeptide activates the immune cell to recognize a target cell expressing the first member of the second specific binding pair.

[0046] In some cases, the antigen-triggered activating polypeptide is a chimeric antigen receptor or a variant thereof. In some cases, the antigen-triggered activating polypeptide is an engineered T cell receptor or a variant thereof. In some cases, the expressed binding-triggered transcriptional switch is a SynNotch polypeptide.

[0047] The present disclosure provides a method for locally regulating an activity of a cell, the method comprising: expressing in a first cell a binding-triggered transcriptional switch comprising a binding-transducer, an intracellular domain, and a first extracellular domain comprising a first adaptor binding domain that specifically binds to a first epitope on a soluble adaptor molecule; and contacting the first cell with: i) a second cell expressing a second extracellular domain comprising a second adaptor binding domain that specifically binds to a second epitope on the soluble adaptor molecule; and ii) an effective concentration of the soluble adaptor molecule, wherein binding of the first adaptor binding domain and the second adaptor binding domain to the adaptor molecule induces the binding-transducer to transduce a binding signal to activate the intracellular domain, thereby producing an activated intracellular domain, and the activated intracellular domain regulates an activity of the first cell selected from the group consisting of expression of a gene product of the cell, proliferation of the cell, apoptosis of the cell, non-apoptotic death of the cell, differentiation of the cell, dedifferentiation of the cell, migration of the cell, secretion of a molecule from the cell, and cell adhesion of the cell. In some examples, the contacting comprises applying the soluble adapter molecule to the cell in vitro or ex vivo or administering the soluble adapter molecule to the cell in vivo. In some examples, contacting the first cell with an effective concentration of the soluble adapter molecule comprises placing the first cell in the presence of a third cell that expresses the adapter molecule, wherein the third cell constitutively or conditionally expresses the adapter molecule. In some examples, the first extracellular domain and the soluble adapter molecule are first and second members of a specific binding pair. In some examples, the second extracellular domain and the soluble adapter molecule are first and second members of a specific binding pair. In some examples, the first extracellular domain and the second extracellular domain are an antibody or a nanobody. In some examples, the intracellular domain is a transcription factor. In some examples, the activated intracellular domain regulates expression of an endogenous or heterologous gene product in the first cell.In some examples, the binding-transducer comprises a ligand-inducible proteolytic cleavage site, and binding of the first extracellular domain and the second extracellular domain to a soluble adaptor molecule induces cleavage of the binding-transducer at the ligand-inducible proteolytic cleavage site, thereby transmitting a binding signal and activating the intracellular domain by proteolytically releasing the intracellular domain.

[0048] The present disclosure provides a host cell comprising: a nucleic acid encoding a first binding-triggered transcriptional switch responsive to a first antigen; a first promoter responsive to the first binding-triggered transcriptional switch and operably linked to a nucleic acid encoding a CAR comprising an extracellular domain that specifically binds to a first member of a specific binding pair; a nucleic acid encoding a second binding-triggered transcriptional switch responsive to a second antigen; and a second promoter responsive to the second binding-triggered transcriptional switch and operably linked to a nucleic acid encoding an intracellular CAR inhibitory domain, wherein in the presence of the second antigen, the intracellular CAR inhibitory domain is expressed and activation of the cell by the CAR is inhibited; and in the presence of the first antigen but absence of the second antigen, the CAR is expressed and activatable by the second member of the specific binding pair.

[0049] The present disclosure provides host cells comprising a nucleic acid encoding a first binding-triggered transcriptional switch responsive to a first antigen; a first promoter responsive to the first binding-triggered transcriptional switch and operably linked to a nucleic acid encoding a first portion of a CAR comprising an extracellular domain that specifically binds to a first member of a specific binding pair; a nucleic acid encoding a second binding-triggered transcriptional switch responsive to a second antigen; and a second promoter responsive to the second binding-triggered transcriptional switch and operably linked to a nucleic acid encoding a second portion of the CAR comprising an intracellular signaling domain, wherein in the presence of the first antigen and the second antigen, the first and second portions of the CAR are expressed and the CAR is activatable by the second member of the specific binding pair. In some examples, the cell further comprises a nucleic acid encoding a third binding-triggered transcriptional switch responsive to a third antigen; and a third promoter responsive to the third binding-triggered transcriptional switch and operably linked to a nucleic acid encoding an intracellular CAR inhibitory domain, wherein in the presence of the third antigen, the intracellular CAR inhibitory domain is expressed and activation of the cell by the CAR is inhibited. [The present invention 1001] From the N-terminus to the C-terminus, covalently bound a) an extracellular domain comprising a first member of a specific binding pair; b) a Notch receptor polypeptide having a length of between 50 amino acids and 1000 amino acids and containing one or more ligand-inducible proteolytic cleavage sites; and c) Intracellular domain Including, a chimeric polypeptide, wherein the first member of the specific binding pair is heterologous to the Notch receptor polypeptide, and wherein binding of the first member of the specific binding pair to a second member of the specific binding pair induces cleavage of the Notch receptor polypeptide at the one or more ligand-inducible proteolytic cleavage sites, thereby releasing the intracellular domain. [The present invention 1002] 1001. The chimeric polypeptide of the present invention, wherein the Notch receptor polypeptide has a length of 300 to 400 amino acids. [The present invention 1003] The chimeric polypeptide of the present invention 1001 or 1002, comprising a linker interposed between said extracellular domain and said Notch receptor polypeptide. [The present invention 1004] The chimeric polypeptide of any one of claims 1001 to 1003, wherein the intracellular domain is a transcriptional activator. [The present invention 1005] The chimeric polypeptide of any one of claims 1001 to 1003, wherein the intracellular domain is a transcriptional repressor. [The present invention 1006] The chimeric polypeptide of any one of claims 1001 to 1003, wherein the intracellular domain is a site-specific nuclease. [The present invention 1007] The chimeric polypeptide of the present invention, wherein the site-specific nuclease is a Cas9 polypeptide. [The present invention 1008] The chimeric polypeptide of any one of claims 1001 to 1003, wherein the intracellular domain is a recombinase. [The present invention 1009] The chimeric polypeptide of any one of claims 1001 to 1003, wherein the intracellular domain is an inhibitory immunoreceptor. [The present invention 1010] The chimeric polypeptide of any one of claims 1001 to 1003, wherein the intracellular domain is an activating immunoreceptor. [The present invention 1011] 1011. The chimeric polypeptide of any of claims 1001 to 1010, wherein said first member of said specific binding pair comprises an antibody-based recognition scaffold. [The present invention 1012] 1010. The chimeric polypeptide of any of claims 1001 to 1010, wherein said first member of said specific binding pair comprises an antibody. [The present invention 1013] The chimeric polypeptide of the present invention, wherein the antibody specifically binds to a tumor-specific antigen, a disease-associated antigen, a pathogen-associated antigen, an autoimmune disease-associated antigen, or an extracellular matrix component. [The present invention 1014] The chimeric polypeptide of the present invention, wherein the antibody specifically binds to a cell surface antigen, a soluble antigen, or an antigen immobilized on an insoluble substrate. [The present invention 1015] The chimeric polypeptide of the present invention, wherein the antibody is a single-chain Fv. [The present invention 1016] 1011. The chimeric polypeptide of the invention, wherein said first member of said specific binding pair is a nanobody, a single domain antibody, a diabody, a triabody, or a minibody. [The present invention 1017] 1011. The chimeric polypeptide of any of claims 1001 to 1010, wherein said first member of said specific binding pair is a non-antibody-based recognition scaffold. [The present invention 1018] 1017. The chimeric polypeptide of the invention, wherein said non-antibody-based recognition scaffold is an avimer, a DARPin, an adnectin, an avimer, an affibody, an anticalin, or an affilin. [The present invention 1019] 1010. The chimeric polypeptide of any of claims 1001 to 1010, wherein said first member of said specific binding pair is an antigen. [The present invention 1020] The chimeric polypeptide of the present invention, wherein the antigen is an endogenous antigen. [The present invention 1021] The chimeric polypeptide of the present invention, wherein the antigen is a foreign antigen. [The present invention 1022] 1010. The chimeric polypeptide of any of claims 1001 to 1010, wherein said first member of said specific binding pair is a ligand for a receptor. [The present invention 1023] 1010. The chimeric polypeptide of any one of claims 1001 to 1010, wherein said first member of said specific binding pair is a receptor. [The present invention 1024] 1010. The chimeric polypeptide of any one of claims 1001 to 1010, wherein said first member of said specific binding pair is a cell adhesion molecule. [The present invention 1025] The chimeric polypeptide of any of claims 1001 to 1010, wherein the first member of the specific binding pair comprises a first dimerization domain and the second member of the specific binding pair comprises a second dimerization domain. [The present invention 1026] 1025. The chimeric polypeptide of the invention, wherein the binding of said first dimerization domain to said second dimerization domain is induced by a small molecule dimerizer. [The present invention 1027] 1025. The chimeric polypeptide of claim 1025, wherein binding of said first dimerization domain to said second dimerization domain is induced by light. [The present invention 1028] The chimeric polypeptide of any one of claims 1001 to 1027, wherein the Notch receptor polypeptide comprises an amino acid sequence having at least 75% amino acid sequence identity to any one of the amino acid sequences shown in Figures 2A to 2G. [The present invention 1029] 1029. The chimeric polypeptide of any of claims 1001 to 1028, wherein said one or more ligand-inducible proteolytic cleavage sites are selected from S1, S2, and S3 proteolytic cleavage sites. [The present invention 1030] 1029. The chimeric polypeptide of the invention, wherein said S1 proteolytic cleavage site is a furin-like protease cleavage site comprising the amino acid sequence Arg-X-(Arg / Lys)-Arg, where X is any amino acid. [The present invention 1031] 1029. The chimeric polypeptide of the present invention, wherein the S2 proteolytic cleavage site is an ADAM-17 type protease cleavage site comprising an Ala-Val dipeptide sequence. [The present invention 1032] 1029. The chimeric polypeptide of the present invention, wherein said S3 proteolytic cleavage site is a γ-secretase cleavage site comprising a Gly-Val dipeptide sequence. [The present invention 1033] A nucleic acid comprising a nucleotide sequence encoding any one of the chimeric polypeptides of the present inventions 1001 to 1032. [The present invention 1034] A recombinant expression vector comprising a nucleotide sequence encoding any one of the chimeric polypeptides of the present inventions 1001 to 1032. [This invention 1035] A host cell genetically modified with a nucleic acid of the invention 1033 or an expression vector of the invention 1034. [The present invention 1036] A host cell of the present invention 1035 that is a eukaryotic cell. [This invention 1037] The host cell of the present invention 1036, which is a mammalian cell. [The present invention 1038] The host cell of the present invention 1037 is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell. [This invention 1039] The host cell of the present invention 1038, wherein the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, or a cytotoxic T cell. [The present invention 1040] 1039. The host cell of any of claims 1035 to 1039, which is genetically modified with a nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR), wherein the intracellular domain of the chimeric polypeptide is a transcriptional activator. [This invention 1041] 1040. The host cell of claim 1040, wherein the nucleotide sequence encoding the CAR or the TCR is operably linked to a transcriptional control element that is activated by the intracellular domain of the chimeric polypeptide. [The present invention 1042] A method for regulating the activity of a cell expressing any one of the chimeric Notch receptor polypeptides of the present inventions 1001 to 1032, comprising: contacting said cell with a second member of said specific binding pair. wherein binding of the first member of the specific binding pair to the second member of the specific binding pair induces cleavage of the Notch receptor polypeptide at the one or more ligand-inducible proteolytic cleavage sites, thereby releasing the intracellular domain, and wherein release of the intracellular domain modulates an activity of the cell. [This invention 1043] 1043. The method of claim 1042, wherein said contacting is carried out in vivo, ex vivo, or in vitro. [This invention 1044] The method of claim 1042, wherein the second member of the specific binding pair is on the surface of a second cell, immobilized on an insoluble substrate, present in an extracellular matrix, present in an artificial matrix, or is soluble. [This invention 1045] 1043. The method of claim 1042, wherein release of said intracellular domain regulates proliferation of said cell. [The present invention 1046] 1043. The method of claim 1042, wherein release of said intracellular domain regulates apoptosis in said cell. [This invention 1047] 1043. The method of claim 1042, wherein release of said intracellular domain induces cell death by a mechanism other than apoptosis. [This invention 1048] The method of claim 1042, wherein release of said intracellular domain regulates gene expression in said cell through transcriptional regulation, chromatin control, translation, trafficking or post-translational processing. [This invention 1049] 1043. The method of claim 1042, wherein release of said intracellular domain regulates differentiation of said cell. [The present invention 1050] 1043. The method of claim 1042, wherein release of said intracellular domain regulates migration of said cell. [This invention 1051] 1043. The method of claim 1042, wherein release of said intracellular domain regulates expression and secretion of a molecule from said cell. [This invention 1052] 1043. The method of claim 1042, wherein release of said intracellular domain modulates adhesion of said cell to a second cell or to an extracellular matrix. [This invention 1053] 1043. The method of claim 1042, wherein release of said intracellular domain induces or modulates de novo expression of a gene product in said cell. [This invention 1054] 1054. The method of claim 1053, wherein said gene product is a transcriptional activator, a transcriptional repressor, a chimeric antigen receptor, a second chimeric Notch receptor polypeptide, a translational regulator, a cytokine, a hormone, a chemokine, or an antibody. [This invention 1055] A method for regulating the activity of a cell expressing any one of the chimeric Notch receptor polypeptides of the present inventions 1001 to 1032, comprising: contacting said cell with a second member of said specific binding pair. wherein binding of the first member of the specific binding pair to the second member of the specific binding pair induces cleavage of the Notch receptor polypeptide at the one or more ligand-inducible proteolytic cleavage sites, thereby releasing the intracellular domain, which is a transcription factor that induces transcription of a nucleic acid encoding an effector polypeptide that modulates an activity of the cell. [This invention 1056] The method of claim 1055, wherein said contacting is carried out in vivo, ex vivo, or in vitro. [This invention 1057] The method of claim 1055, wherein the second member of the specific binding pair is on the surface of a second cell, immobilized on an insoluble substrate, present in an extracellular matrix, present in an artificial matrix, or is soluble. [This invention 1058] 1055. The method of claim 1055, wherein said effector polypeptide is an apoptosis inducer, an apoptosis inhibitor, an activating immunoreceptor, an inhibitory immunoreceptor, a transcriptional activator, a transcriptional repressor, a cytokine, a growth factor, a hormone, a receptor, an antibody, a site-specific nuclease, or a recombinase. [This invention 1059] 1. A method for modulating cellular activity, comprising: contacting said cell with a second member of the first specific binding pair. wherein the cells comprise i) a first chimeric Notch receptor polypeptide of any of claims 1001 to 1032, comprising a first member of a first specific binding pair; and ii) a second chimeric Notch receptor polypeptide of any of claims 1001 to 1032, comprising at least a first member of a second specific binding pair. Expressing the first and second specific binding pairs are different from each other; The method, wherein the intracellular domain of the first chimeric Notch receptor polypeptide provides a first effector function; the intracellular domain of the second chimeric Notch receptor polypeptide provides a second effector function that is different from the first effector function; and the released first and second intracellular domains modulate an activity of the cell. [The present invention 1060] 1059. The method of claim 1059, wherein said contacting is carried out in vivo, ex vivo, or in vitro. [This invention 1061] 1. A method for activating T cells, comprising: Contacting the T cells of the present invention with immobilized antigens Including, the extracellular domain of the chimeric polypeptide comprises an antibody specific for a first antigen; The method, wherein the contact results in release of the transcriptional activator and production of the CAR or the TCR in the cell, and the CAR or TCR provides activation of the T cell after binding of a second antigen. [This invention 1062] 1. A method for modulating cellular activity, comprising: contacting said cell with a second member of the first specific binding pair. wherein the cells comprise i) a first chimeric Notch receptor polypeptide of any of claims 1001 to 1032, comprising a first member of a first specific binding pair; and ii) a second chimeric Notch receptor polypeptide of any of claims 1001 to 1032, comprising at least a first member of a second specific binding pair. Expressing the first and second specific binding pairs are different from each other; The method, wherein the nucleotide sequence encoding the second chimeric Notch receptor is operably linked to a transcriptional control element that is activated or repressed by the intracellular domain of the first chimeric Notch receptor polypeptide. [This invention 1063] The method of claim 1062, wherein said contacting is carried out in vivo, ex vivo, or in vitro. [This invention 1064] 1. A method for activating T cells, comprising: Contacting the T cells of the present invention with immobilized antigens wherein the extracellular domain of the chimeric polypeptide comprises an antibody specific for a first antigen; The method, wherein the contacting results in release of the transcriptional activator and production of the CAR or TCR in the cell, and the CAR or TCR provides activation of the T cell after binding of a second antigen. [This invention 1065] 1. A method for modulating cellular activity, comprising: contacting said cells with an antigen immobilized on a surface; wherein the cell expresses a chimeric Notch receptor polypeptide of the present invention, and wherein the first member of the specific binding pair binds to the antigen; The method, wherein said contacting results in release of the intracellular domain and modulation of an activity of the cell. [The present invention 1066] 1065. The method of claim 105, wherein said intracellular domain is a transcription factor that regulates differentiation of said cell. [This invention 1067] 1. A method for locally modulating cellular activity, comprising: expressing in the cell a binding-triggered transcriptional switch comprising an extracellular domain comprising a first member of a specific binding pair, a binding transducer, and an intracellular domain; and contacting said cell with a second member of said specific binding pair. wherein binding of the first member of the specific binding pair to the second member of the specific binding pair induces the binding transducer to transmit a binding signal to activate the intracellular domain, thereby producing an activated intracellular domain, and the activated intracellular domain modulates an activity of the cell selected from the group consisting of expression of a gene product of the cell, proliferation of the cell, apoptosis of the cell, non-apoptotic death of the cell, differentiation of the cell, dedifferentiation of the cell, migration of the cell, secretion of a molecule from the cell, and cell adhesion of the cell. [The present invention 1068] 1068. The method of claim 1067, wherein said activated intracellular domain regulates expression of an endogenous gene product of said cell. [The present invention 1069] 1068. The method of claim 1068, wherein the endogenous gene product of the cell is selected from the group consisting of chemokines, chemokine receptors, cytokines, cytokine receptors, differentiation factors, growth factors, growth factor receptors, hormones, metabolic enzymes, proliferation inducers, receptors, small molecule second messenger synthesis enzymes, T cell receptors, transcriptional activators, transcriptional repressors, transcriptional activators, transcriptional repressors, translational regulators, translational activators, translational repressors, activating immunoreceptors, apoptosis inhibitors, apoptosis inducers, immunoactivators, immunoinhibitors, and inhibitory immunoreceptors. [The present invention 1070] 1069. The method of any of claims 1068 to 1069, wherein said endogenous gene product of said cell is a secreted gene product. [This invention 1071] 1069. The method of any one of claims 1068 to 1069, wherein said endogenous gene product of said cell is a surface-expressed gene product. [This invention 1072] 1072. The method of any of claims 1068 to 1071, wherein said activated intracellular domains simultaneously regulate the expression of two or more endogenous gene products of said cell. [This invention 1073] 1068. The method of claim 1067, wherein said activated intracellular domain regulates expression of a heterologous gene product in said cell. [This invention 1074] 1073. The method of claim 1073, wherein the heterologous gene product of the cell is selected from the group consisting of 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 site-specific nuclease, a small molecule second messenger synthesis enzyme, a T cell receptor, a toxin-derived protein, a transcriptional activator, a transcriptional repressor, a transcriptional activator, a transcriptional repressor, a translational regulator, a translational activator, a translational repressor, an activating immunoreceptor, an antibody, an apoptosis-inhibiting factor, an apoptosis inducer, an engineered T cell receptor, an immunoactivator, an immunoinhibitor, an inhibitory immunoreceptor, an RNA-guided DNA-binding protein, and a second binding-triggered transcriptional switch. [This invention 1075] 1075. The method of claim 1074, wherein said heterologous gene product of said cell is an antibody. [This invention 1076] The antibody is selected from the group consisting of 806, 9E10, 3F8, 81C6, 8H9, abagovomab, abatacept, abciximab, abituzumab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alefacept, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, AMG 102, anatumomab-mafenatox, anetumab-ravtansine, anifrolumab, anrukinzumab, apolizumab, arcitumomab, ascrinvacumab, aselizumab, atacicept, atezolizumab, atinumab, atlizumab / tocilizumab, atorlimumab, AVE1642, bapine Ozumab, Basiliximab, Bavituximab, Bectumomab, Begelomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab-mertansine, Blinatumomab, Brosozumab, BMS-93655 9, bococizumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, brontiximab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cBR96-doxorubicin immunoconjugate, CC49, CDP791, cedelizumab, certolizumab pegol, cetuximab, cG250, Ch.14.18, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, coltuximab ravtansine ravtansine, Conatumumab, Concizumab, CP 751871, CR6261, Crenezumab, CS-1008, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Derlotuximab biotinBiotin), Detumomab, Dinutuximab, Zilidabumab, Dorlimomab-Alitox, Drozitumab, Durigotumab, Dupilumab, Durvalumab, Ducizitumab, Eclomeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emibetuzumab, Enaba Tuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enotikumab, Ensituximab, Epitumomab situxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etanercept, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, F19, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FB TA05, Felvizumab, Fezakinumab, Ficlatuzumab, Fizitumumab, Filibumarb, Frivumab, Fritikumab, Fletikumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Furlanumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavirimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab, Glembatumumab vedotin, Golimumab, Golimumab Cimab, Guselkumab, HGS-ETR2, hu3S193, huA33, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Idarucizumab, IGN101, IgN311, Igovomab, IIIA4, IM-2C6, IMAB362, Imalumab, IMC-A12, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotinvedotin), infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, ipilimumab, iratumumab, isatuximab, itolizumab, ixekizumab, J591, KB004, keliximab, KW-2871, labetuzumab, lambrolizumab, lampalizumab, lebrikizumab, remaresomab, lenzilumab, lerdelimumab, lexatumumab, ribivirumab, rifastuzumab vedotin, ligelizumab, rilotomab satratetraxetan satetrazole, lintuzumab, lirilumab, roderucizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lurizumab pegol, lumiliximab, lumretuzumab, mapatumumab, margetuximab, maslimomab, matuzumab, mavrilimumab, MEDI4736, mepolizumab, methelimumab, METMAB, milatuzumab, minletumomab, mirvetuximab soravtansinesoravtansine), mitumomab, MK-0646, MK-3475, MM-121, mogamulizumab, MORAb-003, morolimumab, motavizumab, MOv18, moxetumomab-pasudotox, MPDL33280A, muromonab CD3, nacolomab-tafenatox, namilumab, naptumomab-estafenatox Natox, narutuzumab, natalizumab, nebacumab, necitumumab, nemolizumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentan, obiltoxaximab, obinutuzumab, occaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumumab, olokizumab, omalizumab Mab, onartuzumab, ontuxizumab, opicinumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, otreltuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pancomab, panobacumab, palsatuzumab, Pascolizumab, pasotuximab, pateclizumab, patritumab, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placuramab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab, pritumumab, PRO 140, Kirizumab, R1507, Racotumomab, Ladletumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab, Regavirumab, Reslizumab, Rilotumumab, Linucumab, Rituximab, Lobatumumab, Lorezumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Sacituzumab govitecan, Samalizumab, Sarilumab, Satumomab pendetide, SCH900105, secukinumab, seribantumab, cetoxaximab, sevirumab, SGN-CD19A, SGN-CD33A, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tet Laxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab-paptox, Talexuzumab, Tefibazumab, Terimomab-allitox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, Tesidolumab, Tetulomab, TGN1412, Ticilimumab / Tremelimumab, Tigatuzumab, Tildrakizumab, TNX-650, Tocilizumab, Tocilizumab Ralizumab, Tosatoxumab, Tositumomab, Tobetumab, Tralokinumab, Trastuzumab, TRBS07, Tregalizumab, Tremelimumab, Trevogrumab, Tucotuzumab-celmoleukin, Tubilumab, Ublituximab, Ulocuplumab, Urelumab, Urutoxazumab, Ustekinumab, Ban 1075. The method of the present invention, wherein the antibody is selected from the group consisting of dortuzumab vedotin, vanticutumab, vanucizumab, bapaliximab, varlilumab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, diralimumab, and zolimomab alitoxin. [This invention 1077] 1077. The method of any one of claims 1073 to 1076, wherein said heterologous gene product of said cell is a secreted gene product. [This invention 1078] 1077. The method of any one of claims 1073 to 1076, wherein said heterologous gene product of said cell is a surface-expressed gene product. [This invention 1079] 1079. The method of any of claims 1073 to 1078, wherein said activated intracellular domains simultaneously regulate the expression of two or more heterologous gene products in said cell. [The present invention 1080] 1079. The method of any one of claims 1067 to 1079, wherein said contacting is carried out in vivo, ex vivo, or in vitro. [This invention 1081] 1080. The method of any of claims 1067 to 1080, wherein the second member of the specific binding pair is on the surface of a second cell, immobilized on an insoluble substrate, present in an extracellular matrix, present in an artificial matrix, or is soluble. [This invention 1082] 1068. The method of claim 1067, wherein said intracellular domain is a transcription factor that regulates differentiation of said cell. [This invention 1083] 1083. The method of claim 1082, wherein said transcription factor directly regulates differentiation of said cell. [This invention 1084] 1083. The method of claim 1082, wherein said transcription factor indirectly regulates differentiation of said cell by regulating expression of a second transcription factor. [This invention 1085] 1068. The method of claim 1067, wherein said cell is an immune cell and said activity of said cell is differentiation of said immune cell. [The present invention 1086] 1068. The method of claim 1067, wherein said cell is an immune cell, said intracellular domain is a transcription factor that regulates differentiation of said cell, and said activity of said cell is differentiation of said immune cell. [This invention 1087] 1086. The method of claim 1086, wherein said transcription factor directly regulates the differentiation of said immune cells. [This invention 1088] 1086. The method of claim 1086, wherein said transcription factor indirectly regulates the differentiation of said immune cell by regulating said expression of a second transcription factor. [This invention 1089] 1068. The method of claim 1067, wherein said cell is a stem cell and said activity of said cell is differentiation of said stem cell. [The present invention 1090] 1068. The method of claim 1067, wherein said cell is a progenitor or precursor cell, and said activity of said cell is differentiation of said progenitor or precursor cell. [This invention 1091] 1068. The method of claim 1067, wherein activation of said intracellular domain regulates expression of an endogenous gene of said cell through transcriptional regulation, chromatin control, translation, trafficking or post-translational processing. [This invention 1092] 1068. The method of claim 1067, wherein activation of said intracellular domain modulates cell adhesion of said cell to a second cell or to an extracellular matrix. [This invention 1093] The method of claim 1067, wherein the binding transducer comprises a ligand-inducible proteolytic cleavage site, and binding of the first member of the specific binding pair to the second member of the specific binding pair induces cleavage of the binding transducer at the ligand-inducible proteolytic cleavage site, thereby transmitting the binding signal and activating the intracellular domain by proteolytically releasing the intracellular domain. [This invention 1094] Any of the methods of claims 1067 to 1093, further comprising contacting the cell with a soluble inhibitor molecule that competitively inhibits binding of the first member of the specific binding pair to the second member of the specific binding pair, thereby preventing induction of a binding transducer to transmit a binding signal to activate the intracellular domain. [This invention 1095] 1094. The method of claim 1094, wherein contacting said cell with said soluble inhibitory molecule comprises applying or administering said soluble inhibitory molecule to the first cell. [This invention 1096] 1094. The method of claim 1094, wherein contacting said cell with said soluble inhibitory molecule comprises placing said cell in the presence of a second cell that expresses said soluble inhibitory molecule. [This invention 1097] 1096. The method of claim 1096, wherein said second cell constitutively expresses said soluble inhibitor molecule. [This invention 1098] 1096. The method of claim 1096, wherein said second cell conditionally expresses said soluble inhibitory molecule. [This invention 1099] 1. A method for modulating cellular activity, comprising: contacting the cell with a second member of the first specific binding pair and a second member of the second specific binding pair. wherein the cells comprise i) a first binding-triggered transcriptional switch comprising an extracellular domain comprising a first member of the first specific binding pair, a binding transducer, and an intracellular domain; and ii) a second binding-triggered transcriptional switch comprising at least an extracellular domain comprising said first member of a second specific binding pair, a binding transducer, and an intracellular domain. expresses; the intracellular domain of the first binding-triggered transcriptional switch provides a first effector function and the intracellular domain of the second binding-triggered transcriptional switch provides a second effector function different from the first effector function, wherein binding of the first and second members of the first and second specific binding pairs induces the binding-transducer to transduce a binding signal to activate the first and second intracellular domains. [The present invention 1100] 1099. The method of claim 1099, wherein said effector function of said intracellular domain of said first binding-triggered transcriptional switch regulates expression of a gene product in said cell. [The present invention 1101] 110. The method of claim 1100, wherein said gene product of said cell is an endogenous gene product of said cell. [The present invention 1102] 110. The method of claim 1100, wherein said gene product of said cell is a heterologous gene product of said cell. [The present invention 1103] 1100. The method of claim 1100, wherein said gene product of said cell is selected from the group consisting of chemokines, chemokine receptors, cytokines, cytokine receptors, differentiation factors, growth factors, growth factor receptors, hormones, metabolic enzymes, proliferation inducers, receptors, small molecule second messenger synthesis enzymes, T cell receptors, transcriptional activators, transcriptional repressors, transcriptional activators, transcriptional repressors, translational regulators, translational activators, translational repressors, activating immunoreceptors, apoptosis inhibitors, apoptosis inducers, immunoactivators, immunoinhibitors and inhibitory immunoreceptors. [The present invention 1104] 110. The method of claim 1100, wherein said effector function of said intracellular domain of said second binding-triggered transcriptional switch regulates expression of a gene product in said cell. [This invention 1105] 1105. The method of claim 1104, wherein said gene product of said cell is an endogenous gene product of said cell. [The present invention 1106] 1105. The method of claim 1104, wherein said gene product of said cell is a heterologous gene product of said cell. [This invention 1107] 1104. The method of claim 1104, wherein said gene product of said cell is selected from the group consisting of chemokines, chemokine receptors, cytokines, cytokine receptors, differentiation factors, growth factors, growth factor receptors, hormones, metabolic enzymes, proliferation inducers, receptors, small molecule second messenger synthesis enzymes, T cell receptors, transcriptional activators, transcriptional repressors, transcriptional activators, transcriptional repressors, translational regulators, translational activators, translational repressors, activating immunoreceptors, apoptosis inhibitors, apoptosis inducers, immunoactivators, immunoinhibitors and inhibitory immunoreceptors. [This invention 1108] 1099. The method of claim 1099, wherein at least one of the binding-transducers of the first and second binding-triggered transcriptional switches comprises a ligand-inducible proteolytic cleavage site, and binding of the first and second members of each specific binding pair induces cleavage of the binding-transducer at the ligand-inducible proteolytic cleavage site, thereby transmitting the binding signal and activating each intracellular domain by proteolytically releasing the intracellular domain. [This invention 1109] 1109. The method of claim 1108, wherein said binding-transducers of said first and second binding-triggered transcriptional switches both comprise a ligand-inducible proteolytic cleavage site. [The present invention 1110] 1. A method for modulating cellular activity, comprising: contacting said cell with a second member of the first specific binding pair. wherein the cells comprise i) a first binding-triggered transcriptional switch comprising an extracellular domain comprising a first member of the first specific binding pair, a binding transducer, and an intracellular domain; and ii) a second binding-triggered transcriptional switch comprising at least an extracellular domain comprising said first member of a second specific binding pair, a binding transducer, and an intracellular domain. Expressing the method, wherein the nucleotide sequence encoding the second binding-triggered transcriptional switch is operably linked to a transcriptional control element that is activated or repressed by the intracellular domain of the first binding-triggered transcriptional switch. [The present invention 1111] The method of claim 1110, wherein said contacting is carried out in vivo, ex vivo, or in vitro. [The present invention 1112] The method of claim 1110, wherein the second member of the first specific binding pair is on the surface of a second cell, immobilized on an insoluble substrate, present in an extracellular matrix, present in an artificial matrix, or is soluble. [The present invention 1113] The method of claim 1110, wherein activation of the intracellular domain of the second binding-triggered transcriptional switch regulates an activity of the cell selected from the group consisting of expression of a gene product of the cell, proliferation of the cell, apoptosis of the cell, non-apoptotic death of the cell, differentiation of the cell, dedifferentiation of the cell, migration of the cell, secretion of a molecule from the cell, and cell adhesion of the cell. [This invention 1114] 1114. The method of claim 1113, wherein said activity of said cell is expression of a gene product in said cell. [This invention 1115] The method of claim 1114, wherein the gene product of the cell is selected from the group consisting of chemokines, chemokine receptors, cytokines, cytokine receptors, differentiation factors, growth factors, growth factor receptors, hormones, metabolic enzymes, proliferation inducers, receptors, small molecule second messenger synthesis enzymes, T cell receptors, transcriptional activators, transcriptional repressors, transcriptional activators, transcriptional repressors, translational regulators, translational activators, translational repressors, activating immunoreceptors, apoptosis inhibitors, apoptosis inducers, immunoactivators, immunoinhibitors and inhibitory immunoreceptors. [The present invention 1116] 1110. The method of claim 1110, wherein at least one of the binding-transducers of the first and second binding-triggered transcriptional switches comprises a ligand-inducible proteolytic cleavage site, and wherein binding of the first and second members of the respective specific binding pairs induces cleavage of the binding-transducer at the ligand-inducible proteolytic cleavage site, thereby transducing the binding signal and activating the respective intracellular domains by proteolytically releasing the intracellular domains. [This invention 1117] 1. A method for tracking cell-cell contacts, comprising: expressing in each cell of the first plurality of cells a binding-triggered transcriptional switch comprising an extracellular domain comprising a first member of a specific binding pair, a binding transducer, and an intracellular domain; expressing a second member of the specific binding pair in each cell of the second plurality of cells; and contacting said first plurality of cells with said second plurality of cells wherein binding of the first member of the specific binding pair to the second member of the specific binding pair induces a binding transducer to transduce a binding signal of the binding-triggered transcriptional switch, thereby activating the intracellular domain, and wherein activation of the intracellular domain induces expression of a detectable reporter sufficient to track cell-cell contacts in space, time, or a combination thereof. [This invention 1118] 1118. The method of claim 1117, wherein said first plurality of cells, said second plurality of cells, or both, are neurons. [This invention 1119] The method of claim 1117, wherein the binding transducer comprises a ligand-inducible proteolytic cleavage site, and binding of the first member of the specific binding pair to the second member of the specific binding pair induces cleavage of the binding transducer at the ligand-inducible proteolytic cleavage site, thereby transmitting the binding signal and activating the intracellular domain by proteolytically releasing the intracellular domain. [The present invention 1120] 1119. The method of any of claims 1067 to 1119, wherein said binding-triggered transcriptional switch is a SynNotch polypeptide. [This invention 1121] A localized cell activation system comprising: an expressed binding-triggered transcriptional switch comprising an extracellular domain comprising a first member of a first specific binding pair, a binding transducer, and an intracellular domain; and a nucleic acid encoding a binding-triggered activating polypeptide comprising a first member of a second specific binding pair operably linked to a transcriptional control element induced by the intracellular domain of the first binding-triggered transcriptional switch. a cell comprising Upon contact with the second member of the first specific binding pair, the binding-triggered activating polypeptide is expressed, and upon contact with the second member of the second specific binding pair, the binding-triggered activating polypeptide activates the cell. [This invention 1122] The system of the present invention 1121, wherein the cells are selected from the group consisting of immune cells, progenitor or precursor cells, stem cells, and neurons. [This invention 1123] The system of claim 1122, wherein the cell is an immune cell, the binding-triggered transcriptional switch is an antigen-triggered transcriptional switch, the binding-triggered activation polypeptide is an antigen-triggered activation polypeptide, and upon contact with the second member of the first specific binding pair, the antigen-triggered activation polypeptide is expressed, and upon contact with the second member of the second specific binding pair, the antigen-triggered activating polypeptide activates the immune cell to recognize a target cell expressing the first member of the second specific binding pair. [This invention 1124] The system of the present invention 1123, wherein the antigen-induced activating polypeptide is a chimeric antigen receptor or a variant thereof. [This invention 1125] The system of the present invention 1123, wherein said antigen-induced activating polypeptide is an engineered T cell receptor or a variant thereof. [Invention 1126] 1126. The system of any one of claims 1121 to 1125, wherein the expressed binding-triggered transcriptional switch is a SynNotch polypeptide. [This invention 1127] 1. A method for locally modulating cellular activity, comprising: expressing in the first cell a binding-triggered transcriptional switch comprising a binding transducer, an intracellular domain, and a first extracellular domain comprising a first adaptor-binding domain that specifically binds to a first epitope on a soluble adaptor molecule; the first cell; and i) a second cell expressing a second extracellular domain comprising a second adaptor binding domain that specifically binds to a second epitope on the soluble adaptor molecule; and ii) an effective concentration of said soluble adapter molecule; to contact wherein binding of the first adaptor binding domain and the second adaptor binding domain to the adaptor molecule induces the binding transducer to transmit a binding signal to activate the intracellular domain, thereby producing an activated intracellular domain, and the activated intracellular domain regulates an activity of the first cell selected from the group consisting of expression of a gene product of the cell, proliferation of the cell, apoptosis of the cell, non-apoptotic death of the cell, differentiation of the cell, dedifferentiation of the cell, migration of the cell, secretion of a molecule from the cell, and cell adhesion of the cell. [This invention 1128] 1127. The method of claim 1127, wherein said contacting comprises applying said soluble adapter molecule to said cells in vitro or ex vivo. [This invention 1129] 1127. The method of claim 1127, wherein said contacting comprises administering said soluble adapter molecule to said cell in vivo. [The present invention 1130] 1127. The method of claim 1127, wherein contacting the first cell with an effective concentration of the soluble adapter molecule comprises placing the first cell in the presence of a third cell that expresses the adapter molecule. [This invention 1131] 1130. The method of claim 1130, wherein said third cell constitutively expresses said adapter molecule. [This invention 1132] 1130. The method of claim 1130, wherein said third cell conditionally expresses said adapter molecule. [This invention 1133] 1133. The method of any of claims 1127 to 1132, wherein said first extracellular domain and said soluble adaptor molecule are first and second members of a specific binding pair. [This invention 1134] 1133. The method of any of claims 1127 to 1132, wherein said second extracellular domain and said soluble adaptor molecule are first and second members of a specific binding pair. [This invention 1135] The method of any one of claims 1127 to 1134, wherein the first extracellular domain and the second extracellular domain are antibodies. [This invention 1136] 1135. The method of claim 1135, wherein one or both of said first extracellular domain and second extracellular domain is a nanobody. [This invention 1137] The method of any one of claims 1127 to 1136, wherein the intracellular domain is a transcription factor. [This invention 1138] 1138. The method of any of claims 1127 to 1137, wherein said activated intracellular domain regulates expression of an endogenous gene product of said first cell. [This invention 1139] The method of any of claims 1127 to 1137, wherein said activated intracellular domain regulates expression of a heterologous gene product in said first cell. [This invention 1140] 1139. Any of the methods of claims 1127 to 1139, wherein the binding transducer comprises a ligand-inducible proteolytic cleavage site, and binding of the first extracellular domain and the second extracellular domain to the soluble adaptor molecule induces cleavage of the binding transducer at the ligand-inducible proteolytic cleavage site, thereby transmitting the binding signal and activating the intracellular domain by proteolytically releasing the intracellular domain. [This invention 1141] a nucleic acid encoding a first binding-triggered transcriptional switch responsive to a first antigen; a first promoter responsive to the first binding-triggered transcriptional switch and operably linked to a nucleic acid encoding a CAR comprising an extracellular domain that specifically binds to a first member of a specific binding pair; a nucleic acid encoding a second binding-triggered transcriptional switch responsive to a second antigen; and a second promoter responsive to the second binding-triggered transcriptional switch and operably linked to a nucleic acid encoding an intracellular CAR inhibitory domain. wherein in the presence of the second antigen, the intracellular CAR inhibitory domain is expressed to inhibit activation of the cell by the CAR, and in the presence of the first antigen but in the absence of the second antigen, a CAR is expressed and activatable by the second member of the specific binding pair. [This invention 1142] a nucleic acid encoding a first binding-triggered transcriptional switch responsive to a first antigen; a first promoter responsive to the first binding-triggered transcriptional switch and operably linked to a nucleic acid encoding a first portion of a CAR comprising an extracellular domain that specifically binds to a first member of a specific binding pair; a nucleic acid encoding a second binding-triggered transcriptional switch responsive to a second antigen; and a second promoter responsive to the second binding-triggered transcriptional switch and operably linked to a nucleic acid encoding a second portion of the CAR comprising an intracellular signaling domain. wherein in the presence of the first antigen and the second antigen, the first and second portions of the CAR are expressed and the CAR is activatable by the second member of the specific binding pair. [This invention 1143] a nucleic acid encoding a third binding-triggered transcriptional switch responsive to a third antigen; and a third promoter responsive to the third binding-triggered transcriptional switch and operably linked to a nucleic acid encoding an intracellular CAR inhibitory domain. wherein in the presence of the third antigen, the intracellular CAR inhibitory domain is expressed and activation of the cell by the CAR is inhibited. [Brief explanation of the drawings]

[0050] [Figure 1] FIG. 1 is a schematic diagram of a Notch receptor polypeptide. [Figure 2A-1] The amino acid sequences of Notch receptor polypeptides of various species are provided (SEQ ID NO:131). [Figure 2A-2] This is a diagram showing a continuation of Figure 2A-1. [Figure 2B-1] The amino acid sequences of Notch receptor polypeptides of various species are provided (SEQ ID NO:132). [Figure 2B-2] This is a diagram showing a continuation of Figure 2B-1. [Figure 2C] The amino acid sequences of Notch receptor polypeptides of various species are provided (SEQ ID NO:133). [Figure 2D-1] The amino acid sequences of Notch receptor polypeptides of various species are provided (SEQ ID NO:134). [Figure 2D-2] This is a figure showing a continuation of Figure 2D-1. [Figure 2E-1] The amino acid sequences of Notch receptor polypeptides of various species are provided (SEQ ID NO:135). [Figure 2E-2] This is a diagram showing a continuation of Figure 2E-1. [Figure 2F-1] The amino acid sequences of Notch receptor polypeptides of various species are provided (SEQ ID NO:136). [Figure 2F-2] This is a figure showing a continuation of Figure 2F-1. [Figure 2G-1] The amino acid sequences of Notch receptor polypeptides of various species are provided (SEQ ID NO:17). [Figure 2G-2] This is a continuation of Figure 2G-1. [Figure 3] An amino acid sequence alignment of portions of Notch receptor polypeptides from various mammalian species is provided (mouse - SEQ ID NO: 138; human - SEQ ID NO: 139; bovine - SEQ ID NO: 140). [Figure 4A] 1 provides a schematic diagram of an exemplary chimeric Notch receptor polypeptide of the present disclosure. [Figure 4B] 1 provides a schematic diagram of an exemplary chimeric Notch receptor polypeptide of the present disclosure. [Figure 4C] 1 provides a schematic diagram of an exemplary chimeric Notch receptor polypeptide of the present disclosure. [Figure 4D] 1 provides a schematic diagram of an exemplary chimeric Notch receptor polypeptide of the present disclosure. [Figure 4E] 1 provides a schematic diagram of an exemplary chimeric Notch receptor polypeptide of the present disclosure. [Figure 4F] 1 provides a schematic diagram of an exemplary chimeric Notch receptor polypeptide of the present disclosure. [Figure 4G] 1 provides a schematic diagram of an exemplary chimeric Notch receptor polypeptide of the present disclosure. [Figure 5] 1 provides a schematic diagram of direct control of effector function using chimeric Notch receptor polypeptides of the present disclosure. [Figure 6] 1 provides a schematic diagram of an example of direct control of effector function using a chimeric Notch receptor polypeptide of the present disclosure. [Figure 7] 1 provides a schematic diagram of indirect regulation of effector function using chimeric Notch receptor polypeptides of the present disclosure. [Figure 8] 1 provides a schematic diagram of an example of indirect regulation of effector function using a chimeric Notch receptor polypeptide of the present disclosure. [Figure 9A] 1 provides a schematic diagram of the use of multiple chimeric Notch receptor polypeptides in parallel. [Figure 9B] 1 provides a schematic diagram of the use of multiple chimeric Notch receptor polypeptides in parallel. [Figure 10] 1 provides a schematic diagram of the use of a series of multiple chimeric Notch receptor polypeptides. [Figure 11] 1 provides a schematic diagram of a series of chimeric Notch receptor polypeptides and the use of chimeric antigen receptors (CARs). [Figure 12] 1 provides a schematic representation of the use of chimeric Notch receptor polypeptides in two or more cells, demonstrating multicellular cooperation. [Figure 13]1 provides a schematic diagram of the use of chimeric Notch receptor polypeptides in a multicellular environment. [Figure 14] 1 provides a schematic representation of the use of multiple receptor circuits with two or more cells. [Figure 15] 1 provides a schematic diagram of localized / targeted production of biologics in response to specific extracellular structures. [Figure 16A] An example of a Notch receptor polypeptide is shown (SEQ ID NO:141). [Figure 16B] An example of a Notch receptor polypeptide is shown (SEQ ID NO:142). [Figure 16C] An example of a Notch receptor polypeptide is shown (SEQ ID NO:143). [Figure 17A] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:144). [Figure 17B] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:145). [Figure 17C] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:146). [Figure 18] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:147). [Figure 19A] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:148). [Figure 19B] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:149). [Figure 20A] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:150). [Figure 20B] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:151). [Figure 20C]The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:152). [Figure 20D] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:153). [Figure 21] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:154). [Figure 22] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:155). [Figure 23] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:156). [Figure 24] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:157). [Figure 25] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:158). [Figure 26] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:159). [Figure 27] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:160). [Figure 28] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:161). [Figure 29] The amino acid sequence of an exemplary chimeric Notch receptor polypeptide is provided (SEQ ID NO:162). [Figure 30] Figures 30A and 30B show representative results for chimeric Notch with anti-CD19 in a TRE reporter line. [Figure 31] Figures 31A and 31B show representative results for chimeric Notch with anti-mesothelin in a TRE reporter line. [Figure 32]Figures 32A and 32B show representative results for chimeric Notch anti-CD19 in a UAS reporter line. [Figure 33] Figures 33A and 33B depict results with SV40 / UAS reporter cells transduced with an anti-CD19 chimeric Notch whose intracellular domain is a fusion of the Gal4 DNA-binding domain and the transcriptional repressor domain KRAB. [Figure 34] 1 illustrates the use of chimeric Notch receptor polypeptides in a cascade of signaling relays. [Figure 35A] 1 shows the effect of chimeric Notch receptor polypeptides on chimeric antigen receptor (CAR) expression on cancer cells and T cell activation. [Figure 35B] 1 shows the effect of chimeric Notch receptor polypeptides on chimeric antigen receptor (CAR) expression on cancer cells and T cell activation. [Figure 35C] 1 shows the effect of chimeric Notch receptor polypeptides on chimeric antigen receptor (CAR) expression on cancer cells and T cell activation. [Figure 36] The amino acid sequence of the Cas9 polypeptide is provided (SEQ ID NO:163). [Figure 37] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:164). [Figure 38] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:165). [Figure 39] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:165). [Figure 40] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:166). [Figure 41] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:167). [Figure 42]The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:169). [Figure 43] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:170). [Figure 44] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:171). [Figure 45] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:172). [Figure 46A] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:173). [Figure 46B] This is a figure showing a continuation of Figure 46A. [Figure 47] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:174). [Figure 48] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:175). [Figure 49] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:176). [Figure 50] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:177). [Figure 51] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:178). [Figure 52] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:179). [Figure 53] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:180). [Figure 54] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:181). [Figure 55]The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:182). [Figure 56] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:183). [Figure 57] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:184). [Figure 58] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:185). [Figure 59] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:186). [Figure 60] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:187). [Figure 61] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:188). [Figure 62] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:189). [Figure 63] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:190). [Figure 64] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:191). [Figure 65] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:192). [Figure 66] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:193). [Figure 67] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:194). [Figure 68] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:195). [Figure 69]The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:196). [Figure 70] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:197). [Figure 71] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:198). [Figure 72] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:199). [Figure 73] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:200). [Figure 74] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:201). [Figure 75] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:202). [Figure 76] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:203). [Figure 77] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:204). [Figure 78] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:205). [Figure 79] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:206). [Figure 80] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:207). [Figure 81] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:208). [Figure 82] The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:209). [Figure 83]The amino acid sequences of exemplary transcriptional activators and repressors are provided (SEQ ID NO:210). [Figure 84] Figures 84A and 84B show the effect of gamma secretase inhibitors on the activation of chimeric Notch receptor polypeptides. [Figure 85A] 1 shows an exemplary modular organization of a synNotch receptor. [Figure 85B] 1 shows an exemplary modular organization of a synNotch receptor. [Figure 86A] We demonstrate that SynNotch receptors can be used to program contact-dependent transcriptional regulation. [Figure 86B] We demonstrate that SynNotch receptors can be used to program contact-dependent transcriptional regulation. [Figure 86C] We demonstrate that SynNotch receptors can be used to program contact-dependent transcriptional regulation. [Figure 87A-1] Provides additional data for Figures 85A and 85B. [Figure 87A-2] This is a continuation of Figure 87A-1. [Figure 87A-3] This is a continuation of Figure 87A-2. [Figure 87B] Provides additional data for Figures 85A and 85B. [Figure 87C] Provides additional data for Figures 85A and 85B. [Figure 87D] Provides additional data for Figures 85A and 85B. [Figure 88A] Provides additional data for Figures 86A-C. [Figure 88B] Provides additional data for Figures 86A-C. [Figure 88C] Provides additional data for Figures 86A-C. [Figure 89A] We demonstrate that SynNotch receptors function in diverse cell types. [Figure 89B]We demonstrate that SynNotch receptors function in diverse cell types. [Figure 90] Figures 90A-90C provide additional data regarding Figures 89A and 89B. [Figure 91A] We demonstrate that SynNotch receptors exert spatial control over diverse cell behaviors. [Figure 91B] We demonstrate that SynNotch receptors exert spatial control over diverse cell behaviors. [Figure 91C] We demonstrate that SynNotch receptors exert spatial control over diverse cell behaviors. [Figure 91D] We demonstrate that SynNotch receptors exert spatial control over diverse cell behaviors. [Figure 92A-1] Provides additional data for Figures 91A-D. [Figure 92A-2] This is a continuation of Figure 92A-1. [Figure 92B] Provides additional data for Figures 91A-D. [Figure 92C] Provides additional data for Figures 91A-D. [Figure 93A] We demonstrate that SynNotch receptors are orthogonal to each other and can be used for combinatorial regulation. [Figure 93B] We demonstrate that SynNotch receptors are orthogonal to each other and can be used for combinatorial regulation. [Figure 93C] We demonstrate that SynNotch receptors are orthogonal to each other and can be used for combinatorial regulation. [Figure 94A] We demonstrate that multiple synNotch receptors can be used to generate multilayered self-organizing epithelial patterns. [Figure 94B] We demonstrate that multiple synNotch receptors can be used to generate multilayered self-organizing epithelial patterns. [Figure 94C] We demonstrate that multiple synNotch receptors can be used to generate multilayered self-organizing epithelial patterns. [Figure 95A] We demonstrate that the modularity of synNotch receptors expands the sensing / response operations of mammalian cells. [Figure 95B] We demonstrate that the modularity of synNotch receptors expands the sensing / response operations of mammalian cells. [Figure 95C] We demonstrate that the modularity of synNotch receptors expands the sensing / response operations of mammalian cells. [Figure 96A] We demonstrate the feasibility of engineering customized therapeutic T cell responses using synNotch receptors. [Figure 96B] We demonstrate the feasibility of engineering customized therapeutic T cell responses using synNotch receptors. [Figure 96C] We demonstrate the feasibility of engineering customized therapeutic T cell responses using synNotch receptors. [Figure 97A] We show that synNotch receptors can drive antigen-induced transcription in CD4+ and CD8+ human primary T lymphocytes. [Figure 97B] We show that synNotch receptors can drive antigen-induced transcription in CD4+ and CD8+ human primary T lymphocytes. [Figure 97C] We show that synNotch receptors can drive antigen-induced transcription in CD4+ and CD8+ human primary T lymphocytes. [Figure 97D] We show that synNotch receptors can drive antigen-induced transcription in CD4+ and CD8+ human primary T lymphocytes. [Figure 97E] We show that synNotch receptors can drive antigen-induced transcription in CD4+ and CD8+ human primary T lymphocytes. [Figure 97F] We show that synNotch receptors can drive antigen-induced transcription in CD4+ and CD8+ human primary T lymphocytes. [Figure 98A] We demonstrate that synNotch receptors can drive antigen-induced custom cytokine programs. [Figure 98B]We demonstrate that synNotch receptors can drive antigen-induced custom cytokine programs. [Figure 98C] We demonstrate that synNotch receptors can drive antigen-induced custom cytokine programs. [Figure 98D] We demonstrate that synNotch receptors can drive antigen-induced custom cytokine programs. [Figure 98E] We demonstrate that synNotch receptors can drive antigen-induced custom cytokine programs. [Figure 98F] We demonstrate that synNotch receptors can drive antigen-induced custom cytokine programs. [Figure 99A] We demonstrate that synNotch receptors can drive antigen-dependent skewing of T cell differentiation toward an antitumor Th1 fate. [Figure 99B] We demonstrate that synNotch receptors can drive antigen-dependent skewing of T cell differentiation toward an antitumor Th1 fate. [Figure 99C] We demonstrate that synNotch receptors can drive antigen-dependent skewing of T cell differentiation toward an antitumor Th1 fate. [Figure 99D] We demonstrate that synNotch receptors can drive antigen-dependent skewing of T cell differentiation toward an antitumor Th1 fate. [Figure 99E] We demonstrate that synNotch receptors can drive antigen-dependent skewing of T cell differentiation toward an antitumor Th1 fate. [Figure 100A] We demonstrate custom T cell delivery of non-native therapeutic agents—synNotch-driven TRAIL production. [Figure 100B] We demonstrate custom T cell delivery of non-native therapeutic agents—synNotch-driven TRAIL production. [Figure 100C] We demonstrate custom T cell delivery of non-native therapeutic agents—synNotch-driven TRAIL production. [Figure 100D] We demonstrate custom T cell delivery of non-native therapeutic agents—synNotch-driven TRAIL production. [Figure 100E] We demonstrate custom T cell delivery of non-native therapeutic agents—synNotch-driven TRAIL production. [Figure 101A] We demonstrate in vivo localized cytokine expression in solid tumors via synNotch receptor-engineered T cells. [Figure 101B] We demonstrate in vivo localized cytokine expression in solid tumors via synNotch receptor-engineered T cells. [Figure 101C] We demonstrate in vivo localized cytokine expression in solid tumors via synNotch receptor-engineered T cells. [Figure 102] Figures 102A-102B demonstrate that synNotch receptors are pluripotent regulators that enable T cells to monitor and selectively regulate their microenvironment. [Figure 103A] Provides supplemental data for Figures 97A-97F. [Figure 103B] Provides supplementary data for Figures 97A-97F. [Figure 103C] Provides supplemental data for Figures 97A-97F. [Figure 103D] Provides supplemental data for Figures 97A-97F. [Figure 103E] Provides supplemental data for Figures 97A-97F. [Figure 103F] Provides supplementary data for Figures 97A-97F. [Figure 104A] Provides supplementary data for Figures 98A-98F. [Figure 104B] Provides supplementary data for Figures 98A-98F. [Figure 104C] Provides supplementary data for Figures 98A-98F. [Figure 104D] Provides supplementary data for Figures 98A-98F. [Figure 104E] Provides supplementary data for Figures 98A-98F. [Figure 104F] Provides supplementary data for Figures 98A-98F. [Figure 104G] Provides supplementary data for Figures 98A-98F. [Figure 104H] Provides supplementary data for Figures 98A-98F. [Figure 104I] Provides supplementary data for Figures 98A-98F. [Figure 105A] Provides supplementary data for Figures 99A-99E. [Figure 105B] Provides supplementary data for Figures 99A-99E. [Figure 105C] Provides supplementary data for Figures 99A-99E. [Figure 105D] Provides supplementary data for Figures 99A-99E. [Figure 105E] Provides supplementary data for Figures 99A-99E. [Figure 105F] Provides supplementary data for Figures 99A-99E. [Figure 105G] Provides supplementary data for Figures 99A-99E. [Figure 106A] Provides supplementary data for Figures 100A-100E. [Figure 106B] Provides supplementary data for Figures 100A-100E. [Figure 106C] Provides supplementary data for Figures 100A-100E. [Figure 106D] Provides supplementary data for Figures 100A-100E. [Figure 106E] Provides supplementary data for Figures 100A-100E. [Figure 106F] Provides supplementary data for Figures 100A-100E. [Figure 106G] Provides supplementary data for Figures 100A-100E. [Figure 106H] Provides supplementary data for Figures 100A-100E. [Figure 107A] Provides supplementary data for Figures 101A-101C. [Figure 107B] Provides supplementary data for Figures 101A-101C. [Figure 107C] Provides supplementary data for Figures 101A-101C. [Figure 107D-1] Provides supplementary data for Figures 101A-101C. [Figure 107D-2] This is a continuation of Figure 107D-1. [Figure 107E] Provides supplementary data for Figures 101A-101C. [Figure 108A] Embodiments of synNotch receptors for combinatorial antigen sensing in T cells are provided. [Figure 108B] Embodiments of synNotch receptors for combinatorial antigen sensing in T cells are provided. [Figure 108C] Embodiments of synNotch receptors for combinatorial antigen sensing in T cells are provided. [Figure 108D] Embodiments of synNotch receptors for combinatorial antigen sensing in T cells are provided. [Figure 109A] Demonstrates combinatorial antigen requirement for synNotch-gated CAR-expressing Jurkat T cell activation. [Figure 109B] Demonstrates combinatorial antigen requirement for synNotch-gated CAR-expressing Jurkat T cell activation. [Figure 109C] Demonstrates combinatorial antigen requirement for synNotch-gated CAR-expressing Jurkat T cell activation. [Figure 109D] Demonstrates combinatorial antigen requirement for synNotch-gated CAR-expressing Jurkat T cell activation. [Figure 110A] synNotch-gated CAR expression in human primary T cells - demonstrating combinatorial antigen control for therapeutic T cell activation and tumor killing. [Figure 110B] synNotch-gated CAR expression in human primary T cells - demonstrating combinatorial antigen control for therapeutic T cell activation and tumor killing. [Figure 110C]synNotch-gated CAR expression in human primary T cells - demonstrating combinatorial antigen control for therapeutic T cell activation and tumor killing. [Figure 110D] synNotch-gated CAR expression in human primary T cells - demonstrating combinatorial antigen control for therapeutic T cell activation and tumor killing. [Figure 110E] synNotch-gated CAR expression in human primary T cells - demonstrating combinatorial antigen control for therapeutic T cell activation and tumor killing. [Figure 110F] synNotch-gated CAR expression in human primary T cells - demonstrating combinatorial antigen control for therapeutic T cell activation and tumor killing. [Figure 111A] 1 shows the synNotch receptor driving tumor-localized CAR expression in vivo. [Figure 111B] 1 shows the synNotch receptor driving tumor-localized CAR expression in vivo. [Figure 111C] 1 shows the synNotch receptor driving tumor-localized CAR expression in vivo. [Figure 112A] 1 shows selective combined antigen tumor killing in vivo by synNotch-gated CAR expression. [Figure 112B] 1 shows selective combined antigen tumor killing in vivo by synNotch-gated CAR expression. [Figure 112C] 1 shows selective combined antigen tumor killing in vivo by synNotch-gated CAR expression. [Figure 112D] 1 shows selective combined antigen tumor killing in vivo by synNotch-gated CAR expression. [Figure 113A] We show that synNotch receptors regulate and localize CAR T cell responses for precision immunotherapy. [Figure 113B] We show that synNotch receptors regulate and localize CAR T cell responses for precision immunotherapy. [Figure 113C]We show that synNotch receptors regulate and localize CAR T cell responses for precision immunotherapy. [Figure 114A] Provides supplementary data for Figures 109A-109D. [Figure 114B] Provides supplementary data for Figures 109A-109D. [Figure 114C] Provides supplementary data for Figures 109A-109D. [Figure 114D] Provides supplementary data for Figures 109A-109D. [Figure 115A] Provides supplemental data for Figures 110A-110F. [Figure 115B] Provides supplemental data for Figures 110A-110F. [Figure 115C] Provides supplemental data for Figures 110A-110F. [Figure 115D] Provides supplemental data for Figures 110A-110F. [Figure 115E] Provides supplemental data for Figures 110A-110F. [Figure 115F] Provides supplemental data for Figures 110A-110F. [Figure 115G] Provides supplemental data for Figures 110A-110F. [Figure 115H] Provides supplemental data for Figures 110A-110F. [Figure 115I] Provides supplemental data for Figures 110A-110F. [Figure 116A] Provides supplementary data for Figures 111A-111C. [Figure 116B] Provides supplementary data for Figures 111A-111C. [Figure 116C] Provides supplementary data for Figures 111A-111C. [Figure 117A] Provides supplementary data for Figures 112A-112D. [Figure 117B] Provides supplementary data for Figures 112A-112D. [Figure 117C]Provides supplementary data for Figures 112A-112D. [Figure 117D] Provides supplementary data for Figures 112A-112D. [Figure 117E] Provides supplementary data for Figures 112A-112D. [Figure 118] 1 demonstrates the induction of Foxp3 expression in human T cells using synNotch. [Figure 119] 1 provides a schematic antibody construct design for SynNotch-regulated antibody secretion from human T cells. [Figure 120] 1 provides a schematic diagram of the in vitro assay used to examine SynNotch-induced antibody secretion. [Figure 121] 1 provides a schematic diagram of a cell surface "sandwich ELISA" flow cytometry assay. [Figure 122] We demonstrate that SynNotch T cells can be induced to produce xenoantibodies in response to antigenic stimulation. [Figure 123] 1 provides a schematic diagram of the split SynNotch signaling system. [Figure 124] 10 demonstrates receiver cell activation at various concentrations of soluble adaptor molecules according to embodiments of the split-SynNotch signaling system of the present disclosure. [Figure 125] 1 provides a schematic diagram of a three cell division SynNotch signaling system according to an embodiment of the present disclosure. [Figure 126] 1 demonstrates SynNotch receiver cell activation in a three cell division SynNotch signaling system according to embodiments of the present disclosure. [Figure 127] 1 provides a schematic diagram of three cell division SynNotch inhibitory signaling systems according to embodiments of the present disclosure. [Figure 128] 10 demonstrates inhibition of SynNotch receiver cell activation in a three cell division SynNotch inhibitory signaling system according to embodiments of the present disclosure. [Figure 129]129A-129F provide schematic diagrams of specific embodiments of the split CAR systems described herein (FIG. 129F identifies elements of the schematic diagrams depicted in FIGS. 129A-129E). [Figure 130] FIG. 1 shows one embodiment of a two antigen-gated split CAR circuit. [Figure 131] FIG. 1 shows a further embodiment of a two antigen-gated split CAR circuit. [Figure 132] 1 provides a schematic diagram of an embodiment of a three input gate using circuit. [Figure 133] FIG. 1 provides a diagram of one configuration of a three antigen gated SynNotch, split CAR circuit. [Figure 134] 1 provides a schematic diagram of an embodiment of a four input gate using circuit. [Figure 135] 1 provides a schematic diagram of an embodiment of a five input gate using circuit. [Figure 136] 1 provides a schematic diagram of one embodiment of a three input AND+NOT gate of the present disclosure. [Figure 137] FIG. 1 provides a schematic diagram of one embodiment of a multi-input gate with split transcription factor AND functionality and dominant-negative NOT functionality. [Figure 138] FIG. 1 provides a schematic diagram of one embodiment of a multi-input gated CAR T cell activation circuit. [Figure 139] 12 provides the results of an analysis similar to that performed in FIG. 122 of SynNotch CD4 T cells engineered to conditionally secrete pembrolizumab. [Figure 140] 12 provides the results of an analysis similar to that performed in FIG. 122 of SynNotch E6-1 Jurkat cells engineered to secrete pembrolizumab. [Figure 141] 12 provides the results of a similar analysis to that performed in FIG. 122 of SynNotch E6-1 Jurkat cells engineered to secrete pembrolizumab using an alternative construct. [Figure 142]12 provides the results of an analysis similar to that performed in FIG. 122 of SynNotch CD4 T cells engineered to secrete tremelimumab. [Figure 143] 12 provides the results of an analysis similar to that performed in FIG. 122 of SynNotch E6-1 Jurkat cells engineered to secrete tremelimumab. DETAILED DESCRIPTION OF THE INVENTION

[0051] definition The terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine or pyrimidine bases, or other natural, chemically or biologically modified, non-natural, or derivatized nucleotide bases.

[0052] "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.

[0053] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus, or cosmid, to which another DNA segment, or "insert," may be attached so as to bring about the replication of the attached segment in a cell.

[0054] "Heterologous," as used herein, means a nucleotide or polypeptide sequence that is not found in the native (eg, naturally occurring) nucleic acid or protein, respectively.

[0055] The terms "antibody" and "immunoglobulin" include antibodies or immunoglobulins of any isotype, and fragments of antibodies that retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies (scAbs), single-domain antibodies (dAbs), single-domain heavy chain antibodies, single-domain light chain antibodies, nanobodies, bispecific antibodies, multispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody (also referred to herein as antigen-binding) and a non-antibody protein. Antibodies can be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, a fluorescent protein, etc. Antibodies can be further conjugated to other moieties, such as a member of a specific binding pair, e.g., biotin (a member of the biotin-avidin specific binding pair). Antibodies can also be bound to a solid support, including, but not limited to, a polystyrene plate or bead. Further encompassed by the term are Fab', Fv, F(ab')2, and / or other antibody fragments that retain specific binding to an antigen, as well as monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by a group of individual cells, all of which are produced from a single cell by repeated cell replication. That is, a clone of a cell produces only a single antibody species. Monoclonal antibodies can be produced using hybridoma production techniques, although other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries). Antibodies can be monovalent or bivalent. An antibody can be an Ig monomer, which is a "Y-shaped" molecule consisting of four polypeptide chains: two heavy chains and two light chains linked by disulfide bonds.

[0056] The term "humanized immunoglobulin," as used herein, refers to an immunoglobulin comprising immunoglobulin portions of different origins, at least one of which contains an amino acid sequence of human origin. For example, a humanized antibody can comprise a portion derived from an immunoglobulin of non-human origin, such as a mouse, with the requisite specificity, and an immunoglobulin sequence of human origin (e.g., a chimeric immunoglobulin), either chemically joined together by conventional techniques (e.g., synthesis) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portion of the chimeric antibody can be expressed to produce a contiguous polypeptide chain). Another example of a humanized immunoglobulin is an immunoglobulin containing one or more immunoglobulin chains comprising complementarity-determining regions (CDRs) derived from an antibody of non-human origin and framework regions derived from light and / or heavy chains of human origin (e.g., a CDR-grafted antibody with or without framework changes). Chimeric or CDR-grafted single-chain antibodies are also encompassed by the term humanized immunoglobulin. See, for example, Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Patent No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, MS et al., WO 86 / 01533; Neuberger, MS et al., European Patent No. 0,194,276 B1; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, E A et al., European Patent Application No. 0,519,596 A1. See also Ladner et al., U.S. Patent No. 4,946,778; Huston, U.S. Patent No. 5,476,786; and Bird, RE et al., Science, 242:423-426 (1988)) regarding single chain antibodies.

[0057] The term "nanobody" (Nb) as used herein refers to the smallest antigen-binding fragment or single variable domain (V) derived from a naturally occurring heavy chain antibody.HH ) and are known to those skilled in the art. They are derived from heavy chain-only antibodies found in camels (Hamers-Casterman et al., 1993; Desmyter et al., 1996). In the family of "camelid" immunoglobulins, a lack of light polypeptide chains has been found. "Camelid" includes the archaic term camelus (Bactrian camel and dromedary) and the neologism camelid (e.g., alpaca, llama, guanaco, and vicuna). Single variable domain heavy chain antibodies are herein referred to as nanobodies or V HH These are called antibodies.

[0058] An "antibody fragment" comprises a portion of an intact antibody, e.g., the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); domain antibodies (dAbs; Holt et al. (2003) Trends Biotechnol. 21:484); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments: termed "Fab" fragments, each containing a single antigen-binding site; and a residual "Fc" fragment, a name reflecting its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment, which contains two antigen-combining sites and is still capable of cross-linking antigen.

[0059] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three CDRs of each variable domain interact to form the V H -V LThe six CDRs form an antigen-binding site on the surface of the dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to antigen, albeit with lower affinity than the entire binding site.

[0060] The "Fab" fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0061] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Subclasses can be further divided into types, e.g., IgG2a and IgG2b.

[0062] "Single-chain Fv" or "sFv" or "scFv" antibody fragments are fragments of the V of an antibody. H and V L In some embodiments, an Fv polypeptide comprises a V domain, and these domains are present in a single polypeptide chain. H and V LThe sFv further comprises a polypeptide linker between the domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994).

[0063] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which are bound to the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) linked to a heavy chain variable domain (V H ). Using a linker that is too short to allow pairing between the two domains on the same chain forces the domains to pair with complementary domains on another chain and create two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0064] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two substances (e.g., an antibody and an antigen), and is also referred to as the dissociation constant (K D) Affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1,000-fold greater, or more, than the affinity of the antibody for an unrelated amino acid sequence. The affinity of an antibody for a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM), or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more substances to dissociation after dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0065] The term "binding" refers to a direct association between two molecules, e.g., by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bonding interactions, including interactions such as salt bridges and water bridges. In some cases, a first member of a specific binding pair present in the extracellular domain of a chimeric Notch receptor polypeptide of the present disclosure specifically binds to a second member of the specific binding pair. "Specific binding" refers to a binding of at least about 10 -7 M or more, e.g., 5 x 10 -7 M, 10 -8 M, 5 x 10 -8 "Non-specific binding" refers to binding with an affinity of about 10 M or greater. -7 Binding with an affinity of less than 10 M, e.g., -6 M, 10 -5 M, 10 -4 It refers to binding with an affinity such as M.

[0066] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which can include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The term encompasses fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences with or without an N-terminal methionine residue; immunologically tagged proteins; and the like.

[0067] An "isolated" polypeptide is one that has been identified, separated, and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that may interfere with diagnostic or therapeutic uses of the polypeptide, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, the polypeptide will be purified (1) to greater than 90%, greater than 95%, or greater than 98%, e.g., greater than 99%, by weight, of the antibody as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. Because at least one component of the polypeptide's natural environment will not be present, an isolated polypeptide includes an in situ polypeptide within a recombinant cell. In some examples, an isolated polypeptide will be prepared by at least one purification step.

[0068] The terms "chimeric antigen receptor" and "CAR" are used interchangeably herein and generally, but not exclusively, refer to an artificial, multi-module molecule capable of inducing or inhibiting immune cell activation, comprising an extracellular domain (e.g., a ligand / antigen-binding domain), a transmembrane domain, and one or more intracellular signaling domains. The term CAR is not specifically limited to CAR molecules but also includes CAR variants. CAR variants include split CARs, in which the extracellular portion (e.g., a ligand-binding portion) and the intracellular portion (e.g., an intracellular signaling portion) of the CAR are present on two separate molecules. CAR variants also include on-switch CARs, which are conditionally activatable CARs, including, for example, split CARs, in which the conditional heterodimerization of the two portions of the split CAR is pharmacologically controlled. CAR variants also include bispecific CARs, which contain a second CAR-binding domain that can amplify or inhibit the activity of the primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs), which may be used, for example, as components of bispecific CAR systems, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation.CAR molecules and their derivatives (i.e., CAR variants) have been described, for example, in PCT Application No. US2014 / 016527; Fedorov et al. Sci Transl Med (2013); 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al. Cancer J (2014) 20(2):141-4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1):91-106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304, the disclosures of which are incorporated herein by reference in their entireties.

[0069] As used herein, GFP nanobodies may be referred to herein according to their "LaG" (llama antibody against GFP) nomenclature as described in Fridy et al. (2014) Nat. Methods. 11(12):1253-1260; the disclosure of which, including any associated supplemental material, is incorporated herein by reference in its entirety. Thus, for example, in the example where GFP (or mutants of GFP or other cnidarian fluorescent proteins related to GFP (e.g., AmCFP, DsRed, etc.) is used as the adapter molecule, various combinations of LaG nanobodies may be used, provided that, for example, if the members of a LaG nanobody pair bind to different epitopes of GFP, the members of the LaG nanobody pair do not interfere with each other in their binding to GFP. LaG nanobodies include, for example, LaG-2, LaG-3, LaG-6, LaG-9, LaG-10, LaG-12, LaG-14, LaG-16, LaG-17, LaG-19, LaG-20, LaG-21, LaG-22, LaG-23, LaG-24, LaG-25, LaG-26, LaG-27, LaG-28, LaG-29, LaG-30, LaG-31, LaG-32, LaG-33, LaG-34, LaG-35, LaG-36, LaG-37, LaG-38, LaG-39, LaG-40, LaG-41, LaG-42, LaG-43, LaG-44, LaG-45, LaG-46, LaG-47, LaG-48, LaG-49, LaG-50, LaG-51, LaG-52, LaG-53, LaG-54, LaG-55, LaG-56, LaG-57, LaG-58, LaG-59, LaG-60, LaG-61, LaG-62, LaG-63, LaG-64, LaG-65, LaG-66, LaG-67, LaG-68, LaG-69, LaG-70, LaG-71, LaG-72, LaG-73, LaG-74, Examples of suitable nanobodies include, but are not limited to, LaG-21, LaG-24, LaG-26, LaG-27, LaG-29, LaG-30, LaG-35, LaG-37, LaG-41, LaG-42, LaG-43, LaG-5, LaG-8, LaG-11, LaG-18, LaG16-G4S-2, LaG16-3xFLAG-2, LaG41-G4S-2, etc. In some examples, llama antibodies against mCherry (LaM) may also be used in the systems and devices described herein, where, for example, LaM nanobodies include, but are not limited to, LaM-1, LaM-2, LaM-3, LaM-4, LaM-6, LaM-8, etc.

[0070] As used herein, the terms "treatment," "treating," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a partial or complete cure is provided for the disease and / or the deleterious effects attributable to the disease. "Treatment," as used herein, encompasses all treatments of disease in mammals, particularly humans, and includes (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed as having it; (b) suppressing the disease, i.e., arresting its progression; and (c) alleviating the disease, i.e., causing the disease to regress.

[0071] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to mammals, including, but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., horses, cattle, sheep, pigs, goats), lagomorphs, etc. In some cases, the individual is a human. In some cases, the individual is a non-human primate. In some cases, the individual is a rodent, e.g., a rat or mouse. In some cases, the individual is a lagomorph, e.g., a rabbit.

[0072] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

[0073] Where a range of values is provided, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the invention, unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded value in the stated range. Where a stated range includes one or both of its limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0075] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "chimeric Notch receptor polypeptide" includes a plurality of such chimeric Notch receptor polypeptides; a reference to a "genetically modified host cell" includes one or more genetically modified host cells and equivalents thereof known to those skilled in the art; and so forth. It should be further noted that the claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a predicate to the use of exclusive terminology such as "solely," "only," and the like, or the use of a "negative" limitation in connection with the recitation of claim elements.

[0076] It is understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention that are described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0077] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0078] Detailed Description The present disclosure provides chimeric Notch receptor polypeptides, nucleic acids comprising nucleotide sequences encoding the chimeric Notch receptor polypeptides, and host cells genetically modified with the nucleic acids. The chimeric Notch receptor polypeptides are useful in a variety of applications, which are also provided.

[0079] The present disclosure includes binding-triggered transcriptional switches and methods of using binding-triggered transcriptional switches. As used herein, a "binding-triggered transcriptional switch" generally refers to a synthetic modular polypeptide or system of interacting polypeptides having an extracellular domain comprising a first member of a specific binding pair, a binding transducer, and an intracellular domain. Upon binding of the second member of the specific binding pair to the binding-triggered transcriptional switch, a binding signal is transmitted to the intracellular domain, thereby activating the intracellular domain and performing some function within the cell that would not occur in the absence of the signal.

[0080] The components of a binding-triggered transcriptional switch and the arrangement of the components of the switch relative to each other can vary depending on many factors, including, but not limited to, the desired binding trigger, the activity of the intracellular domain, the overall function of the binding-triggered transcriptional switch, the broader arrangement of the molecular circuitry that comprises the binding-triggered transcriptional switch, etc. The first binding member can include, but is not limited to, the first and / or second binding members of a specific binding pair described herein. The intracellular domain can include, but is not limited to, the intracellular domain and / or domain that has a biological function as described herein.

[0081] The binding transducer of a binding-triggered transcriptional switch will also vary depending on the desired method of transduction of the binding signal. Generally, the binding transducer may comprise a polypeptide and / or domain of a polypeptide that transduce an extracellular signal into an intracellular signal, such as is done by receptors of various signaling pathways. Transduction of the binding signal may be achieved through various mechanisms, including, but not limited to, binding-induced proteolytic cleavage, binding-induced phosphorylation, binding-induced conformational change, etc. In some examples, the binding transducer may contain a ligand-inducible proteolytic cleavage site such that, upon binding, the binding signal is transduced by cleavage of the binding-triggered transcriptional switch, e.g., to release the intracellular domain. For example, in some examples, the binding-triggered transcriptional switch may comprise a cleavable binding transducer from Notch, e.g., a chimeric Notch receptor polypeptide described herein.

[0082] In other examples, the binding signal can be transmitted in the absence of inducible proteolytic cleavage. Any signaling component or component of a signaling pathway, whether or not proteolytic cleavage is required for signal propagation, can be used in a binding-triggered transcriptional switch. For example, in some examples, phosphorylation-based binding transducers, including, but not limited to, one or more signaling components of the Jak-Stat pathway, can be used in a non-proteolytic binding-triggered transcriptional switch.

[0083] For simplicity, binding-triggered transcriptional switches, including but not limited to chimeric Notch receptor polypeptides, are described primarily as single polypeptide chains. However, as will be apparent from the present disclosure, binding-triggered transcriptional switches, including chimeric Notch receptor polypeptides, may be separated or split across two or more separate polypeptide chains, in which case joining of the two or more polypeptide chains to form a functional binding-triggered transcriptional switch, e.g., a chimeric Notch receptor polypeptide, may be constitutively or conditionally controlled. For example, constitutive joining of the two portions of a split binding-triggered transcriptional switch may be achieved by inserting a constitutive heterodimerization domain between the first and second portions of the split polypeptides, such that the split portions are functionally joined upon heterodimerization.

[0084] In some examples, the junction of and / or signaling from a split-binding-triggered transcriptional switch may be conditionally controlled, e.g., through the use of an "adapter" that mediates functional junction of the first and second parts of the split-binding-triggered transcriptional switch. To mediate signaling through the split-binding-triggered transcriptional switch, the adapter may be added or directly administered, or may be indirectly produced, e.g., through expression of the adapter, either conditionally or constitutively, e.g., from a cell configured for such expression. Useful adapters include proteins with first and second binding surfaces that can be simultaneously used by two different binding molecules. In some examples, the adapter may comprise a protein in which two antibodies bind to two different epitopes of the protein.

[0085] For example, in some instances, an antigen may be used as an adapter, where the binding molecules used may be two different antibodies that bind to two different epitopes of the antigen. In such a configuration, attachment of the antibodies, or portions thereof, to the first and second parts of the split-binding-triggered transcriptional switch is mediated by simultaneous binding of both antibodies to a single molecule of antigen, resulting in functional joining of the parts in the presence of the antigen. Antigens that can function as adapters include, but are not limited to, pathogen antigens, cancer-associated antigens, disease-associated antigens, antibodies, etc. In some cases, the adapter antigen is soluble (e.g., not bound to the surface of a cell). In some cases, the adapter antigen is bound to the surface of a cell.

[0086] For example, in some instances, GFP may be used as an adaptor where the binding molecules used may be two different antibodies that bind to two different surfaces of GFP. In such a configuration, attachment of the antibodies, or portions thereof, to the first and second parts of the split-binding-triggered transcriptional switch is mediated by the simultaneous binding of both antibodies to a single molecule of GFP, resulting in functional joining of the parts in the presence of GFP.

[0087] In some examples, the split binding-triggered transcriptional switch allows for detection of the presence of an antigen in proximity to the first and second parts of the split binding-triggered transcriptional switch, e.g., when binding of the first and second parts of the split binding-triggered transcriptional switch is mediated by an antigen adaptor, resulting in functional joining of the parts. For example, in certain embodiments, the first part of the binding-triggered transcriptional switch is expressed on the surface of a first cell and the second part of the binding-triggered transcriptional switch is expressed on a second cell, and the first and second parts are configured such that when soluble antigen is present in the vicinity of the first and second cells, the first and second parts are functionally joined by the antigen, resulting in activation of the reporter by the activated binding-triggered transcriptional switch.

[0088] Both portions of the split binding-triggered transcriptional switch do not necessarily need to be anchored to a cell to function in detecting an antigen. For example, in some instances, a first portion of the binding-triggered transcriptional switch is solubly expressed and a second portion of the binding-triggered transcriptional switch is expressed on a cell, such that when a soluble antigen is present near the first and second portions, the portions are functionally joined by the antigen, resulting in priming of the binding-triggered transcriptional switch, enabling the primed binding-triggered transcriptional switch to respond to, e.g., report a second event, including the presence of a second antigen that activates the binding-triggered transcriptional switch. Such a second antigen may be present on the surface of the cell or may not be attached to the cell (i.e., may be soluble).

[0089] Conditional control of the joining of portions of a split-binding-triggered transcriptional switch provides further control of signaling from the split-binding-triggered transcriptional switch. For example, mediating joining by providing or expressing an adapter signaling from the split-binding-triggered transcriptional switch creates a condition that allows signaling from the switch. Conversely, inhibiting joining by providing a competitive inhibitor that prevents joining of portions of the split-binding-triggered transcriptional switch can prevent signaling from the switch. In some instances, such effects are dose-dependent, i.e., can be further controlled based on the amount of adapter and / or competitive inhibitor provided.

[0090] Thus, those skilled in the art will readily appreciate that the description of split binding-triggered transcriptional switches and the description of single polypeptide binding-triggered transcriptional switches provided herein may be exploited to provide additional constitutive and / or conditional control over signaling from such switches and molecular circuits containing such switches.

[0091] Chimeric NOTCH receptor polypeptides The present disclosure provides chimeric Notch receptor polypeptides. The chimeric Notch receptor polypeptides of the present disclosure comprise: a) an extracellular domain comprising a first member of a specific binding pair; b) a Notch receptor polypeptide, wherein the Notch receptor polypeptide has a length of 50 to 1000 amino acids and comprises one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain. Binding of the first member of the specific binding pair to a second member of the specific binding pair induces cleavage of the Notch receptor polypeptide at the one or more ligand-inducible proteolytic cleavage sites, thereby releasing the intracellular domain. Release of the intracellular domain regulates the activity of a cell producing the chimeric Notch receptor polypeptide. The extracellular domain comprises the first member of the specific binding pair; and the first member of the specific binding pair comprises an amino acid sequence heterologous to the Notch receptor polypeptide. The intracellular domain comprises an amino acid sequence heterologous to the Notch receptor polypeptide.

[0092] A schematic diagram of a Notch receptor polypeptide is presented in Figure 1. The Notch receptor polypeptide shown in Figure 1 includes: a) an epidermal growth factor (EGF) repeat; ii) a ligand-binding site; iii) three Lin-12 Notch repeats (LNRs), designated LNR-A, LNR-B, and LNR-C; iv) an extracellular portion containing two heterodimerization domains (HD-N and HD-C); b) a transmembrane (TM) portion; and c) an intracellular portion containing: i) a RAM domain; ii) ankyrin repeats; iii) a transcriptional activation domain; and iv) a PEST region. The Notch receptor polypeptide contains three proteolytic sites designated S1, S2, and S3. S1 is a furin cleavage site located between HD-N and HD-C; S2 is an ADAM17 cleavage site located within HD-C; and S3 is a gamma secretase cleavage site within the TM portion. Notch receptor polypeptides mediate intercellular communication, e.g., communication between contacting cells, where one contacting cell is a "receiving" cell and the other contacting cell is a "sending" cell. Engagement of a Notch receptor polypeptide present on a receiving cell by a Delta polypeptide ("ligand") present on a sending cell results in ligand-induced cleavage of the Notch receptor polypeptide, resulting in release of the intracellular portion of the receptor from the membrane into the cytoplasm. The released portion alters the behavior of the receiving cell by functioning as a transcriptional regulator.

[0093] Extracellular domain As described above, the chimeric Notch receptor polypeptide of the present disclosure comprises an extracellular domain. The extracellular domain comprises a first member of a specific binding pair. The first member of the specific binding pair binds to a second member of the specific binding pair, where the second member of the specific binding pair is on a different polypeptide from the chimeric Notch receptor polypeptide of the present disclosure. The second member of the specific binding pair is separate (e.g., not covalently linked) from the chimeric Notch receptor polypeptide comprising the extracellular domain that comprises the first member of the specific binding pair. The second member of the specific binding pair can be present on the surface of a cell. The second member of the specific binding pair can be immobilized on an insoluble support. The second member of the specific binding pair can be soluble. The second member of the specific binding pair can be present in an extracellular environment (e.g., an extracellular matrix). The second member of the specific binding pair can be present in an artificial matrix. The second member of the specific binding pair can be present in a cell-free environment.

[0094] The extracellular domain comprises a first member of a specific binding pair that is heterologous to the Notch receptor polypeptide, in other words, the first member of the specific binding pair present in the extracellular domain does not naturally occur in the Notch receptor polypeptide.

[0095] Suitable first members of specific binding pairs include, but are not limited to, antibody-based recognition scaffolds; antibodies (i.e., antibody-based recognition scaffolds comprising antigen-binding antibody fragments); non-antibody-based recognition scaffolds; antigens (e.g., endogenous antigens; exogenous antigens; etc.); ligands for receptors; receptors; targets of non-antibody-based recognition scaffolds; Fc receptors (e.g., FcγRIIIa; FcγRIIIb; etc.); extracellular matrix components; and the like.

[0096] Specific binding pairs include, for example: antigen-antibody specific binding pairs, where a first member is an antibody (or antibody-based recognition scaffold) that specifically binds to a second member that is an antigen, or where the first member is an antigen and the second member is an antibody (or antibody-based recognition scaffold) that specifically binds to an antigen; a ligand-receptor specific binding pair, where the first member is a ligand and the second member is a receptor to which the ligand binds, or where the first member is a receptor and the second member is a ligand that binds to the receptor; non-antibody-based recognition scaffold-target-specific binding pairs, where the first member is a non-antibody-based recognition scaffold and the second member is a target that binds to the non-antibody-based recognition scaffold, or where the first member is a target and the second member is a non-antibody-based recognition scaffold that binds to the target; adhesion molecule-extracellular matrix binding pairs; an Fc receptor-Fc binding pair, wherein a first member is an immunoglobulin Fc that binds to a second member that is an Fc receptor, or wherein the first member is an Fc receptor that binds to a second member that comprises an immunoglobulin Fc; and A receptor-co-receptor binding pair, where a first member is a receptor that specifically binds to a second member that is a co-receptor, or where a first member is a co-receptor that specifically binds to a second member that is a receptor.

[0097] Non-limiting examples of suitable extracellular domains include, for example, cadherins (CDH1-20), integrins (alpha and beta isoforms), ephrins, NCAM, connexins, CD44, syndecans, CD47, DG alpha / beta, SV2, protocadherins, Fas, dectin-1, CD7, CD40, neuregulin, KIR, BTLA, Tim-2, Lag-3, CD19, CTLA4, CD28, TIGIT, and ICOS.

[0098] In some cases, the extracellular domain comprises a Toll-like receptor (TLR). In some cases, the extracellular domain comprises a dectin that recognizes N-glycans present on the surface of pathogenic fungi and cancer cells. See, e.g., Xie (2012) Glycoconj. 29:273; and Brown et al. (2007) Protein Sci. 16:1042. In some cases, the extracellular domain comprises a polypeptide that recognizes a bacterial surface molecule.

[0099] In some cases, the extracellular domain of a chimeric Notch polypeptide of the present disclosure has the following amino acid sequence: It comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000003.tif17150.

[0100] Those skilled in the art can select an extracellular domain based on the desired localization or function of the cell that has been genetically modified to express the chimeric Notch receptor polypeptide of the present disclosure. For example, the extracellular domain can target cells to estrogen-dependent breast cancer cells that have an increased number of estrogen receptors on the cell surface, where the first member of the specific binding pair binds to the estrogen receptor (the second member of the specific binding pair). Other non-limiting examples of ligand / receptor interactions include CCRI (e.g., for targeting inflamed joint tissue or the brain in rheumatoid arthritis, and / or multiple sclerosis), CCR7, CCR8 (e.g., for targeting lymph node tissue), CCR6, CCR9, CCRIO (e.g., for targeting intestinal tissue), CCR4, CCRIO (e.g., for targeting the skin), CXCR4 (e.g., for general enhancement of transmigration), HCELL (e.g., for targeting inflammation and inflammatory disorders, bone marrow), alpha4beta7 (e.g., for targeting the intestinal mucosa), VLA-4 / VCAM-I (e.g., for targeting the endothelium). Generally, any receptor involved in targeting (e.g., cancer metastasis) can be used as the extracellular domain of a chimeric Notch receptor polypeptide of the present disclosure.

[0101] Antibody-based recognition scaffolds In some cases, the first member of the specific binding pair is an antibody. The antibody can be any antigen-binding antibody-based polypeptide, a wide variety of which are known in the art. In some examples, the antigen-binding domain is a single-chain Fv (scFv). Other antibody-based recognition domains are suitable for use, including cAb VHH (camelid antibody variable domains) and humanized versions, IgNAR VH (shark antibody variable domains) and humanized versions, sdAb VH (single-domain antibody variable domains), and "camelized" antibody variable domains. In some examples, T cell receptor (TCR)-based recognition domains, such as single-chain TCRs (scTvs, single-chain two-domain TCRs containing VαVβ), are also suitable for use.

[0102] When the member of the specific binding pair in a chimeric Notch receptor polypeptide of the present disclosure is an antibody-based recognition scaffold, the chimeric Notch receptor polypeptide can be activated in the presence of a second member of the specific binding pair, where the second member of the specific binding pair is an antigen that binds to the antibody-based recognition scaffold.

[0103] Antibodies suitable for inclusion in the chimeric Notch polypeptides of the present disclosure can have a variety of antigen binding specificities.

[0104] In some cases, the antigen-binding domain is specific for an antigen expressed (synthesized by) by a cancer cell, i.e., an epitope present in a cancer cell-associated antigen. The cancer cell-associated antigen can be, for example, an antigen associated with breast cancer cells, B-cell lymphoma, pancreatic cancer, Hodgkin's lymphoma cells, ovarian cancer cells, prostate cancer, mesothelioma, lung cancer cells (e.g., small cell lung cancer cells), non-Hodgkin's B-cell lymphoma (B-NHL) cells, ovarian cancer cells, prostate cancer, mesothelioma cells, lung cancer cells (e.g., small cell lung cancer cells), melanoma cells, chronic lymphocytic leukemia cells, acute lymphocytic leukemia cells, neuroblastoma cells, glioma, glioblastoma, medulloblastoma, colorectal cancer cells, etc. The cancer cell-associated antigen may also be expressed by a non-cancerous cell.

[0105] In some cases, the antigen-binding domain is specific for an epitope present in a tissue-specific antigen. In some cases, the antigen-binding domain is specific for an epitope present in a disease-associated antigen.

[0106] Non-limiting examples of antigens to which the antigen-binding domain of a subject chimeric Notch receptor polypeptide can bind include, for example, CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like.

[0107] Non-limiting examples of antigens to which the antigen-binding domain of a subject chimeric Notch receptor polypeptide can bind include, for example, cadherins (CDH1-20), integrins (alpha and beta isoforms), ephrins, NCAM, connexins, CD44, syndecans, CD47, DG alpha / beta, SV2, protocadherins, Fas, dectin-1, CD7, CD40, neuregulin, KIR, BTLA, Tim-2, Lag-3, CD19, CTLA4, CD28, TIGIT, and ICOS.

[0108] In some cases, the antibody is specific for a cytokine. In some cases, the antibody is specific for a cytokine receptor. In some cases, the antibody is specific for a growth factor. In some cases, the antibody is specific for a growth factor receptor. In some cases, the antibody is specific for a cell surface receptor.

[0109] In some cases, the antibody is specific for a cell surface target, where non-limiting examples of cell surface targets include CD19, CD30, Her2, CD22, ENPP3, EGFR, CD20, CD52, CD11a, and alpha-integrin.

[0110] In some cases, the antigen (second member of the specific binding pair) bound by the antibody-based scaffold is soluble. In some cases, the antigen is membrane-bound, e.g., in some cases, the antigen is present on the surface of a cell. In some cases, the antigen is immobilized on an insoluble support, which can include any of a variety of materials (e.g., polyethylene, polystyrene, polyvinylpyrrolidone, polycarbonate, nitrocellulose, etc.); the insoluble support can take a variety of forms, e.g., a plate, a tissue culture dish, a column, etc. In some cases, the antigen is present in the extracellular matrix (ECM) (e.g., the antigen is an ECM component). In some cases, the antigen is present in an artificial matrix. In some cases, the antigen is present in a cell-free environment.

[0111] Non-antibody-based recognition scaffolds In some cases, the first member of the specific binding pair is a non-antibody-based recognition scaffold. When a member of a specific binding pair in a chimeric Notch receptor polypeptide of the present disclosure is a non-antibody-based recognition scaffold, the chimeric Notch receptor polypeptide can be activated in the presence of a second member of the specific binding pair, where the second member of the specific binding pair is a target that binds to the non-antibody-based recognition scaffold.

[0112] Non-antibody-based recognition scaffolds include, for example, affibodies, engineered Kunitz domains, monobodies (adnectins), anticalins, designed ankyrin repeat domains (DARPins), binding sites for cysteine-rich polypeptides (e.g., cysteine-rich knottin peptides), avimers, affilins, etc. See, e.g., Gebauer and Skerra (2009) Curr. Opin. Chem. Biol. 13:245.

[0113] Non-antibody-based scaffolds (also referred to herein as "antibody mimetic molecules") can be identified by selecting or isolating target-binding variants from a library of binding molecules with artificially diversified binding sites. Diversified libraries can be generated using a completely random approach (e.g., error-prone polymerase chain reaction (PCR), exon shuffling, or directed evolution) or can be aided by art-recognized design strategies. For example, amino acid positions normally involved in the interaction of a binding site with its cognate target molecule can be randomized by inserting degenerate codons, trinucleotides, random peptides, or entire loops at corresponding positions in the nucleic acid encoding the binding site (see, e.g., U.S. Publication No. 20040132028). The location of the amino acid positions can be identified by examining the crystal structure of the binding site in a protein entity bearing the target molecule. Candidate positions for randomization include loops, planes, helices, and the binding cavity of the binding site. In certain embodiments, amino acids within the binding site that may be candidates for diversification can be identified by their homology with the immunoglobulin fold. For example, residues within the CDR-like loops of fibronectin can be randomized to generate a library of fibronectin-binding molecules (see, for example, Koide et al., J. Mol. Biol., 284:1141-1151 (1998)). Other parts of the binding site that may be randomized include planar surfaces. After randomization, the diversified library can be subjected to selection or screening procedures to obtain binding molecules with desired binding properties. For example, selection can be achieved by art-recognized methods, such as phage display, yeast display, or ribosome display.

[0114] For example, in some cases, non-antibody-based scaffolds contain binding sites derived from fibronectin-binding molecules. Fibronectin-binding molecules (e.g., molecules containing fibronectin type I, II, or III domains) exhibit CDR-like loops that, in contrast to immunoglobulins, do not rely on intrachain disulfide bonds. The FnIII loop contains a region that can be subjected to random mutation and directed evolution schemes that iteratively target binding, selection, and further mutation to develop useful therapeutic tools. Fibronectin-based "addressable" therapeutic binding molecules ("FATBIMs") can be developed to specifically bind to target antigens or epitopes. Methods for producing fibronectin-binding polypeptides are described, for example, in WO01 / 64942 and U.S. Patent Nos. 6,673,901, 6,703,199, 7,078,490, and 7,119,171.

[0115] As another example, in some cases, non-antibody-based scaffolds contain binding sites derived from affibodies. Affibodies are derived from the immunoglobulin-binding domain of Staphylococcus aureus protein A (SPA) (see, e.g., Nord et al., Nat. Biotechnol., 15:772-777 (1997)). Affibodies are antibody mimics with unique binding sites that bind to specific targets. Affibodies can be small (e.g., consisting of three alpha helices with 58 amino acids and a molar mass of approximately 6 kDa), have an inactive form (no Fc function), and have been successfully tested in humans as targeting moieties. Affibody binding sites can be synthesized by mutagenizing an SPA-related protein (e.g., protein Z) derived from a domain (e.g., domain B) of SPA and selecting for mutant SPA-related polypeptides with binding affinity for the target antigen or epitope. Other methods for generating affibody binding sites are described in US Pat. Nos. 6,740,734 and 6,602,977 and WO 00 / 63243.

[0116] As another example, in some cases, non-antibody-based scaffolds contain binding sites derived from anticalins. Anticalins are antibody functional mimics derived from human lipocalins. Lipocalins are a family of naturally occurring binding proteins that bind and transport small hydrophobic molecules, such as steroids, bilins, retinoids, and lipids. The primary structure of anticalins is similar to wild-type lipocalins. The central element of this protein structure is a beta-barrel structure of eight antiparallel strands supporting four loops at its open ends. These loops form the natural binding site of lipocalins and can be remodeled in vitro by extensive amino acid substitutions, thus resulting in novel binding specificities. Anticalins have high affinity and specificity for their ligands and rapid binding kinetics, so their functional properties resemble those of antibodies. Anticalins are described, for example, in U.S. Patent No. 7,723,476.

[0117] As another example, in some cases, non-antibody-based scaffolds include binding sites from cysteine-rich polypeptides. The cysteine-rich domain, in some cases, does not form an alpha-helix, beta-sheet, or beta-barrel structure. In some cases, disulfide bonds facilitate folding of the domain into a three-dimensional structure. In some cases, the cysteine-rich domain has at least two disulfide bonds, e.g., at least three disulfide bonds. An exemplary cysteine-rich polypeptide is an A domain protein. An A domain (sometimes called a "complement-type repeat") contains approximately 30-50 or 30-65 amino acids. In some cases, the domain contains approximately 35-45 amino acids, and in some cases, approximately 40 amino acids. Within the 30-50 amino acids, approximately six cysteine residues are present. Of the six cysteines, disulfide bonds are typically found between the following cysteines: C1 and C3, C2 and C5, and C4 and C6. The A domain constitutes the ligand-binding portion. The cysteine residues in the domain are disulfide-linked to form a compact, stable, and functionally independent portion. Clusters of these repeats constitute the ligand-binding domain, and differential clustering can confer specificity with respect to ligand binding. Exemplary proteins containing A domains include, for example, complement components (e.g., C6, C7, C8, C9, and factor I), serine proteases (e.g., enteropeptidase, matriptase, and corin), transmembrane proteins (e.g., ST7, LRP3, LRP5, and LRP6), and endocytic receptors (e.g., sortilin-related receptor, LDL receptor, VLDLR, LRP1, LRP2, and ApoER2). Methods for generating A domain proteins of desired binding specificity are disclosed, for example, in WO02 / 088171 and WO04 / 044011.

[0118] As another example, in some cases, non-antibody-based scaffolds include binding sites derived from repeat proteins. Repeat proteins are proteins containing consecutive copies of small (e.g., about 20 to about 40 amino acid residues) structural units or repeats that stack to form a continuous domain. Repeat proteins can be modified to suit specific target binding sites by adjusting the number of repeats in the protein. Exemplary repeat proteins include designed ankyrin repeat proteins (i.e., DARPins) (see, e.g., Binz et al., Nat. Biotechnol., 22:575-582 (2004)) or leucine-rich repeat proteins (i.e., LRRPs) (see, e.g., Pancer et al., Nature, 430:174-180 (2004)). As another example, in some cases, non-antibody-based scaffolds include DARPins.

[0119] As used herein, the term "DARPin" refers to genetically engineered antibody-mimetic proteins that typically exhibit highly specific and high-affinity target protein binding. DARPins were originally derived from natural ankyrin proteins. In some cases, DARPins contain three, four, or five repeat motifs of ankyrin proteins. In some cases, ankyrin repeat units consist of 30 to 34 amino acid residues and function to mediate protein-protein interactions. In some cases, each ankyrin repeat exhibits a helix-turn-helix conformation, and a series of such tandem repeats packs into a nearly linear array to form a helix-turn-helix bundle connected by relatively flexible loops. In some cases, the globular structure of ankyrin repeat proteins is stabilized by intra- and inter-repeat hydrophobic and hydrogen-bonding interactions. The repetitive and elongated nature of ankyrin repeats provides the molecular basis for the unique characteristics of ankyrin repeat proteins in protein stability, folding and unfolding, and binding specificity. The molecular mass of a DARPin domain can range from approximately 14 or 18 kDa for four- or five-repeat DARPins, respectively. DARPins are described, for example, in U.S. Pat. No. 7,417,130. In some cases, the tertiary structure of ankyrin repeat units shares the characteristic of consisting of a beta-hairpin followed by two antiparallel alpha helices, terminating in a loop connecting the repeat unit to the next. Domains made of ankyrin repeat units can be formed by stacking the repeat units into an elongated, curved structure. LRRP binding sites, derived from part of the adaptive immune system of sea lampreys and other jawless fish, resemble antibodies in that they are formed by the recombination of a series of leucine-rich repeat genes during lymphocyte maturation. Methods for generating DARpin or LRRP binding sites are described in WO02 / 20565 and WO06 / 083275.

[0120] As another example, in some cases, the non-antibody-based scaffold comprises a binding site derived from a Src homology domain (e.g., an SH2 or SH3 domain), a PDZ domain, a beta-lactamase, a high-affinity protease inhibitor, or a small disulfide-bonded protein scaffold, e.g., a scorpion venom. Methods for generating binding sites from these molecules have been disclosed in the art, see, e.g., Panni et al., J. Biol. Chem., 277:21666-21674 (2002); Schneider et al., Nat. Biotechnol., 17:170-175 (1999); Legendre et al., Protein Sci., 11:1506-1518 (2002); Stoop et al., Nat. Biotechnol., 21:1063-1068 (2003); and Vita et al., PNAS, 92:6404-6408 (1995). Still other binding sites may be derived from a binding domain selected from the group consisting of an EGF-like domain, a kringle-domain, a PAN domain, a Gla domain, an SRCR domain, a Kunitz / bovine pancreatic trypsin inhibitor domain, a Kazal-type serine protease inhibitor domain, a trefoil (P-type) domain, a von Willebrand factor type C domain, an anaphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat, an LDL-receptor class A domain, a sushi domain, a link domain, a thrombospondin type I domain, an immunoglobulin-like domain, a C-type lectin domain, a MAM domain, a von Willebrand factor type A domain, a somatomedin B domain, a WAP-type four disulfide core domain, an F5 / 8 C-type domain, a hemopexin domain, a laminin-type EGF-like domain, a C2 domain, a binding domain derived from tetranectin in its monomeric or trimeric form, and other such domains known to those skilled in the art, as well as derivatives and / or variants thereof.Exemplary non-antibody-based scaffolds, and methods for making the same, can also be found in Stemmer et al., "Protein scaffolds and uses thereof," U.S. Patent Publication No. 20060234299 (October 19, 2006) and Hey, et al., Artificial, Non-Antibody Binding Proteins for Pharmaceutical and Industrial Applications, TRENDS in Biotechnology, Vol. 23, No. 10, Table 2 and pp. 514-522 (October 2005).

[0121] As another example, in some cases, non-antibody-based scaffolds contain Kunitz domains. The term "Kunitz domain" as used herein refers to a conserved protein domain that inhibits certain proteases, e.g., serine proteases. Kunitz domains are relatively small, typically about 50-60 amino acids in length, with a molecular weight of about 6 kDa. Kunitz domains typically feature an arrangement of two, four, six, or eight or more disulfide bonds that carry a basic charge and contribute to the compact and stable nature of the folded peptide. For example, many Kunitz domains have six conserved cysteine residues that form three disulfide bonds. The disulfide-rich α / β fold of Kunitz domains can contain two, three (typically), or four or more disulfide bonds.

[0122] Kunitz domains have a pear-shaped structure containing a stabilized reactive site region characterized by, for example, three disulfide bonds and a P1 residue that is a key determinant of tight confirmation. These inhibitors competitively block the access of target proteins (e.g., serine proteases) to their physiologically relevant macromolecular substrates through the insertion of the P1 residue into the active site cleft. The P1 residue in the proteinase inhibitory loop provides a key specificity determinant and dictates much of the inhibitory activity that a particular Kunitz protein possesses against a targeted proteinase. In general, the N-terminal side of the reactive site (P) is energetically more important than the C-terminal side of P'. In most cases, lysine or arginine occupies the P1 position, inhibiting proteinases that cleave adjacent to those residues in the protein substrate. Other residues, particularly those in the inhibitor loop region, contribute to the strength of binding. Typically, approximately 10-12 amino acid residues in the target protein and 20-25 residues in the proteinase directly contact each other to form a stable proteinase inhibitor entity, providing a buried region of approximately 600-900 Å. By modifying residues in the P site and surrounding residues, Kunitz domains can be engineered to target proteins of choice. Kunitz domains are described, for example, in U.S. Patent No. 6,057,287.

[0123] As another example, in some cases, the non-antibody-based scaffold is an affilin. Affilins are small, antibody-mimetic proteins designed for specific affinities to proteins and small molecules. New affilins can be very rapidly selected from two libraries, each based on a different human-derived scaffold protein. Affilins do not display any structural homology to immunoglobulin proteins. There are two commonly used affilin scaffolds: one is a gamma-crystal human eye lens structural protein, and the other is a "ubiquitin" superfamily protein. Both human scaffolds are very small, exhibit high temperature stability, and are largely resistant to pH changes and denaturing agents. This high stability is primarily due to the extensive beta-sheet structure of these proteins. Examples of gamma-crystal-derived proteins are described in WO200104144, and examples of "ubiquitin-like" proteins are described in WO2004106368.

[0124] As another example, in some cases, non-antibody-based scaffolds are avimers. Avimers are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display to generate multidomain proteins with binding and inhibitory properties. Linking multiple independent binding domains has been shown to create avidity, resulting in improved affinity and specificity compared to traditional single-epitope binding proteins. In certain embodiments, avimers consist of two or more peptide sequences of 30-35 amino acids, each linked by a spacer region peptide. These individual sequences are derived from the A domains of various membrane receptors and have a rigid structure stabilized by disulfide bonds and calcium. Each A domain can bind to a specific epitope of the target protein. Combining domains that bind different epitopes of the same protein increases affinity for that protein (hence the name), a process known as avidity. Avimers with subnanomolar affinities have been obtained for a variety of targets. Alternatively, the domains can be directed against epitopes on different target proteins. Further information regarding avimers can be found in U.S. Patent Application Publication Nos. 2006 / 0286603, 2006 / 0234299, 2006 / 0223114, 2006 / 0177831, 2006 / 0008844, 2005 / 0221384, 2005 / 0164301, 2005 / 0089932, 2005 / 0053973, 2005 / 0048512, and 2004 / 0175756.

[0125] Suitable targets for non-antibody-based scaffolds include any of the antigens mentioned above that can be bound by antibody-based scaffolds.

[0126] In some cases, the target (second member of the specific binding pair) bound by the non-antibody-based scaffold is soluble. In some cases, the target is membrane-bound, e.g., in some cases, the target is present on the surface of a cell. In some cases, the target is immobilized on an insoluble support, which can include any of a variety of materials (e.g., polyethylene, polystyrene, polyvinylpyrrolidone, polycarbonate, nitrocellulose, etc.); the insoluble support can take a variety of forms, e.g., a plate, a tissue culture dish, a column, etc. In some cases, the target is present in the extracellular matrix (ECM) (e.g., the antigen is an ECM component). In some cases, the target is present in an artificial matrix. In some cases, the target is present in a cell-free environment.

[0127] cell adhesion molecules In some cases, the first member of the specific binding pair is a cell adhesion molecule (CAM), i.e., a polypeptide that binds to a component of the extracellular matrix (ECM) or to a cell surface molecule. For example, in some cases, the first member of the specific binding pair is an extracellular region of a CAM. In some cases, the CAM is a calcium-independent adhesion molecule; for example, in some cases, the CAM is an immunoglobulin superfamily CAM. In some cases, the CAM is a calcium-dependent adhesion molecule; for example, the CAM is an integrin, cadherin, or selectin. In some cases, the first member of the specific binding pair is an integrin. In some cases, the first member of the specific binding pair is a cadherin, such as E-cadherin, P-cadherin, N-cadherin, R-cadherin, M-cadherin, etc. In some cases, the first member of the specific binding pair is a selectin, such as E-selectin, L-selectin, or P-selectin. A binding fragment of a CAM can be used as the first member of a specific binding pair.

[0128] When the first member of the specific binding pair is a CAM, the second member of the specific binding pair is a component of the ECM or a cell surface molecule that binds to the CAM. For example, when the first member of the specific binding pair is an integrin, the second member of the specific binding pair is a component of collagen, fibrinogen, fibronectin, or vitronectin. As another example, when the first member of the specific binding pair is a cadherin, the second member of the specific binding pair is a cell surface antigen bound by the cadherin. As another example, when the first member of the specific binding pair is a selectin, the second member of the specific binding pair is a fucosylated carbohydrate.

[0129] Ligand In some cases, the first member of the specific binding pair is a ligand for the receptor. Ligands include polypeptides, nucleic acids, glycoproteins, small molecules, carbohydrates, lipids, glycolipids, lipoproteins, lipopolysaccharides, etc. In some cases, the ligand is soluble.

[0130] Ligands include, but are not limited to, cytokines (e.g., IL-13, etc.); growth factors (e.g., heregulin; vascular endothelial growth factor (VEGF); etc.); peptide hormones; integrin-binding peptides (e.g., peptides containing the sequence Arg-Gly-Asp); N-glycans; and the like.

[0131] When the member of the specific binding pair in the chimeric Notch receptor polypeptide of the present disclosure is a ligand, the chimeric Notch receptor polypeptide can be activated in the presence of a second member of the specific binding pair, where the second member of the specific binding pair is a receptor for the ligand. For example, when the ligand is VEGF, the second member of the specific binding pair can be a VEGF receptor, including a soluble VEGF receptor. Alternatively, the first member of the specific binding pair can be a VEGF receptor; the first member of the specific binding pair can be VEGF. As another example, when the ligand is heregulin, the second member of the specific binding pair can be Her2.

[0132] When the first member of the specific binding pair is a ligand, the second member of the specific binding pair is a molecule that binds to the ligand, e.g., the second member of the specific binding pair is an antibody that specifically binds to the ligand, a receptor for the ligand, etc.

[0133] When the first member of the specific binding pair is a ligand, in some cases, the second member of the specific binding pair (the molecule that binds to the ligand) is soluble. In some cases, the second member of the specific binding pair is membrane-bound, e.g., in some cases, the second member of the specific binding pair is present on the surface of a cell. In some cases, the second member of the specific binding pair is immobilized on an insoluble support, which can include any of a variety of materials (e.g., polyethylene, polystyrene, polyvinylpyrrolidone, polycarbonate, nitrocellulose, etc.); the insoluble support can take a variety of forms, e.g., a plate, a tissue culture dish, a column, etc. In some cases, the second member of the specific binding pair is present in a cell-free environment.

[0134] antigen In some cases, the first member of the specific binding pair is an antigen to which the antibody specifically binds. The antigen can be any antigen, e.g., a naturally occurring (endogenous) antigen; a synthetic (e.g., modified so that it is no longer identical to a naturally occurring antigen; modified from its native state; etc.) antigen; etc.

[0135] When the member of a specific binding pair in a chimeric Notch receptor polypeptide of the present disclosure is an antigen, the chimeric Notch receptor polypeptide can be activated in the presence of a second member of the specific binding pair, where the second member of the specific binding pair is an antibody (antibody-based recognition scaffold) that binds to the antigen.

[0136] In some cases, the antigen is a disease-associated antigen, e.g., a cancer-associated antigen, an autoimmune disease-associated antigen, a pathogen-associated antigen, an inflammation-associated antigen, etc.

[0137] For example, where the second member of the specific binding pair is an antibody specific for a cancer-associated antigen, the antigen can be a cancer-associated antigen, including, for example, CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, CA125, MUC-1, prostate-specific membrane antigen (PSMA), CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight melanoma-associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, and the like. Cancer-associated antigens include, for example, 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, and CD44. v6, CD51, CD52, CD56, CD74, CD80, CEA, CNTO888, CTLA-4, DRS, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2 / neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgG1, L1-CAM, IL-13, IL-6, insulin-like growth factor I receptor, integrin α5β1, integrin αvβ3, MORAb-009, MS4A1, MUC1, mucin CanAg, N-glycolylneuraminic acid, NPC-1C, PDGF-Rα, PDL192, phosphatidylserine, prostate carcinoma cells, RANKL, RON, ROR1, SCH Also included are 900105, SDC1, SLAMF7, TAG-72, tenascin C, TGF beta 2, TGF-β, TRAIL-R1, TRAIL-R2, tumor antigen CTAA16.88, VEGF-A, VEGFR-1, VEGFR2, and vimentin.

[0138] The antigen may be associated with an inflammatory disease. Non-limiting examples of antigens associated with an inflammatory disease include, for example, AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (alpha chain of the IL-2 receptor), CD3, CD4, CD5, IFN-α, IFN-γ, IgE, IgE These include the Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin α4, integrin α4β7, LFA-1 (CD11a), myostatin, OX-40, scleroscin, SOST, TGF beta 1, TNF-α, and VEGF-A.

[0139] When the first member of the specific binding pair is an antigen, the second member of the specific binding pair can be an antibody-based scaffold (e.g., an antibody) or a non-antibody-based scaffold. In some cases, the second member of the specific binding pair is present on the surface of a cell. In some cases, the second member of the specific binding pair is immobilized on an insoluble support. In some cases, the second member of the specific binding pair is soluble. In some cases, the second member of the specific binding pair is present in an extracellular environment (e.g., an extracellular matrix). In some cases, the second member of the specific binding pair is present in an artificial matrix. In some cases, the second member of the specific binding pair is present in an acellular environment.

[0140] Targeting non-antibody-based recognition scaffolds In some cases, the first member of the specific binding pair is a target of the non-antibody-based scaffold, including, for example, polypeptides, nucleic acids, glycoproteins, small molecules, carbohydrates, lipids, glycolipids, lipoproteins, lipopolysaccharides, etc.

[0141] When the first member of the specific binding pair is the target of the non-antibody-based scaffold, the second member of the specific binding pair is a non-antibody-based scaffold.

[0142] receptor In some cases, the first member of the specific binding pair is a receptor. In some cases, the receptor is a growth factor receptor. In some cases, the receptor is a cytokine receptor. In some cases, the receptor is a cell surface receptor that binds to a co-receptor on a cell. In some cases, the receptor is a neurotransmitter receptor. In some cases, the receptor binds to an extracellular matrix component. In some cases, the receptor is an immunoglobulin Fc receptor.

[0143] Suitable receptors include growth factor receptors (e.g., VEGF receptors); killer cell lectin-like receptor subfamily K, member 1 (NKG2D) polypeptides (receptors for MICA, MICB, and ULB6); cytokine receptors (e.g., IL-13 receptor; IL-2 receptor; etc.); epidermal growth factor (EGF) receptor; Her2; CD27; natural cytotoxicity receptors (NCRs) (e.g., NKP30 (NCR3 / CD337) polypeptides (receptors for HLA-B associated transcript 3 (BAT3) and B7-H6); etc.); T cell antigen receptors; dihydrofolate receptors; chimeric cytokine receptors; Fc receptors; extracellular matrix receptors (e.g., integrins); cell adhesion receptors (e.g., cadherins); immunomodulatory receptors, including both positive co-receptors (e.g., CD28) and negative (immunosuppressive) co-receptors (e.g., PD1); cytokine receptors; and receptors for immunomodulatory molecules (e.g., TGFβ). In some cases, the receptor is shortened compared to the wild-type receptor.

[0144] When the first member of the specific binding pair is a receptor, the second member of the specific binding pair is a target of the receptor, in which case the target can be a ligand for the receptor or a co-receptor. In some cases, the second member of the specific binding pair is present on the surface of a cell. In some cases, the second member of the specific binding pair is immobilized on an insoluble support. In some cases, the second member of the specific binding pair is soluble. In some cases, the second member of the specific binding pair is present in an extracellular environment (e.g., an extracellular matrix). In some cases, the second member of the specific binding pair is present in an artificial matrix. In some cases, the second member of the specific binding pair is present in a cell-free environment.

[0145] Notch receptor polypeptides As described above, chimeric Notch receptor polypeptides of the present disclosure include Notch receptor polypeptides having a length of between 50 amino acids and 1000 amino acids and containing one or more ligand-inducible proteolytic cleavage sites.

[0146] In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure is between 50 amino acids (aa) and 1000 aa, e.g., between 50 aa and 75 aa, between 75 aa and 100 aa, between 100 aa and 150 aa, between 150 aa and 200 aa, between 200 aa and 250 aa, between 250 aa and 300 aa, between 300 aa and 350 aa, between 350 aa and 400 aa, In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure has a length of 300 aa to 400 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure has a length of 300 aa to 350 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure has a length of 300 aa to 325 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure has a length of 350 aa to 400 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure has a length of 750 aa to 850 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure has a length of 50 aa to 75 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure has a length of 310 aa to 320 aa, e.g., 310 aa, 311 aa, 312 aa, 313 aa, 314 aa, 315 aa, 316 aa, 317 aa, 318 aa, 319 aa, or 320 aa. In some cases, a Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure has a length of 315 aa.In some cases, a Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure has a length of 360 aa to 370 aa, e.g., 360 aa, 361 aa, 362 aa, 363 aa, 364 aa, 365 aa, 366 aa, 367 aa, 368 aa, 369 aa, or 370 aa. In some cases, a Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure has a length of 367 aa.

[0147] Notch receptor polypeptides containing a TM domain In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure has the following amino acid sequence: TIFF0007719836000004.tif11149; wherein the TM domain is underlined; the Notch receptor polypeptide comprises an S2 proteolytic cleavage site and an S3 proteolytic cleavage site; and the Notch receptor polypeptide has a length of 50 amino acids (aa) to 65 aa, e.g., 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 aa. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure has the following amino acid sequence: The Notch receptor polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000005.tif11149; wherein the TM domain is underlined; the Notch receptor polypeptide comprises an S2 proteolytic cleavage site and an S3 proteolytic cleavage site; and the Notch receptor polypeptide has a length of 56 amino acids.

[0148] Notch receptor polypeptides comprising an LNR segment, an HD-N segment, an HD-C segment, and a TM domain In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, i) an LNR-A segment; ii) an LNR-B segment; iii) an LNR-C segment; iv) an HD-N segment, v) an HD-C segment; and vi) a TM domain. The LNR-A segment, LNR-B segment, and LNR-C segment can be collectively referred to as "LNR segments." Such a Notch receptor polypeptide is shown schematically in Figure 4A.

[0149] An LNR segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1442 to 1562 of the amino acid sequence shown in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 90 amino acids to 150 amino acids, e.g., 90 amino acids (aa) to 100 aa, 100 aa to 110 aa, 110 aa to 120 aa, 120 aa to 130 aa, 130 aa to 140 aa, or 140 aa to 150 aa. In some cases, the LNR segment comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1442 to 1562 of the amino acid sequence shown in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and has a length of 115 aa to 125 aa, e.g., 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125 aa.

[0150] The LNR segment has the following amino acid sequence: It can include an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000006.tif17151; and can have a length of 118 to 122 amino acids (e.g., 118, 119, 120, 121, or 122 amino acids).

[0151] The HD-N segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1563-1664 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and can have a length of 90 amino acids (aa) to 110 aa, e.g., 90 aa to 95 aa, 95 aa to 100 aa, 100 aa to 105 aa, or 105 aa to 110 aa. In some cases, the HD-N segment comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1563-1664 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and has a length of 95 aa to 105 aa, e.g., 95, 96, 98, 98, 99, 100, 101, 102, 103, 104, or 105 aa.

[0152] The HD-C segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1665-1733 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and can have a length of 60 amino acids (aa) to 80 aa, e.g., 60 aa to 65 aa, 65 aa to 70 aa, 70 aa to 75 aa, or 75 aa to 80 aa. In some cases, the HD-C segment comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1665 to 1733 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and has a length of 65 amino acids to 75 amino acids, e.g., 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 amino acids.

[0153] The HD segments (HD-N and HD-C) have the following amino acid sequences: It may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000007.tif17150; and may have a length of 150, 151, 152, 153, or 154 amino acids.

[0154] The transmembrane segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1736 to 1756 of the amino acid sequence shown in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 15 amino acids (aa) to 25 amino acids, e.g., 15, 16, 17, 18, 29, 20, 21, 22, 23, 24, or 25 amino acids.

[0155] The transmembrane segment has the following amino acid sequence: It may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000008.tif4128; and may have a length of 21, 22, 23, 24, or 25 amino acids.

[0156] In some cases, the Notch receptor polypeptide has a length of about 310 amino acids (aa) to about 320 aa (e.g., 310 aa, 311 aa, 312 aa, 313 aa, 314 aa, 315 aa, 316 aa, 317 aa, 318 aa, 319 aa, or 320 aa) and comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1442 to 1756 of the amino acid sequence depicted in FIG. 2A , or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are depicted in FIGS. 2B-2G.

[0157] In some cases, the Notch receptor polypeptide has the following amino acid sequence: It comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000009.tif37151; and has a length of 300 amino acids to 310 amino acids (e.g., 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, or 310 amino acids).

[0158] In some cases, the Notch receptor polypeptide has the following amino acid sequence: It comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000010.tif44151; and has a length of 350 amino acids to 370 amino acids (e.g., 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, or 370 amino acids).

[0159] Notch receptor polypeptides containing a single EGF repeat, an LNR segment, an HD-N segment, an HD-C segment, and a TM domain In some cases, a Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, i) a single EGF repeat; ii) an LNR segment; iii) an HD-N segment; iv) an HD-C segment; and v) a TM domain. Such a Notch receptor polypeptide is shown schematically in Figure 4B.

[0160] An EGF repeat can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1390 to 1430 of the amino acid sequence shown in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 35 amino acids (aa) to 45 aa (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 aa).

[0161] The EGF repeat has the following sequence: It can include an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000011.tif4128; and can have a length of 35 amino acids to 40 amino acids (e.g., 35, 36, 37, 38, 39, or 40 amino acids).

[0162] An LNR segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1442 to 1562 of the amino acid sequence shown in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 90 amino acids to 150 amino acids, e.g., 90 amino acids (aa) to 100 aa, 100 aa to 110 aa, 110 aa to 120 aa, 120 aa to 130 aa, 130 aa to 140 aa, or 140 aa to 150 aa. In some cases, the LNR segment comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1442 to 1562 of the amino acid sequence shown in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and has a length of 115 aa to 125 aa, e.g., 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125 aa.

[0163] The LNR segment has the following amino acid sequence: It can include an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000012.tif17151; and can have a length of 118 to 122 amino acids (e.g., 118, 119, 120, 121, or 122 amino acids).

[0164] The HD-N segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1563-1664 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and can have a length of 90 amino acids (aa) to 110 aa, e.g., 90 aa to 95 aa, 95 aa to 100 aa, 100 aa to 105 aa, or 105 aa to 110 aa. In some cases, the HD-N segment comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1563-1664 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and has a length of 95 aa to 105 aa, e.g., 95, 96, 98, 98, 99, 100, 101, 102, 103, 104, or 105 aa.

[0165] The HD-C segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1665-1733 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and can have a length of 60 amino acids (aa) to 80 aa, e.g., 60 aa to 65 aa, 65 aa to 70 aa, 70 aa to 75 aa, or 75 aa to 80 aa. In some cases, the HD-C segment comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1665 to 1733 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and has a length of 65 amino acids to 75 amino acids, e.g., 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 amino acids.

[0166] The HD segments (HD-N and HD-C) have the following amino acid sequences: It may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000013.tif17150; and may have a length of 150, 151, 152, 153, or 154 amino acids.

[0167] The transmembrane segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1736 to 1756 of the amino acid sequence shown in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 15 amino acids (aa) to 25 amino acids, e.g., 15, 16, 17, 18, 29, 20, 21, 22, 23, 24, or 25 amino acids.

[0168] The transmembrane segment has the following amino acid sequence: It may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000014.tif4128; and may have a length of 21, 22, 23, 24, or 25 amino acids.

[0169] In some cases, the Notch receptor polypeptide has a length of about 360 amino acids (aa) to about 375 aa (e.g., 360 aa, 361 aa, 362 aa, 363 aa, 364 aa, 365 aa, 366 aa, 367 aa, 368 aa, 369 aa, 370 aa, 371 aa, 372 aa, 373 aa, 374 aa, or 375 aa) and comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1390 to 1756 of the amino acid sequence depicted in FIG. 2A , or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are depicted in FIGS. 2B-2G.

[0170] In some cases, a Notch receptor polypeptide comprises a synthetic linker. For example, in some cases, a Notch receptor polypeptide comprises, in order from N-terminus to C-terminus: i) a synthetic linker; ii) an EGF repeat; iii) an LNR segment; iv) an HD-N segment, v) an HD-C segment; and vi) a TM domain. Such a Notch receptor polypeptide is shown schematically in Figure 4C.

[0171] The synthetic linker can have a length of about 10 amino acids (aa) to about 200 aa, for example, 10 aa to 25 aa, 25 aa to 50 aa, 50 aa to 75 aa, 75 aa to 100 aa, 100 aa to 125 aa, 125 aa to 150 aa, 150 aa to 175 aa, or 175 aa to 200 aa. The synthetic linker can have a length of 10 aa to 30 aa, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 aa. The synthetic linker can have a length of 30 aa to 50 aa, for example, 30 aa to 35 aa, 35 aa to 40 aa, 40 aa to 45 aa, or 45 aa to 50 aa.

[0172] In some examples, the synthetic linker, as described herein, may comprise an extracellular protein structural domain or a portion thereof. Suitable extracellular protein structural domains for use as synthetic linkers include, but are not limited to, Ig-like extracellular structural domains, Fc extracellular structural domains, fibronectin extracellular structural domains, etc. In some examples, the synthetic linker may comprise multiple extracellular protein structural domains, where multiple may comprise multiple identical domains or multiple different domains.

[0173] Notch receptor polypeptides containing 2 to 11 EGF repeats, an LNR segment, an HD-N segment, an HD-C segment, and a TM domain In some cases, a Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, i) 2 to 11 EGF repeats; ii) an LNR segment; iii) an HD-N segment; iv) an HD-C segment; and v) a TM domain. Such a Notch receptor polypeptide is shown schematically in Figure 4D.

[0174] In some cases, a Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, i) two EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases, a Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, i) three EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases, a Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, i) four EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, i) five EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, i) six EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, i) seven EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases, the Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, i) eight EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain.In some cases, a Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, i) nine EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases, a Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, i) ten EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain. In some cases, a Notch receptor polypeptide present in a chimeric Notch receptor polypeptide of the present disclosure comprises, in order from N-terminus to C-terminus, i) eleven EGF repeats; ii) an LNR segment; iii) an HD-N segment, iv) an HD-C segment; and v) a TM domain.

[0175] An EGF repeat can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1390 to 1430 of the amino acid sequence shown in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 35 amino acids (aa) to 45 aa (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 aa).

[0176] The EGF repeats are amino acids 869 to 905 of the amino acid sequence shown in Figure 2A. TIFF0007719836000015.tif4128, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 35 amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa).

[0177] The EGF repeats are amino acids 907 to 943 of the amino acid sequence shown in Figure 2A. TIFF0007719836000016.tif4128, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 35 amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa).

[0178] The EGF repeats are amino acids 945 to 981 of the amino acid sequence shown in Figure 2A. TIFF0007719836000017.tif4128, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 35 amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa).

[0179] The EGF repeats are amino acids 988 to 1019 of the amino acid sequence shown in Figure 2A. TIFF0007719836000018.tif4128, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 30 amino acids (aa) to 35 aa (e.g., 30, 31, 32, 33, 34, or 35 aa).

[0180] The EGF repeats are amino acids 1021 to 1057 of the amino acid sequence shown in Figure 2A. TIFF0007719836000019.tif4128, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 35 amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa).

[0181] The EGF repeats are amino acids 1064 to 1090 of the amino acid sequence shown in Figure 2A. TIFF0007719836000020.tif4128, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 25 amino acids (aa) to 30 aa, e.g., 25, 26, 27, 28, 29, or 30 aa.

[0182] The EGF repeats are amino acids 1146 to 1180 of the amino acid sequence shown in Figure 2A. TIFF0007719836000021.tif4128, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 35 amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa).

[0183] The EGF repeats are amino acids 1184 to 1219 of the amino acid sequence shown in Figure 2A. TIFF0007719836000022.tif4128, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 35 amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa).

[0184] The EGF repeats are amino acids 1238 to 1265 of the amino acid sequence shown in Figure 2A. TIFF0007719836000023.tif4128, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 25 amino acids (aa) to 30 aa, e.g., 25, 26, 27, 28, 29, or 30 aa.

[0185] The EGF repeats are amino acids 1267 to 1305 of the amino acid sequence shown in Figure 2A. TIFF0007719836000024.tif4133, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 35 amino acids to about 40 amino acids (aa) (e.g., 35, 36, 37, 38, 39, or 40 aa).

[0186] The EGF repeat has the following sequence: It can include an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000025.tif4128; and can have a length of 35 amino acids to 40 amino acids (e.g., 35, 36, 37, 38, 39, or 40 amino acids).

[0187] An LNR segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1442 to 1562 of the amino acid sequence shown in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 90 amino acids to 150 amino acids, e.g., 90 amino acids (aa) to 100 aa, 100 aa to 110 aa, 110 aa to 120 aa, 120 aa to 130 aa, 130 aa to 140 aa, or 140 aa to 150 aa. In some cases, the LNR segment comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1442 to 1562 of the amino acid sequence shown in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and has a length of 115 aa to 125 aa, e.g., 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125 aa.

[0188] The LNR segment has the following amino acid sequence: It can include an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000026.tif17151; and can have a length of 118 to 122 amino acids (e.g., 118, 119, 120, 121, or 122 amino acids).

[0189] The HD-N segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1563-1664 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and can have a length of 90 amino acids (aa) to 110 aa, e.g., 90 aa to 95 aa, 95 aa to 100 aa, 100 aa to 105 aa, or 105 aa to 110 aa. In some cases, the HD-N segment comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1563-1664 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and has a length of 95 aa to 105 aa, e.g., 95, 96, 98, 98, 99, 100, 101, 102, 103, 104, or 105 aa.

[0190] The HD-C segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1665-1733 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and can have a length of 60 amino acids (aa) to 80 aa, e.g., 60 aa to 65 aa, 65 aa to 70 aa, 70 aa to 75 aa, or 75 aa to 80 aa. In some cases, the HD-C segment comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1665 to 1733 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and has a length of 65 amino acids to 75 amino acids, e.g., 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 amino acids.

[0191] The HD segments (HD-N and HD-C) have the following amino acid sequences: It may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000027.tif17150; and may have a length of 150, 151, 152, 153, or 154 amino acids.

[0192] The transmembrane segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1736 to 1756 of the amino acid sequence shown in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 15 amino acids (aa) to 25 amino acids, e.g., 15, 16, 17, 18, 29, 20, 21, 22, 23, 24, or 25 amino acids.

[0193] The transmembrane segment has the following amino acid sequence: It may comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000028.tif4128; and may have a length of 21, 22, 23, 24, or 25 amino acids.

[0194] In some cases, the Notch receptor polypeptide has a length of from about 490 amino acids (aa) to about 900 aa and comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to i) amino acids 1267-1756; ii) 1238-1756; iii) 1184-1756; iv) 1146-1756; v) 1064-1756; vi) 1021-1756; vii) 988-1756; viii) 945-1756; ix) 907-1756; or x) 869-1756 of the amino acid sequence shown in FIG. 2A , or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G.

[0195] In some cases, a Notch receptor polypeptide comprises a synthetic linker. For example, in some cases, a Notch receptor polypeptide comprises, from N-terminus to C-terminus, i) 2 to 11 EGF repeats; ii) a synthetic linker; iii) an LNR segment; iv) an HD-N segment; v) an HD-C segment; and vi) a TM domain. Such a Notch receptor polypeptide is shown schematically in Figure 4E.

[0196] The synthetic linker can have a length of about 10 amino acids (aa) to about 200 aa, for example, 10 aa to 25 aa, 25 aa to 50 aa, 50 aa to 75 aa, 75 aa to 100 aa, 100 aa to 125 aa, 125 aa to 150 aa, 150 aa to 175 aa, or 175 aa to 200 aa. The synthetic linker can have a length of 10 aa to 30 aa, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 aa. The synthetic linker can have a length of 30 aa to 50 aa, for example, 30 aa to 35 aa, 35 aa to 40 aa, 40 aa to 45 aa, or 45 aa to 50 aa.

[0197] Notch receptor polypeptide comprising an HD-C segment and a TM domain In some cases, the Notch receptor polypeptide comprises, in order from N-terminus to C-terminus, i) an HD-C segment; and ii) a TM domain, wherein the Notch receptor polypeptide does not comprise an LNR segment. In some cases, the LNR segment is replaced with a heterologous polypeptide. Such a Notch receptor polypeptide is shown schematically in Figure 4F.

[0198] The HD-C segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1665-1733 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and can have a length of 60 amino acids (aa) to 80 aa, e.g., 60 aa to 65 aa, 65 aa to 70 aa, 70 aa to 75 aa, or 75 aa to 80 aa. In some cases, the HD-C segment comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1665 to 1733 of the amino acid sequence shown in FIG. 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in FIGS. 2B-2G; and has a length of 65 amino acids to 75 amino acids, e.g., 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 amino acids.

[0199] The transmembrane segment can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1736 to 1756 of the amino acid sequence shown in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are shown in Figures 2B-2G; and can have a length of 15 amino acids (aa) to 25 amino acids, e.g., 15, 16, 17, 18, 29, 20, 21, 22, 23, 24, or 25 amino acids.

[0200] The transmembrane segment has the following amino acid sequence: It may comprise amino acids having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000029.tif4128; and may have a length of 21, 22, 23, 24, or 25 amino acids.

[0201] In some cases, the Notch receptor polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to amino acids 1665-1756 of the amino acid sequence depicted in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are depicted in Figures 2B-2G; having a length of 85 amino acids (aa) to 95 aa (e.g., 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 aa).

[0202] In some cases, the Notch receptor polypeptide comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 1665-1756 of the amino acid sequence depicted in Figure 2A, or a corresponding segment of another Notch receptor polypeptide, where examples of corresponding segments are depicted in Figures 2B-2G; including a heterologous polypeptide fused in-frame at the N-terminus of the Notch receptor polypeptide.

[0203] Ligand-induced proteolytic cleavage site As described above, the chimeric Notch receptor polypeptides of the present disclosure include a Notch receptor polypeptide having a length of 50 to 1000 amino acids and comprising one or more ligand-inducible proteolytic cleavage sites. As discussed above, the chimeric Notch receptor polypeptides of the present disclosure include: a) an extracellular domain comprising a first member of a specific binding pair; b) a Notch receptor polypeptide having a length of 50 to 1000 amino acids and comprising one or more ligand-inducible proteolytic cleavage sites; and c) an intracellular domain, wherein binding of the first member of the specific binding pair to a second member of the specific binding pair induces cleavage of the Notch receptor polypeptide at the one or more ligand-inducible proteolytic cleavage sites, thereby releasing the intracellular domain. The second member of the specific binding pair ("ligand") can be present on the contacting (e.g., "transmitting") cell.

[0204] In some cases, a Notch receptor polypeptide comprises only one ligand-inducible proteolytic cleavage site. In some cases, a Notch receptor polypeptide comprises two ligand-inducible proteolytic cleavage sites. In some cases, a Notch receptor polypeptide comprises three ligand-inducible proteolytic cleavage sites. For simplicity, the ligand-inducible cleavage sites will be referred to herein as "S1," "S2," and "S3" ligand-inducible proteolytic cleavage sites.

[0205] In some cases, the Notch receptor polypeptide comprises an S1 ligand-inducible proteolytic cleavage site. The S1 ligand-inducible proteolytic cleavage site can be located between the HD-N segment and the HD-C segment. In some cases, the S1 ligand-inducible proteolytic cleavage site is a furin-like protease cleavage site. The furin-like protease cleavage site can have the canonical sequence Arg-X-(Arg / Lys)-Arg, where X is any amino acid; the protease cleaves immediately C-terminal to the canonical sequence. For example, in some cases, the amino acid sequence comprising the S1 ligand-inducible proteolytic cleavage site is the amino acid sequence TIFF0007719836000030.tif4128, in which case cleavage occurs between the "RE" sequences. As another example, an amino acid sequence comprising an S1 ligand-inducible proteolytic cleavage site can have the amino acid sequence TIFF0007719836000031.tif4128, where cleavage occurs between the "RE" sequences.

[0206] In some cases, the Notch receptor polypeptide comprises an S2 ligand-inducible proteolytic cleavage site. The S2 ligand-inducible proteolytic cleavage site can be located within the HD-C segment. In some cases, the S2 ligand-inducible proteolytic cleavage site is an ADAM-17 type protease cleavage site. The ADAM-17 type protease cleavage site can comprise an Ala-Val dipeptide sequence, where the enzyme cleaves between the Ala and Val. For example, in some cases, the amino acid sequence comprising the S2 ligand-inducible proteolytic cleavage site is the amino acid sequence TIFF0007719836000032.tif4128, in which case cleavage occurs between the "AV" sequences. As another example, the amino acid sequence comprising the S2 ligand-inducible proteolytic cleavage site can have the amino acid sequence TIFF0007719836000033.tif4128, where the cleavage occurs between the "AV" sequences.

[0207] In some cases, the Notch receptor polypeptide comprises an S3 ligand-inducible proteolytic cleavage site. The S3 ligand-inducible proteolytic cleavage site can be located within the TM domain. In some cases, the S3 ligand-inducible proteolytic cleavage site is a gamma-secretase (γ-secretase) cleavage site. The γ-secretase cleavage site can comprise a Gly-Val dipeptide sequence, where the enzyme cleaves between Gly and Val. For example, in some cases, the S3 ligand-inducible proteolytic cleavage site is the amino acid sequence TIFF0007719836000034.tif4128, in which case cleavage occurs between the "GV" sequences. In some cases, the S3 ligand-inducible proteolytic cleavage site has the amino acid sequence Contains TIFF0007719836000035.tif4128.

[0208] In some cases, the Notch receptor polypeptide lacks an S1 ligand-inducible proteolytic cleavage site. In some cases, the Notch receptor polypeptide lacks an S2 ligand-inducible proteolytic cleavage site. In some cases, the Notch receptor polypeptide lacks an S3 ligand-inducible proteolytic cleavage site. In some cases, the Notch receptor polypeptide lacks both an S1 ligand-inducible proteolytic cleavage site and an S2 ligand-inducible proteolytic cleavage site. In some cases, the Notch receptor polypeptide includes an S3 ligand-inducible proteolytic cleavage site; it lacks both an S1 ligand-inducible proteolytic cleavage site and an S2 ligand-inducible proteolytic cleavage site. An example is shown schematically in Figure 4G.

[0209] Intracellular domain As described above, the chimeric Notch receptor polypeptides of the present disclosure comprise an intracellular domain that is released following binding of the chimeric Notch receptor polypeptide to a second member of the specific binding pair, wherein binding of the chimeric Notch receptor polypeptide to the second member of the specific binding pair induces cleavage of the proteolytic cleavage site described above.

[0210] The intracellular domain comprises an amino acid sequence that is heterologous to the Notch receptor polypeptide, in other words, the intracellular domain comprises an amino acid sequence that does not naturally occur within the Notch receptor polypeptide.

[0211] The intracellular domain, when released from the chimeric Notch receptor polypeptide, provides an effector function, where effector functions include, for example, increased production of one or more cytokines by the cell; decreased production of one or more cytokines by the cell; increased or decreased production of a hormone by the cell; production of an antibody by the cell; a change in organelle activity; a change in polypeptide trafficking within the cell; a change in transcription of a target gene; a change in protein activity; a change in cellular behavior, e.g., cell death; cell proliferation; an effect on cell differentiation; an effect on cell survival; modulation of a cellular signaling response; etc. In some cases, the intracellular domain, when released from the chimeric Notch receptor polypeptide, provides a change in transcription of a target gene. In some cases, the intracellular domain, when released from the chimeric Notch receptor polypeptide, provides an increase in transcription of a target gene. In some cases, the intracellular domain, when released from the chimeric Notch receptor polypeptide, provides a decrease in target gene expression.

[0212] The intracellular domain can be any of a wide variety of polypeptides, including, but not limited to, transcriptional activators; transcriptional repressors; transcriptional co-activators; transcriptional co-repressors; DNA-binding polypeptides; RNA-binding polypeptides; translational regulatory polypeptides; hormones; cytokines; toxins; antibodies; chromatin regulators; suicide proteins; organelle-specific polypeptides (e.g., nuclear pore regulators, mitochondrial regulators, endoplasmic reticulum regulators, etc.); pro-apoptotic polypeptides; anti-apoptotic polypeptides; other polypeptides that promote cell death through other mechanisms; pro-growth polypeptides; anti-proliferative polypeptides; immune co-stimulatory polypeptides; site-specific nucleases; recombinases; inhibitory immunoreceptors; activating immunoreceptors; mutants of Cas9 and RNA-targeted nucleases; as well as DNA-recognition polypeptides; dominant-negative mutants of polypeptides; signal transduction polypeptides; receptor tyrosine kinases; non-receptor tyrosine kinases; polypeptides that promote differentiation; and the like.

[0213] In some cases, the intracellular domain comprises a signaling polypeptide. Suitable signaling polypeptides include, for example, STAT3 / 5, Akt, Myc, etc. In some cases, the signaling polypeptide is part of a PI3K / mTOR-, NFκB-, MAPK-, STAT-, FAK-, MYC, or TGF-β-mediated signaling pathway. In some cases, the signaling polypeptide is part of a Ras / Raf / Mek / Erk1 / 2, JAK / STAT3, or PI3K / Akt signaling pathway.

[0214] In some cases, the intracellular domain comprises a dominant-negative mutant of a polypeptide, for example, a dominant-negative mutant of a signal transduction polypeptide. Examples of dominant-negative mutants include, for example, a dominant-negative TGF-β receptor; a dominant-negative mutant of STAT3 that contains one or more mutations affecting the DNA-binding domain of STAT3, which functions as a dominant-negative mutant; and the like.

[0215] In some cases, the intracellular domain is an antibody-based or non-antibody-based scaffold that, when released from the chimeric Notch receptor polypeptide, blocks or alters an activity of a cell.

[0216] In some cases, the intracellular domain comprises an immunoreceptor, e.g., an activating immunoreceptor or an inhibitory immunoreceptor. A suitable activating immunoreceptor can comprise an immunoreceptor tyrosine-based activation motif (ITAM). The ITAM motif is YX1X2L / I, where X1 and X2 are independently any amino acid. A suitable intracellular signaling domain can be an ITAM motif-containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12; FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3Z (CD3 zeta); and CD79A (antigen receptor complex-associated protein alpha chain). As one non-limiting example, a suitable ITAM motif-containing polypeptide has the following amino acid sequence: TIFF0007719836000036.tif4128, where the ITAM motif is bolded and underlined. As another example, a suitable ITAM motif-containing polypeptide can include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000036.tif4128, where the ITAM motif is bolded and underlined. The polypeptide can comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000037.tif4128, where the ITAM motif is bold and underlined. The polypeptide can comprise an ITAM motif-containing portion of the full-length CD3 zeta amino acid sequence. As another example, a suitable ITAM motif-containing polypeptide has the following amino acid sequence: The amino acid sequence may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to any of TIFF0007719836000038.tif31157, where the ITAM motif is bold and underlined.

[0217] Intracellular signaling domains suitable for use in the chimeric Notch polypeptides of the present disclosure include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. The ITAM motif is YX1X2L / I, where X1 and X2 are independently any amino acid. In some cases, the intracellular signaling domain of a chimeric Notch polypeptide contains one, two, three, four, or five ITAM motifs. In some cases, the ITAM motif is repeated twice in the intracellular signaling domain, where the first and second instances of the ITAM motif are separated from each other by 6 to 8 amino acids, e.g., (YX1X2L / I)(X3). n (YX1X2L / I), where n is an integer from 6 to 8, and each of the 6-8X3 can be any amino acid. In some cases, the intracellular signaling domain of the chimeric Notch polypeptide comprises three ITAM motifs.

[0218] A suitable intracellular signaling domain can be an ITAM motif-containing portion derived from a polypeptide containing an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12; FCER1G (Fc epsilon receptor 1 gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3Z (CD3 zeta); and CD79A (antigen receptor complex-associated protein alpha chain).

[0219] In some cases, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX-activating protein 12; KAR-associated protein; TYRO protein tyrosine kinase binding protein; killer activating receptor-associated protein; killer activating receptor-associated protein; etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence (four isoforms): TIFF0007719836000039.tif64151, wherein the ITAM motif is bold and underlined.

[0220] Similarly, a suitable intracellular signaling domain polypeptide can comprise an ITAM motif-containing portion of the full-length DAP12 amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide can comprise the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000040.tif4128, where the ITAM motif is bold and underlined.

[0221] In some cases, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc-epsilon RI-gamma; fcR gamma; fceRI gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence: The amino acid sequence may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000041.tif11150, where the ITAM motif is bold and underlined.

[0222] Similarly, a suitable intracellular signaling domain polypeptide can comprise an ITAM motif-containing portion of the full-length FCER1G amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide can comprise the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000042.tif4128, where the ITAM motif is bold and underlined.

[0223] In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-delta; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain; etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence (two isoforms): The amino acid sequence may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 170 aa of any of TIFF0007719836000043.tif44151, where the ITAM motif is bold and underlined.

[0224] Similarly, a suitable intracellular signaling domain polypeptide can comprise an ITAM motif-containing portion of the full-length CD3 delta amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide can comprise the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000044.tif4128, where the ITAM motif is bold and underlined.

[0225] In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence: The ITAM motif may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to a contiguous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 205 aa of TIFF0007719836000045.tif25153, where the ITAM motif is bolded and underlined.

[0226] Similarly, a suitable intracellular signaling domain polypeptide can comprise an ITAM motif-containing portion of the full-length CD3 epsilon amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide can comprise the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000046.tif4128, where the ITAM motif is bold and underlined.

[0227] In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-gamma, T3G, gamma polypeptide (TiT3 complex), etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence: The ITAM motif may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 180 aa of TIFF0007719836000047.tif24151, where the ITAM motif is bolded and underlined.

[0228] Similarly, a suitable intracellular signaling domain polypeptide can comprise an ITAM motif-containing portion of the full-length CD3 gamma amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide can comprise the following amino acid sequence: The amino acid sequence may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000048.tif4128, where the ITAM motif is bold and underlined.

[0229] In some cases, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3 zeta, CD3H, CD3Q, T3Z, TCRZ, etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence (two isoforms): The ITAM motif may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to a contiguous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, or about 150 aa to about 160 aa, of any of TIFF0007719836000049.tif37161, where the ITAM motif is bolded and underlined.

[0230] Similarly, a suitable intracellular signaling domain polypeptide can comprise an ITAM motif-containing portion of the full-length CD3 zeta amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide can comprise the following amino acid sequence: TIFF0007719836000050.tif31151, wherein the ITAM motif is bold and underlined.

[0231] In some cases, the intracellular signaling domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig-alpha; membrane-associated immunoglobulin-associated protein; surface IgM-associated protein; etc.). For example, a suitable intracellular signaling domain polypeptide has the following amino acid sequence (two isoforms): The ITAM motifs may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100%, amino acid sequence identity to a contiguous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 150 aa, about 150 aa to about 200 aa, or about 200 aa to about 220 aa of any of TIFF0007719836000051.tif58151, where the ITAM motifs are bolded and underlined.

[0232] Similarly, a suitable intracellular signaling domain polypeptide can comprise an ITAM motif-containing portion of the full-length CD79A amino acid sequence. Thus, a suitable intracellular signaling domain polypeptide can comprise the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000052.tif4128, where the ITAM motif is bold and underlined.

[0233] DAP10 / CD28 Suitable intracellular signaling domains for use in the chimeric Notch polypeptides of the present disclosure include DAP10 / CD28-type signaling chains.

[0234] An example of a DAP10 signaling chain is the amino acid sequence: TIFF0007719836000053.tif4128. In some embodiments, a suitable intracellular signaling domain has the amino acid sequence It comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity to the entire length of TIFF0007719836000054.tif4128.

[0235] An example of a CD28 signaling chain is the amino acid sequence: TIFF0007719836000055.tif11151. In some embodiments, a suitable intracellular signaling domain has the amino acid sequence It comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity with the entire length of TIFF0007719836000056.tif11151.

[0236] ZAP70 Suitable intracellular signaling domains for use in chimeric Notch polypeptides of the present disclosure include ZAP70 polypeptides, such as those having the following amino acid sequence: These include polypeptides comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a contiguous stretch of about 300 amino acids to about 400 amino acids, about 400 amino acids to about 500 amino acids, or about 500 amino acids to 619 amino acids of TIFF0007719836000057.tif71151.

[0237] Costimulatory domains derived from receptors are suitable for use as the intracellular domain of the chimeric Notch polypeptides of the present disclosure. The costimulatory domain can be the intracellular portion of a transmembrane protein (i.e., the costimulatory domain can be derived from a transmembrane protein). Non-limiting examples of suitable costimulatory polypeptides include, but are not limited to, 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0238] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD28 (also known as Tp44). For example, a suitable costimulatory domain has the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000058.tif4142.

[0239] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein 4-1BB (also known as TNFRSF9; CD137; 4-1BB; CDw137; ILA; etc.). For example, a suitable costimulatory domain has the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000059.tif4133.

[0240] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein OX-40 (also known as TNFRSF4, RP5-902P8.3, ACT35, CD134, OX40, TXGP1L). For example, a suitable costimulatory domain has the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000060.tif4128.

[0241] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein BTLA (also known as BTLA1 and CD272). For example, a suitable costimulatory domain has the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000061.tif17150.

[0242] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD27 (also known as S152, T14, TNFRSF7, and Tp55). For example, a suitable costimulatory domain has the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000062.tif4148.

[0243] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein CD30 (also known as TNFRSF8, D1S166E, and Ki-1). For example, a suitable costimulatory domain has the following amino acid sequence: The amino acid sequence may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to a contiguous stretch of about 100 amino acids to about 110 amino acids (aa), about 110 aa to about 115 aa, about 115 aa to about 120 aa, about 120 aa to about 130 aa, about 130 aa to about 140 aa, about 140 aa to about 150 aa, about 150 aa to about 160 aa, or about 160 aa to about 185 aa of TIFF0007719836000063.tif24151.

[0244] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D). For example, a suitable costimulatory domain has the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000064.tif4166.

[0245] In some cases, the costimulatory domain is derived from the intracellular portion of the transmembrane protein HVEM (also known as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2). For example, a suitable costimulatory domain has the following amino acid sequence: The amino acid sequence may include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000065.tif11149.

[0246] Suitable inhibitory immunoreceptors may include an immunoreceptor tyrosine-based inhibitory motif (ITIM), an immunoreceptor tyrosine-based switch motif (ITSM), an NpxY motif, or a YXXΦ motif. Suitable inhibitory immunoreceptors include PD1; CTLA4; BTLA; CD160; KRLG-1; 2B4; Lag-3; and Tim-3. See, e.g., Odorizzi and Wherry (2012) J. Immunol. 188:2957; and Baitsch et al. (2012) PLoS One 7:e30852.

[0247] In some cases, a suitable inhibitory immunoreceptor has the following PD1 amino acid sequence: It comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000066.tif17159.

[0248] In some cases, a suitable inhibitory immunoreceptor has the following CTLA4 amino acid sequence: It comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000067.tif24153.

[0249] In some cases, a suitable inhibitory immunoreceptor has the following CD160 amino acid sequence: It comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000068.tif24151.

[0250] In some cases, a suitable inhibitory immunoreceptor has the following T cell immunoglobulin and mucin domain-3 (Tim-3) amino acid sequence: It comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000069.tif37150.

[0251] In some cases, a suitable inhibitory immunoreceptor has the following lymphocyte activation gene 3 (Lag-3) amino acid sequence: It includes an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to amino acids 23 to 525 of TIFF0007719836000070.tif57161.

[0252] In some cases, the intracellular domain is a Siglec. See, e.g., Varki and Angata (2006) Glycobiol. 16:1R. In some cases, the Siglec is Siglec-15. In some cases, the intracellular domain is KIR2DL4. Miah et al. (2008) J. Immunol. 180:2922.

[0253] In some cases, the intracellular domain is a recombinase. Suitable recombinases include Cre recombinase; Flp recombinase; Dre recombinase; etc. Suitable recombinases are FLPe recombinase (see, for example, Akbudak and Srivastava (2011) Mol. Biotechnol. 49:82). Suitable recombinases are Flpo recombinase.

[0254] Recombinases as described herein can be intact recombinases or split recombinases. Portions of the split recombinase can be expressed from the same or different expression constructs. In some examples, the two portions of the split recombinase can be operably linked to different binding-triggered transcriptional switches. In other examples, a first portion of the split recombinase can be operably linked to a binding-triggered transcriptional switch, and a second portion of the split recombinase can be expressed separately from an expression construct.

[0255] When the split recombinase is used, for example, in a logic-gated SynNotch circuit, portions of the split recombinase may be arranged in and expressed from one or more expression cassettes with other components in a variety of ways essentially as described below for split transcription factors.

[0256] Thus, activation of one or more binding-triggered transcriptional switches may induce expression of portions of a split recombinase, which may result in heterodimerization and / or complex formation of the split recombinase portions, resulting in the formation of a functional recombinase. Alternatively, activation of one or more binding-triggered transcriptional switches may result in release of the recombinase portions from one or more binding-triggered transcriptional switches, which may result in heterodimerization and / or complex formation of the split recombinase portions, resulting in the formation of a functional recombinase. In addition, induction and release of split recombinase portions may be combined, for example, in which case activation of one or more binding-triggered transcriptional switches may induce expression of portions of the split recombinase and release of the split recombinase portions from one or more binding-triggered transcriptional switches, which may result in heterodimerization and / or complex formation of the split recombinase portions, resulting in the formation of a functional recombinase.

[0257] Suitable split recombinases include, but are not limited to, split Cre recombinases as described, for example, in Beckervordersandforth R et al., Stem Cell Reports. 2014; 2(2): 153-62 Wen M et al., PLoS One. 2014; 9(10): e110290 O'Brien SP et al., Biotechnol J. 2014; 9(3): 355-61 Wang P et al., Sci Rep. 2012; 2: 497 Hirrlinger J et al., PLoS One. 2009; 4(12): e8354 Hirrlinger J et al., PLo SOne. 2009; 4(1): e4286; the disclosures of which are incorporated herein by reference in their entireties.

[0258] A suitable Cre recombinase has the following amino acid sequence: It may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000071.tif37162; and may have a length of 335 amino acids (aa) to 350 aa.

[0259] A suitable FLPe recombinase has the following amino acid sequence: It may comprise an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to TIFF0007719836000072.tif51151; and may have a length of 430 amino acids to 445 amino acids.

[0260] Suitable site-specific nucleases include, but are not limited to, RNA-guided DNA-binding proteins with nuclease activity, such as Cas9 polypeptides; transcription activator-like effector nucleases (TALENs); zinc finger nucleases; and the like.

[0261] Cas9 polypeptides are known in the art; see, e.g., Fonfara et al. (2014) Nucl. Acids Res. 42:2577; and Sander and Joung (2014) Nat. Biotechnol. 32:347. Cas9 polypeptides can include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 36.

[0262] In some cases, the intracellular domain is a Cas9 mutant that lacks nuclease activity but retains DNA target binding activity. Such Cas9 mutants are referred to herein as "inactivated Cas9" or "dCas9." See, e.g., Qi et al. (2013) Cell 152:1173. The dCas9 polypeptide can include a D10A and / or H840A amino acid substitution of the amino acid sequence shown in Figure 36 or the corresponding amino acid in another Cas9 polypeptide.

[0263] In some cases, the intracellular domain is a chimeric dCas9, e.g., a fusion protein comprising dCas9 and a fusion partner, where a suitable fusion partner includes, for example, a non-Cas9 enzyme that provides an enzymatic activity, where the enzymatic activity is a methyltransferase activity, a demethylase activity, an acetyltransferase activity, a deacetylase activity, a kinase activity, a phosphatase activity, a ubiquitin ligase activity, a deubiquitinating activity, an adenylating activity, a deadenylating activity, a sumoylating activity, a desumoylating activity, a ribosylation activity, a deribosylation activity, a myristoylating activity, or a demyristoylating activity. In some cases, the intracellular domain is a chimeric dCas9, e.g., a fusion protein comprising dCas9 and a fusion partner, where suitable fusion partners include, for example, a non-Cas9 enzyme that provides an enzymatic activity, where the enzymatic activity is a nuclease activity, a methyltransferase activity, a demethylase activity, a DNA repair activity, a DNA damage activity, a deamination activity, a dismutase activity, an alkylation activity, a depurination activity, an oxidation activity, a pyrimidine dimer formation activity, an integrase activity, a transposase activity, a recombinase activity, a polymerase activity, a ligase activity, a helicase activity, a photolyase activity, or a glycosylase activity.

[0264] In some cases, the intracellular domain is a chimeric dCas9, e.g., a fusion protein comprising dCas9 and a fusion partner, where suitable fusion partners include, for example, a transcriptional activator or transcriptional repressor domain (e.g., Kruppel-associated box (KRAB or SKD); Mad mSIN3-interacting domain (SID); ERF repressor domain (ERD), etc.); zinc finger-based artificial transcription factors (see, e.g., Sera (2009) Adv. Drug Deliv. 61:513); TALE-based artificial transcription factors (see, e.g., Liu et al. (2013) Nat. Rev. Genetics 14:781); etc.

[0265] In some cases, the intracellular domain is an apoptosis inducer. Suitable apoptosis inducers include tBID. The term "tBID" refers to a C-terminal cleavage fragment of the BH3-coupled death agonist (BID) protein resulting from enzymatic cleavage of cytoplasmic BID (e.g., by activated caspases). During the early stages of apoptosis, tBID translocates to mitochondria and mediates the release of Cyt c therefrom. Non-limiting examples of tBID proteins include human tBID (amino acids 61-195 of the amino acid sequence provided in GenBank Accession No. CAG30275).

[0266] Human tBID has the following amino acid sequence: gnrsshsrlgrieadsesqediirniarhlaqvgdsmdrsippglvnglaedrnrdlataleqllqayprdmekektmlvlalllakkvashtpsllrdvfhttvnfinqnlrtyvrslarngmd (SEQ ID NO:66).

[0267] In some embodiments, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human tBID amino acid sequence presented above; and has a length of about 120 amino acids (aa) to 150 aa, e.g., 120 aa to 125 aa, 125 aa to 130 aa, 130 aa to 135 aa, 135 aa to 140 aa, 140 aa to 145 aa, or 145 aa to 150 aa. In some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the human tBID amino acid sequence presented above; and has a length of 135 aa.

[0268] In some cases, the intracellular domain is a transcription factor. Examples of suitable transcription factors are provided in Table 1 of U.S. Patent Application No. 2014 / 0308746. Non-limiting examples of suitable transcription factors are shown in Figures 37-66. Non-limiting examples of suitable transcription activators and transcription repressors are shown in Figures 37-83. In some cases, the intracellular domain is a transcription regulator. Non-limiting examples of suitable transcription regulators include, for example, Examples include TIFF0007719836000073.tif94151TIFF0007719836000074.tif230151TIFF0007719836000075.tif228151TIFF0007719836000076.tif230151TIFF0007719836000077.tif72151.

[0269] In some cases, the intracellular domain is a transcription factor. Suitable transcription factors include, for example, ASCL1, BRN2, CDX2, CDX4, CTNNB1, EOMES, JUN, FOS, HNF4a, HOXA (e.g., HOXA1, HOXA2, HOXA3, HOXA4, HOXA5, HOXA10, HOXA11, HOXA13), HOXB (e.g., HOXB9), HOXC (e.g., HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXC10, HOXC11, HOXC12, HOXC13), HOXD (e.g., HOXD1, HOXD3, HOXD4, HOXD8, HOXD9, HOXD10, HOXD1), and the like. 1, HOXD12, HOXD13), SNAI1-3, MYOD1, MYOG, NEUROD1-6 (e.g., NEUROD1, NEUROD2, NEUROD4, NEUROD6), PDX1, PU.1, SOX2, Nanog, Klf4, BCL-6, SOX9, STAT1-6, TBET, TCF, TEAD1-4 (e.g., TEAD1, TEAD2, TEAD3, TEAD4), TAF6L, CLOCK, CREB, GATA3, IRF7, MycC, NFkB, RORyt, RUNX1, SRF, TBX21, NFAT, MEF2D, and FoxP3.

[0270] In some cases, the intracellular domain is a transcription factor that has a regulatory function in one or more immune cells (i.e., an immune cell regulatory transcription factor). Suitable immune cell regulatory transcription factors include, for example, Includes TIFF0007719836000078.tif131150, etc.

[0271] In some cases, the transcription factor may be an artificial transcription factor (ATF), including, but not limited to, for example, zinc finger-based artificial transcription factors (e.g., including those described in Sera T. Adv Drug Deliv Rev. 2009 61(7-8):513-26; Collins et al. Curr Opin Biotechnol. 2003 14(4):371-8; Onori et al. BMC Mol Biol. 2013 14:3, the disclosures of which are incorporated herein by reference in their entireties).

[0272] For example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the apoptosis antagonistic transcription factor (AATF) amino acid sequence shown in FIG. 37.

[0273] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the activator of basal transcription (ABT1) amino acid sequence shown in FIG. 38.

[0274] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the adipocyte enhancer binding protein 2 amino acid sequence shown in FIG. 39.

[0275] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the activating transcription factor 1 (ATF1) amino acid sequence shown in FIG. 40.

[0276] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the transcriptional regulator protein BACH1 amino acid sequence shown in FIG. 41.

[0277] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the class E basic helix-loop-helix protein 41 amino acid sequence shown in Figure 42.

[0278] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to a bromodomain-containing protein amino acid sequence shown in Figure 43.

[0279] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the CCAAT / enhancer-binding protein zeta amino acid sequence shown in FIG. 44.

[0280] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the chromodomain-helicase-DNA binding protein 1 amino acid sequence shown in FIG. 45.

[0281] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the death inducer obliterator 1 isoform c amino acid sequence shown in FIG. 46.

[0282] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the protein Dr1 amino acid sequence shown in FIG. 47.

[0283] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the early growth response protein 1 amino acid sequence shown in FIG. 48.

[0284] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the ETS-related transcription factor Elf-2 amino acid sequence shown in FIG.

[0285] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the estrogen receptor amino acid sequence shown in Figure 50.

[0286] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the zinc finger and BTB domain-containing protein 7A amino acid sequence shown in FIG. 51.

[0287] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the four and a half LIM domain protein 1 amino acid sequence shown in Figure 52.

[0288] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the forkhead box protein P3 amino acid sequence shown in Figure 53.

[0289] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the GA-binding protein alpha chain amino acid sequence shown in FIG. 54.

[0290] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the hepatic leukemia factor amino acid sequence shown in Figure 55.

[0291] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the HOP amino acid sequence shown in FIG. 56.

[0292] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA binding protein inhibitor ID-1 amino acid sequence shown in FIG. 57.

[0293] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the DNA binding protein inhibitor ID-2 (dominant negative helix-loop-helix) amino acid sequence shown in Figure 58.

[0294] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the interferon regulatory factor 1 amino acid sequence shown in FIG.

[0295] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Krueppel-like factor 12 amino acid sequence shown in Figure 60.

[0296] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the LIM domain-binding protein 1 amino acid sequence shown in Figure 61.

[0297] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the LIM / homeobox protein Lhx1 amino acid sequence shown in Figure 62.

[0298] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the zinc finger transcription factor E2S-VP64 amino acid sequence shown in Figure 63. As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the zinc finger transcription factor E2S-VP64 amino acid sequence shown in Figure 63. It comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000079.tif11163; and has a length of 105 to 115 amino acids (e.g., 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115 amino acids).

[0299] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the GAL4 DNA binding domain amino acid sequence shown in Figure 64. As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the GAL4 DNA binding domain amino acid sequence shown in Figure 64. It comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000080.tif24151; and has a length of 200 amino acids to 210 amino acids (e.g., 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, or 210 amino acids).

[0300] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the signal transducer and activator of transcription 3 (STAT3) amino acid sequence shown in FIG.

[0301] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Myc amino acid sequence shown in Figure 66.

[0302] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the ASCL1 amino acid sequence shown in Figure 67.

[0303] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the CDX2 amino acid sequence shown in Figure 68.

[0304] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the CREB1 amino acid sequence shown in Figure 69.

[0305] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the CTNNB1 amino acid sequence shown in Figure 70.

[0306] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the EOMES amino acid sequence shown in Figure 71.

[0307] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Fos amino acid sequence shown in Figure 72.

[0308] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the GATA3 amino acid sequence shown in Figure 73.

[0309] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the HOXA1 amino acid sequence shown in FIG. 74.

[0310] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the interferon regulatory factor 7 (IRF7) amino acid sequence shown in Figure 75.

[0311] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the Jun amino acid sequence shown in Figure 76.

[0312] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the myocyte enhancer factor 2D (MEF2D) amino acid sequence shown in FIG. 77.

[0313] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the neural differentiation factor 1 (NEUROD1) amino acid sequence shown in FIG. 78.

[0314] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the NFAT amino acid sequence shown in Figure 79.

[0315] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the NFκB amino acid sequence shown in Figure 80.

[0316] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the SNAI1 amino acid sequence shown in Figure 81.

[0317] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the STAT1 amino acid sequence shown in Figure 82.

[0318] As another example, in some cases, the intracellular domain comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the TEAD1 amino acid sequence shown in Figure 83.

[0319] In some embodiments, the intracellular domain is a transcriptional activator. In some cases, the intracellular domain has the following tetracycline-controlled transcriptional activator (tTA) amino acid sequence: It comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000081.tif31151; and has a length of about 245 amino acids to 252 amino acids (e.g., 248, 249, 250, 251, or 252 amino acids).

[0320] In some embodiments, the intracellular domain is a transcriptional activator. In some cases, the transcriptional activator is GAL4-VP16. In some cases, the transcriptional activator is GAL4-VP64. In some cases, the transcriptional activator is Tbx21. In some cases, the transcriptional activator is an engineered protein, for example, a zinc finger or TALE-based DNA binding domain fused to an effector domain such as VP64 (transcriptional activation) or KRAB (transcriptional repression). A variety of other transcriptional transactivators are known in the art and suitable for use.

[0321] In some cases, the intracellular domain has the following GAL4-VP64 sequence: It comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000082.tif24150; and has a length of 208 to 214 amino acids (e.g., 208, 209, 210, 211, 212, 213, or 214 amino acids).

[0322] In some cases, the intracellular domain has the following Tbx21 sequence: It comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to TIFF0007719836000083.tif64151; and has a length of 530 amino acids to 540 amino acids (e.g., 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, or 540 amino acids).

[0323] In some cases, the intracellular domain has the following MyoD amino acid sequence: TIFF0007719836000084.tif37151; and having a length of 305 to 325 amino acids (e.g., 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, or 325 amino acids).

[0324] In some cases, the intracellular domain comprises a toxin. Examples of toxins include, for example, diphtheria toxin A fragment, a non-binding active fragment of diphtheria toxin, exotoxin A (from Pseudomonas ...

Claims

1. (a) (i) an extracellular binding domain comprising the antigen-binding region of an antibody; (ii) a Notch regulatory region comprising a transmembrane domain containing Lin-12 Notch repeats, an S2 proteolytic cleavage site, and an S3 proteolytic cleavage site; and (iii) an intracellular domain comprising a DNA-binding domain, wherein the intracellular domain does not comprise an immunoreceptor activation domain or a costimulatory domain; wherein binding of the antigen-binding region of (a)(i) to an antigen induces proteolytic cleavage of the Notch regulatory region of (a)(ii) and releases the DNA-binding domain of (a)(iii); and (b) an expression cassette comprising a transcriptional control element operably linked to a nucleic acid encoding a therapeutic agent; A cell comprising: A cell wherein the released DNA binding domain binds to the transcriptional control element and induces expression of the nucleic acid encoding the therapeutic agent.

2. The cell of claim 1 , wherein the therapeutic agent is secreted or expressed on the surface of the cell.

3. The cell of claim 1 , wherein the therapeutic agent is a recombinant therapeutic agent.

4. The cell of claim 1 , wherein the therapeutic substance is an endogenous therapeutic substance.

5. The cell of claim 1 , wherein the antigen is a cancer antigen.

6. The cell of claim 1, wherein the antigen-binding region is an scFv or a nanobody.

7. The cell of claim 1 , wherein the therapeutic agent is a cancer therapeutic agent.

8. The cell of claim 1 , wherein the therapeutic agent is immunosuppressive.

9. The cell of claim 1 , wherein the therapeutic agent is immunostimulatory.

10. The cell of claim 1, wherein the therapeutic agent is selected from the group consisting of an antibody, a chemokine, a chemokine receptor, a cytokine, a cytokine receptor, a chimeric antigen receptor (CAR), a T cell receptor (TCR), an activating immunoreceptor, an inhibitory immunoreceptor, an immunoactivator, an immunosuppressant, an apoptosis inducer, an apoptosis inhibitor, a toxin-derived protein, and a site-specific nuclease.

11. The cell of claim 10, wherein the antibody is a therapeutic antibody for the treatment of cancer.

12. The antigen to which the antigen-binding region binds is present on the surface of a cell or on a solid support, or a soluble adaptor molecule that is bound to the surface of a cell or a solid support, The cell of claim 1.

13. (a) (i) an extracellular binding domain comprising the antigen-binding region of an antibody; (ii) a Notch regulatory region comprising a transmembrane domain containing Lin-12 Notch repeats, an S2 proteolytic cleavage site, and an S3 proteolytic cleavage site; and (iii) an intracellular domain comprising a DNA-binding domain, wherein the intracellular domain does not comprise an immunoreceptor activation domain or a costimulatory domain; wherein binding of the antigen-binding region of (a)(i) to an antigen induces proteolytic cleavage of the Notch regulatory region of (a)(ii) and releases the DNA-binding domain of (a)(iii); and (b) an expression cassette comprising a first transcriptional control element operably linked to a nucleic acid encoding a transcription factor; A cell comprising: A cell wherein the released DNA binding domain binds to the transcriptional control element and induces expression of the nucleic acid encoding the transcription factor.

14. The cell of claim 13 , wherein the transcription factor regulates cell differentiation or dedifferentiation.

15. The cell of claim 13 , wherein the transcription factor is an artificial transcription factor.

16. The cell of claim 13 , wherein the transcription factor is the first half of a split transcription factor.

17. 17. The cell of claim 16, further comprising a second transcriptional control element operably linked to the nucleic acid encoding the second half of the split transcription factor.

18. 18. The cell of claim 17, wherein the second transcriptional control element is constitutive.

19. 18. The cell of claim 17, wherein the second transcriptional control element is inducible.

20. The cell of claim 19, further comprising a nucleic acid encoding a second chimeric Notch receptor, wherein the second transcriptional control element is responsive to binding-induced proteolytic cleavage of the second chimeric Notch receptor.

21. (a) (i) an extracellular binding domain comprising the antigen-binding region of an antibody; (ii) a Notch regulatory region comprising a transmembrane domain containing Lin-12 Notch repeats, an S2 proteolytic cleavage site, and an S3 proteolytic cleavage site; and (iii) an intracellular domain heterologous to a Notch receptor comprising the first half of a split transcription factor, wherein the intracellular domain does not comprise an immunoreceptor activation domain or a costimulatory domain; wherein binding of the antigen-binding region to an antigen induces proteolytic cleavage of the Notch regulatory region and releases the first half of the split transcription factor. and (b) a second nucleic acid encoding the second half of the split transcription factor; including, cells.

22. 22. The cell of claim 21 , wherein the second nucleic acid is operably linked to a transcriptional control element.

23. 23. The cell of claim 22, wherein the transcriptional control element is constitutive.

24. 23. The cell of claim 22, wherein the transcriptional control element is inducible.

25. 25. The cell of claim 24, wherein the second nucleic acid further encodes a second chimeric Notch receptor comprising an intracellular domain that includes the second half of the split transcription factor.

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