Methods and compositions for reducing immunogenicity of chimeric notch receptors

Abstract

To provide methods and compositions for reducing the immunogenicity of chimeric Notch receptors, and in particular to provide transcription factors useful for regulating gene expression delivered to tissues by such chimeric Notch receptors.SOLUTION: Provided is a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising, from N- to C-terminus: a) an antigen binding domain; b) a human Notch2 or human Notch3 core domain; c) a human NHF1 alpha or human EGR1DNA binding domain; and d) a transactivation domain derived from human RelA, human WWT1 (TAZ) or human CREB3 (LZIP).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Patent Application No. 62 / 603,993, filed June 19, 2017, and U.S. Provisional Patent Application No. 62 / 556,765, filed September 11, 2017, the contents of both of which are incorporated herein by reference in their entireties.

[0002] The present invention relates to molecular biology, and in particular to methods and compositions for reducing the immunogenicity of certain receptors useful for controlling selective gene expression in cells of the monocyte / macrophage lineage, and their applications. [Background technology]

[0003] A significant challenge limiting the development of gene therapy in humans is regulating therapeutic gene expression so that gene expression or the vehicle used to achieve expression does not result in enhanced immunogenicity leading to host rejection. One method for achieving gene expression is described in Patent Document 1 and Non-Patent Document 1, which describe the activation of gene expression using a chimeric Notch receptor.

[0004] Notch receptors are single-pass transmembrane proteins that mediate cell-cell contact signaling and play a central role in development and other aspects of intercellular communication between two contacting cells, one of which bears a Notch receptor and the other of which displays a ligand on its surface that binds to the corresponding Notch receptor. Engagement of native Notch with its native ligand, Delta, leads to two stages of proteolysis of the Notch receptor, ultimately releasing the intracellular portion of the receptor from the membrane into the cytoplasm, where it translocates to the nucleus. There, the released domain alters cell behavior by functioning as a transcriptional regulator. Notch receptors are involved in and required for a variety of cellular functions during development and are crucial for the function of numerous cell types across species.

[0005] Patent Literature 1 describes chimeric Notch receptors, demonstrating that Notch-expressing cells possess one or more different binding moieties on their cell surface that recognize cell-associated ligands, such as scFVs, nanobodies, and single-chain T cell receptors, ultimately leading to the release of the receptor's intracellular transcriptional regulatory portion from the cell membrane into the cytoplasm, resulting in transcriptional regulation. When engineered cells bearing chimeric Notch receptors encounter their specific target antigens, the cytosolic fragments are then cleaved so that they can freely migrate to the cell nucleus and regulate the transcription of any open reading frame (ORF) under the control of a synthetic promoter. The expressed ORFs can be cytokines that induce local immune activity and recruit target antigens to the site of target antigen detection. Furthermore, the expressed ORFs can be chimeric antigen T cell receptors (CAR-Ts), which target distinct target antigens for target cell killing only after the priming target antigen detected by the chimeric Notch receptor is detected. This enables highly specific combinatorial antigen pattern recognition, allowing for clearer differentiation between diseased or cancerous cells and healthy cells. This could greatly enhance the application of engineered CAR-T cells to safely target a wider range of tumors with fewer side effects on healthy tissue.

[0006] To date, the transcription machinery used in chimeric Notch constructs has been GAL4-VP16. The DNA-binding fragment, GAL4, is of yeast origin, and VP16 is a simple Being a highly acidic portion of the herpesvirus protein, GAL4-VP16 is highly immunogenic, thus limiting the use of chimeric Notch receptors in the treatment of human disease.

[0007] Another major obstacle to the efficacy of many immunotherapy-based approaches for solid tumors, including cell therapy, is the delivery of drugs or activation of immune cells in solid tumors. Cells of the monocyte / macrophage lineage constitute the major component of immune cells infiltrating solid tumors (Non-Patent Document 2). These cell types are actively recruited and retained in solid tumors, making them potentially important cell types for gene therapy delivery.

[0008] Genetic manipulation of macrophages using clinically approved vectors, such as those carrying HIV-1-based lentiviruses, has been difficult due to the inhibition of HIV-1 infection in macrophages. (Non-Patent Document 3) demonstrated that the addition of the virion-associated Vpx accessory protein found in HIV-2 and simian immunodeficiency virus relieves the inhibition of HIV-1 infection in macrophages by degrading the macrophage restriction factor SAMHD1. Subsequently, it has been demonstrated that monocyte-derived macrophages can be efficiently transduced with Vpx+ lentiviruses encoding macrophage-produced cytokines for the purpose of regulating the tumor microenvironment (Non-Patent Document 4). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 9,670,281 [Non-patent literature]

[0010] [Non-Patent Document 1] Roybal et al., Cell, Feb. 11, 2016 [Non-patent document 2] Long et al., Oncoimmunology 2:e26860, 2013 doi:10.4161 / onci26860 [Non-patent document 3] Hrecka et al. ("Vpx relieves the inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein," Nature 474(7353):658-661, 2011) [Non-patent document 4] Moyes et al., Human Gene Therapy 28(2):200-215, 2017 Summary of the Invention

[0011] The present invention relates to methods and compositions for reducing the immunogenicity of chimeric Notch receptors. The Notch receptors described herein can be engineered in cells of the monocyte / macrophage lineage.

[0012] Another embodiment of the present invention relates to methods and compositions for reducing the immunogenicity of chimeric Notch receptors by humanizing transcription factors useful for regulating gene expression delivered to tissues by the chimeric Notch receptors.

[0013] Yet another embodiment of the present invention is a method and composition for reducing the immunogenicity of a chimeric Notch receptor by humanizing the transcription factors used to express the gene in cells containing the chimeric Notch receptor, wherein such transcription factors include transcription factors from the hepatocyte nuclear factor family of transcription factors.

[0014] The present invention also relates to the use of DNA binding domains (DBDs) of HNF1 transcription factors, such as HNF1 alpha and vHNF1 beta, for the generation of chimeric transcription factors with reduced immunogenicity, preferably useful for transgene delivery via chimeric Notch receptors to tissues that do not express endogenous HNF1 or vHNF1. No. 301096678.

[0015] A further embodiment of the present invention is a human HNF1 DNA binding domain used in conjunction with a human transcriptional activator domain (TAD) or repressor domain, and optionally a human regulatory domain.

[0016] A further embodiment of the present invention is the human HNF1 DNA binding domain used in conjunction with the human transcriptional activator domain (TAD) from the WWTR1 (TAZ) protein.

[0017] A further embodiment of the present invention is the human HNF1 DNA binding domain used in conjunction with the human transcriptional activator domain (TAD) derived from the CREB3 (LZIP) protein.

[0018] A further embodiment of the present invention is the human HNF1 DNA binding domain used in conjunction with a human transcriptional activator domain (TAD) derived from the NF-κB system factor p65 (RelA).

[0019] The present invention also relates to nucleic acid molecules and proteins useful for regulating gene expression in eukaryotic cells and organisms using chimeric Notch receptors with reduced immunogenicity.

[0020] The present invention further provides low-immunogenic chimeric Notch receptor polypeptides, nucleic acids comprising nucleotide sequences encoding the chimeric Notch receptor polypeptides, and host cells genetically modified with the nucleic acids, wherein low immunogenicity is achieved by using a transcription factor comprising a human HNF1 DNA-binding domain in combination with a human transcriptional activator domain (TAD) derived from the NF-κB system factor p65 (RelA).

[0021] In one particular embodiment of the invention, the humanized chimeric notch receptor is composed 5' to 3' of the following sequence: Human CD8a signal peptide 1-22 (NP_001139345 amino acids 1-22 (MALPVTALLLPLALLLHAARPS) (SEQ ID NO: 1)) - directs the protein to the surface of expressing cells. Myc tag (EQKLISEEDL) (SEQ ID NO: 2) - a peptide tag for antibody labeling of surface-expressed synthetic receptors. Myc antibody: Cell Signaling Technology, Myc tag (9B11) mouse mAb (AlexaFluor™ 647 conjugate, catalog number 2233). Anti-human B cell (CD19) antibody, clone FMC63. A short cytosolic fragment containing three NLR domains, the transmembrane domain, and the native nuclear localization sequence (NLS) of human Notch3 and human Notch3 core (gi|134244285|NP_000426.2 amino acids 1374 to 1738). GS flexible linker (GSAAAGGSGGSGGS) (SEQ ID NO: 3). Human HNF1 alpha (gi|807201167|NP_001293108.1 amino acids 1-283) containing the dimerization and DNA-binding domain (DBD) of Homo sapiens hepatocyte nuclear factor 1-alpha isoform 1. GS flexible linker (GGGSGGGS) (SEQ ID NO: 4). Human Rel-A(p65) (gi|223468676|NP_068810.3 amino acids 1–551) containing the transactivation domain of transcription factor p65 isoform 1 [Homo sapiens].

[0022] Also provided herein are methods of treating a disease, including cancer, in a subject (e.g., a human), comprising administering to the subject a mammalian cell comprising a humanized chimeric Notch receptor. In some embodiments, the mammalian cell can be a monocyte / macrophage cell.

[0023] Other features and advantages of the present invention will be apparent from the following detailed description of the invention and the claims. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 is a schematic diagram of a synthetic Notch receptor and the constituent domains it comprises. [Figure 2] 1 shows experimental data showing the relative potency of four human Notch homologs in releasing GAL4-vp64 upon stimulation of myc-loaded beads with external myc-tagged antigen. hsNotch2 and hsNotch3 are the only homologs that show potent activity. [Figure 3A] FIG. 1 shows experimental data demonstrating the functional behavior of a human DNA-binding domain fused to the p65 transactivation domain to upregulate GFP expression. [Figure 3B]FIG. 1 shows experimental data demonstrating the functional behavior of two working synthetic Notch human DNA binding domains with the p65 transactivation domain to upregulate GFP expression. [Figure 4] This figure shows experimental data demonstrating the expression of chimeric Notch receptors in human monocyte-derived macrophage cells, showing the transduction rates of mouse Notch1 protein / Gal4 and VP64 transcription factors (top) and human Notch3 protein / HNF1α and p65 transcription factors (bottom) relative to untransduced monocyte-derived macrophages (right). [Figure 5A] FIG. 1 shows experimental data demonstrating the functional behavior of human Notch3 and human DNA-binding domains fused to the p65 transactivation domain to upregulate GFP expression in human monocyte-derived macrophages. [Figure 5B] FIG. 1 shows experimental data demonstrating the functional behavior of mouse Notch1 and non-human Gal4 binding domains fused to VP64 transactivation to upregulate GFP expression in human myeloid cells. DETAILED DESCRIPTION OF THE INVENTION

[0026] Incorporation by Reference: All publications mentioned herein, including patents, published patent applications, and scientific articles, are hereby incorporated by reference in their entirety.

[0027] definition A "chimeric Notch polypeptide," also referred to as a "chimeric Notch receptor polypeptide" or "chimeric Notch" or "synNotch," is described in U.S. Patent No. 5,999,233 and comprises, covalently linked from the N-terminus to the C-terminus, a) an extracellular domain comprising a first member of a specific binding pair; b) an intracellular domain, 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, 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 is mediated by the one or more ligand-inducible proteolytic cleavage sites of the Notch receptor polypeptide. In some cases, the Notch receptor polypeptide is between 300 and 400 amino acids in length.

[0028] Furthermore, a "chimeric Notch receptor polypeptide" comprises a linker inserted 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 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 where the first member of the specific binding pair is a non-antibody-based recognition scaffold, the non-antibody-based recognition scaffold is an avimer, DARPin, adnectin, avimer, affibody, anticalin, or affilin. In some cases, the first member of the specific binding pair is an antigen. In some cases where the first member of the specific binding pair is an antigen, the antigen is an endogenous antigen. In some cases where 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 part of the extracellular region of a cell adhesion molecule).

[0029] The term "transmembrane domain" refers to a domain of a polypeptide comprising at least one contiguous amino acid sequence that spans a lipid bilayer when expressed in a mammalian cell and present in the corresponding endogenous polypeptide. For example, a transmembrane domain may comprise one, two, three, four, five, six, seven, eight, nine, or ten contiguous amino acid sequences that each span a lipid bilayer when expressed in a mammalian cell and present in the corresponding endogenous polypeptide. As known in the art, a transmembrane domain may comprise, for example, at least one (e.g., two, three, four, five, six, seven, eight, nine, or ten) contiguous amino acid sequence that has an α-helical secondary structure in the lipid bilayer (which spans the lipid bilayer when expressed in a mammalian cell and present in the corresponding endogenous polypeptide). In some embodiments, a transmembrane domain may comprise two or more contiguous amino acid sequences that form a β-barrel secondary structure in the lipid bilayer (which each spans the lipid bilayer when expressed in a mammalian cell and present in the corresponding endogenous polypeptide). Non-limiting examples of transmembrane domains are described herein. Further examples of transmembrane domains are known in the art.

[0030] The phrase "extracellular side of the plasma membrane," when used to describe the location of a polypeptide, means that the polypeptide contains at least one transmembrane domain that crosses the plasma membrane and at least one domain (e.g., at least one antigen-binding domain) that is located in the extracellular space.

[0031] "GFP" or green fluorescent protein (GFP) It is a commonly used reporter of gene expression. Arun et al., J. Pharmacol. Toxicol. Methods 51(1):1-23, 2005.

[0032] By "HNF1 binding site" is intended any specific binding site for any of the known forms of HNF. HNF1 (also called LF-B1 or HNF1 alpha) , a 628 aa long DNA-binding protein that has been implicated as a major determinant of hepatocyte-specific transcription of several genes (Frain, Cell 59, 145-157, 1990).

[0033] In some embodiments, the DNA-binding domain of human origin is the DNA-binding domain of an HNF1 transcription factor (e.g., any of the HNF1 transcription factors described herein or known in the art), and the transactivation domain is a human RelA protein or portion thereof.

[0034] In some embodiments, the amino acid sequence of HNF1 alpha is NCBI number NP_001293108.1, NP_000536.5, or XP_005253988.1. In some embodiments, the amino acid sequence of the transcriptional regulator of a humanized chimeric Notch receptor comprises hepatocyte nuclear factor 1-alpha isoform 1 (NP_001293108.1), hepatocyte nuclear factor 1-alpha isoform 1 (NP_000536.5), or hepatocyte nuclear factor 1-alpha isoform X1 (XP_005253988.1), or a portion thereof. In some embodiments, the amino acid sequence of the transcriptional regulator of a humanized Notch receptor comprises all or a portion of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0035] As used herein, a "portion" of a polypeptide or protein refers to at least 10 amino acids of a reference sequence, e.g., 10-200, 25-300, 50-400, 100-500, 200-600, 300-700, 400-800, 500-900, or 600-1000 or more amino acids of a reference sequence. In some embodiments, the portion of the polypeptide or protein is functional. In some embodiments, the transcriptional regulator is or includes the dimerization and DNA binding domain (DBD) of hepatocyte nuclear factor 1-alpha isoform 1 (NP_001293108.1), hepatocyte nuclear factor 1-alpha isoform 1 (NP_000536.5), or hepatocyte nuclear factor 1-alpha isoform X1 (XP_005253988.1). In some embodiments, the amino acid sequence of the transcriptional regulator of a humanized Notch receptor is or comprises the dimerization and DNA binding domain (DBD) of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some embodiments, the amino acid sequence of the transcriptional regulator of a humanized Notch receptor is or comprises amino acids 1 to 283 of SEQ ID NO: 5.

[0036] Human hepatocyte nuclear factor 1-alpha isoform 1 NP_001293108.1 (SEQ ID NO: 5) MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGELAELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWRVAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKR AALYTWYVRKQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNPSKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHKLAMDTYSGPPPGPGPGPALPAHSSPGLPPPALSPSKVHG VRYGQPATSETAEVPSSSGGPLVTVSTPLHQVSPTGLEPSHSLLSTEAKLVSAAGGPLPPVSTLTALHSLEQTSPGLNQQPQNLIMASLPGVMTIGPGEPASLGPTFTNTGASTLVIGLASTQAQSVPVINSMGSSLTTLQPVQFSQPLHPSYQQPLMP PVQSHVTQSPFMATMAQLQSPHALYSHKPEVAQYTHTGLLPQTMLITDTTNLSALASLTPTKQEAALLPQVFTSDTEASSESGLHTPASQATTLHVPSQDPAGIQHLQPAHRLSASPTVSSSSLVLYQSSDSSNGQSHLLPSNHSVIETFISTQMASSSQ Human hepatocyte nuclear factor 1-alpha isoform 2 NP_000536.5 (SEQ ID NO: 6) MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGELAELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWRVAKMVKSYLQQHNIPQREVVDTTGLN QSHLSQHLNKGTPMKTQKRAALYTWYVRKQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNPSKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHK LAMDTYSGPPPGPGPGPALPAHSSPGLPPPALSPSKVHGVRYGQPATSETAEVPSSSGGPLVTVSTPLHQVSPTGLEPSHSLLSTEAKLVSAAGGPLPPVSTLTALHSLEQTSPGLNQQPQNLIMASLPGVMTIGPGEPA SLGPTFTNTGASTLVIGLASTQAQSVPVINSMGSSLTTLQPVQFSQPLHPSYQQPLMPPVQSHVTQSPFMATMAQLQSPHALYSHKPEVAQYTHTGLLPQTMLITDTTNLSALASLTPTKQVFTSDTEASSESGLHTPAS QATTLHVPSQDPAGIQHLQPAHRLSASPTVSSSSLVLYQSSDSSNGQSHLLPSNHSVIETFISTQMASSSQ Human hepatocyte nuclear factor 1-alpha isoform X1 (predicted) XP_005253988.1 (SEQ ID NO: 7) MVSKLSQLQTELLAALLESGLSKEALIQALGEPGPYLLAGEGPLDKGESCGGGRGELAELPNGLGETRGSEDETDDDGEDFTPPILKELENLSPEEAAHQKAVVETLLQEDPWRVAKMVKSYLQQHNIPQREVVDTTGLNQSHLSQHLNKGTPMKTQKRAALYTWYVRKQREVAQQFTHAGQGGLIEEPTGDELPTKKGRRNRFKWGPASQQILFQAYERQKNPSKEERETLVEECNRAECIQRGVSPSQAQGLGSNLVTEVRVYNWFANRRKEEAFRHKLAMDTYSGPPPGPGPGPALPAHSSPGLPPPALSPSKVHGVRYGQPATSETAEVPSSSGGPLVTVSTPLHQVSPTGLEPSHSLLSTEAKLVSAAGGPLPPVSTLTALHSLEQTSPGLNQQPQNLIMASLPGVMTIGPGEPASLGPTFTNTGASTLVIGLASTQAQSVPVINSMGSSLTTLQPVQFSQPLHPSYQQPLMPPVQSHVTQSPFMATMAQLQSPHALYSHKPEVAQYTHTGLLPQTMLITDTTNLSALASLTPTKQVRSRPAGPPLACDRAPHPHIPRAQEAALLPQVFTSDTEASSESGLHTPASQATTLHVPSQDPASIQHLQPAHRLSASPTVSSSSLVLYQSSDSSNGQSHLLPSNHSVIETFISTQMASSSQ

[0037] In some embodiments, the amino acid sequence of HNF1 alpha or a portion thereof described herein is at least 80% identical to the corresponding amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In some embodiments, the amino acid sequence of HNF1 alpha or a portion thereof is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In some embodiments, the amino acid sequence of HNF1 alpha or a portion thereof described herein may differ from the corresponding amino acid sequence of SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0038] In some embodiments, the HFN1 alpha mRNA sequence is NCBI number NM_001306179.1, NM_00545.6, or XM_005253931.3. In some embodiments, the HFN1 alpha mRNA sequence is SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10.

[0039] Human HNF1 homeobox A (HNF1A), transcript variant 1, mRNA NM_001306179.1 (SEQ ID NO: 8) Human HNF1 homeobox A (HNF1A), transcript variant 2, mRNA NM_000545.6 (SEQ ID NO: 9) Human HNF1 homeobox A (HNF1A), transcript variant X1, mRNA XM_005253931.3 (SEQ ID NO: 10)

[0040] In some embodiments, HNF1 alpha binds to the reverse palindrome 5-GTTAATNATTAAC-3 (SEQ ID NO: 11).

[0041] In some embodiments, the nucleic acid sequence encoding HNF1 alpha described herein is at least 80% identical to the sequence of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In some embodiments, the nucleic acid sequence encoding HNF1 alpha is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10. In some embodiments, the nucleic acid nucleotide sequence encoding HNF1 alpha described herein may differ from the sequence of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides.

[0042] In some embodiments, the amino acid sequence of Rel-A(p65) is NCBI number NP_068810.3, NP_001138610.1, NP_001230913.1, NP_001230914.1, XP_011543508.1, or XP_011543509.1. In some embodiments, the amino acid sequence of Rel-A(p65) is or includes all or a portion of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17. In some embodiments, the amino acid sequence of the transactivation domain of the humanized chimeric Notch receptor comprises all or a portion of transcription factor p65 isoform 1 (NP_068810.3), transcription factor p65 isoform 2 (NP_001138610.1), transcription factor p65 isoform 3 (NP_001230913.1), transcription factor p65 isoform 4 (NP_001230914.1), transcription factor p65 isoform X1 (XP_011543508.1), or transcription factor p65 isoform X2 (XP_011543509.1). In some embodiments, the amino acid sequence of the transactivation domain of the humanized Notch receptor comprises all or a portion of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17. In some embodiments, the amino acid sequence of the transactivation domain of the humanized Notch receptor is or comprises amino acids 1 to 551 of SEQ ID NO:12.

[0043] Human transcription factor p65 isoform 1 NP_068810.3 (SEQ ID NO: 12) MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTDTTKTHPTIKINGYTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFYEAELCPDRCIHSFQNLGIQCVKKRDLEQAISQRIQTNNNPFQVPIEEQRGDYDLNAVRLCFQVTVRDPSGRPLRLPPVLSHPIFDNRAPNTAELKICRVNRNSGSCLGGDEIFLLCDKVQKEDIEVYFTGPGWEARGSFSQADVHRQVAIVFRTPPYADPSLQAPVRVSMQLRRPSDRELSEPMEFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS Human transcription factor p65 isoform 2 NP_001138610.1 (SEQ ID NO: 13) MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTDTTKTHPTIKINGYTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFYEAELCPDRCIHSFQNLGIQCVKKRDLEQAISQRIQTNNNPFQEEQRGDYDLNAVRLCFQVTVRDPSGRPLRLPPVLSHPIFDNRAPNTAELKICRVNRNSGSCLGGDEIFLLCDKVQKEDIEVYFTGPGWEARGSFSQADVHRQVAIVFRTPPYADPSLQAPV RVSMQLRRPSDRELSEPMEFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPA PVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS Human transcription factor p65 isoform 3 NP_001230913.1 (SEQ ID NO: 14) MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTDTTKTHPTIKINGYTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFYEAELCPDRCIHSFQNLGIQCVKKRDLEQAISQRIQTNNNPFQVPIEEQRGDYDLNAVRLCFQVTVRDPSGRPLRLPPVLSHPIFDNRAPNTAELKICRVNRNSGSCLGGDEIFLLCDKVQKEDIEVYFTG PGWEARGSFSQADVHRQVAIVFRTPPYADPSLQAPVRVSMQLRRPSDRELSEPMEFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPGPPQAVAPPA PKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS Human transcription factor p65 isoform 4 NP_001230914.1 (SEQ ID NO: 15) MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTDTTKTHPTIKINGYTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFYEAELCPDRCIHSFQNLGIQCVKKRDLEQAISQRIQTNNNPFQVPIEEQRGDYDLNAVRLCFQVTVRDPSGRPLRLPPVLSHPIFDNRAPNTAELKICRVNRNSGSCLGGDEIF LLCDKVQKEDIEVYFTGPGWEARGSFSQADVHRQVAIVFRTPPYADPSLQAPVRVSMQLRRPSDRELSEPMEFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRR IAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS Human transcription factor p65 isoform X1 XP_011543508.1 (SEQ ID NO: 16) MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTDTTKTHPTIKINGYTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFYEAELCPDRCIHSFQNLGIQCVKKR DLEQAISQRIQTNNNPFQVPIEEQRGDYDLNAVRLCFQVTVRDPSGRPLRLPPVLSHPIFDNRAPNTAELKICRVNRNSGSCLGGDEIFLLCDKVQKDDRHRIEEKRKRTYETFKSIMKKSPFSG PTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS Human transcription factor p65 isoform X2 XP_011543509.1 (SEQ ID NO: 17) MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTDTTKTHPTIKINGYTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFYEAELCPDRCIH SFQNLGIQCVKKRDLEQAISQRIQTNNNPFQVPIEEQRGDYDLNAVRLCFQVTVRDPSGRPLRLPPVLSHPIFDNHDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPP RRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEA LLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS

[0044] In some embodiments, the amino acid sequence of Rel-A(p65) described herein is SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or the sequence The amino acid sequence of Rel-A(p65) is at least 80% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the amino acid sequence of Rel-A(p65) is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the amino acid sequence of Rel-A(p65) described herein may differ from the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0045] In some embodiments, the nucleic acid sequence encoding Rel-A(p65) is provided by NCBI number NM_021975.3, NM_001145138.1, NM_001243984.1, NM_001243985.1, XM_011545206.1, or XM_011545207.1. In some embodiments, the nucleic acid sequence encoding Rel-A(p65) is or includes SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23.

[0046] Human RELA proto-oncogene, NF-kB subunit (RELA), transcript variant 1, mRNA NM_021975.3 (SEQ ID NO: 18) CTCCAGTCAGGAGGCATAGTTTTTACTGAACAATCAAAGCACTTGGACTCTTGCTCTTTCTACTCTGAACTAATAAATCTGTTGCCAAGCTGGCTAGAAAAAAAAAAAAAAAAAA Human RELA proto-oncogene, NF-kB subunit (RELA), transcript variant 2, mRNA NM_001145138.1 (SEQ ID NO: 19) Human RELA proto-oncogene, NF-kB subunit (RELA), transcript variant 3, mRNA NM_001243984.1 (SEQ ID NO: 20) AGCGCGCAGGCGCGGCCGGATTCCGGGCAGTGACGCGACGGCGGGCCGCGCGGCGCATTTCCGCCTCTGGCGAATGGCTCGTCTGTAGTGCACGCCGCGGGCCCAGCTGCGACCCCGGCCCCGCCCCCGGGACCCCGGCCATGGACGAACTGTTCCCCCTCATCTTCCCGGCAGAGCCAGCCCAGGCCTCTGGCCCCTATGTGGAGATCATTGAGCAGCCCAAGCAGCGGGGCATGCGCTTCCGCTACAAGTGCGAGGGGCGCTCCGCGGGCAGCATCCCAGGCGAGAGGAGCACAGATACCACCAAGACCCACCCCACCATCAAGATCAATGGCTACACAGGACCAGGGACAGTGCGCATCTCCCTGGTCACCAAGGACCCTCCTCACCGGCCTCACCCCCACGAGCTTGTAGGAAAGGACTGCCGGGATGGCTTCTATGAGGCTGAGCTCTGCCCGGACCGCTGCATCCACAGTTTCCAGAACCTGGGAATCCAGTGTGTGAAGAAGCGGGACCTGGAGCAGGCTATCAGTCAGCGCATCCAGACCAACAACAACCCCTTCCAAGTTCCTATAGAAGAGCAGCGTGGGGACTACGACCTGAATGCTGTGCGGCTCTGCTTCCAGGTGACAGTGCGGGACCCATCAGGCAGGCCCCTCCGCCTGCCGCCTGTCCTTTCTCATCCCATCTTTGACAATCGTGCCCCCAACACTGCCGAGCTCAAGATCTGCCGAGTGAACCGAAACTCTGGCAGCTGCCTCGGTGGGGATGAGATCTTCCTACTGTGTGACAAGGTGCAGAAAGAGGACATTGAGGTGTATTTCACGGGACCAGGCTGGGAGGCCCGAGGCTCCTTTTCGCAAGCTGATGTGCACCGACA Human RELA proto-oncogene, NF-kB subunit (RELA), transcript variant 4, mRNA NM_001243985.1 (SEQ ID NO: 21) Human RELA proto-oncogene, NF-kB subunit (RELA), transcript variant X1, mRNA XM_011545206.1 (SEQ ID NO: 22) Human RELA proto-oncogene, NF-kB subunit (RELA), transcript variant X2, mRNA XM_011545207.1 (SEQ ID NO: 23) TGATGGAGTACCCTGAGGCTATAACTCGCCTAGTGACAGGGGCCCAGAGGCCCCCCGACCCAGCTCCTGCTCCACTGGGGGCCCCGGGGCTCCCCAATGGCCTCCTTTCAGGAGATGAAGACTTCTCCTCCATTGCGGACATGGACTTCTCAGCCCTGCTGAGTCAGATCAGCTCCTAAGGGGGTGACGCCTGCCCTCCCCAGAGCACTGGGTTGCAGGGGATTGAAGCCCTCCAAAAGCACTTACGGATTCTGGTGGGGTGTGTTCCAACTGCCCCCAACTTTGTGGATGTCTTCCTTGGAGGGGGGAGCCATATTTTATTCTTTTATTGTCAGTATCTGTATCTCTCTCTCTTTTTGGAGGTGCTTAAGCAGAAGCATTAACTTCTCTGGAAAGGGGGGAGCTGGGGAAACTCAAACTTTTCCCCTGTCCTGATGGTCAGCTCCCTTCTCTGTAGGGAACTCTGGGGTCCCCCATCCCCATCCTCCAGCTTCTGGTACTCTCCTAGAGACAGAAGCAGGCTGGAGGTAAGGCCTTTGAGCCCACAAAGCCTTATCAAGTGTCTTCCATCATGGATTCATTACAGCTTAATCAAAATAACGCCCCAGATACCAGCCCCTGTATGGCACTGGCATTGTCCCTGTGCCTAACACCAGCGTTTGAGGGGCTGGCCTTCCTGCCCTACAGAGGTCTCTGCCGGCTCTTTCCTTGCTCAACCATGGCTGAAGGAAACCAGTGCAACAGCACTGGCTCTCTCCAGGATCCAGAAGGGGTTTGGTCTGGGACTTCCTTGCTCTCCCTCTTCTCAAGTGCCTTAATAGTAGGGTAAGTTGTTAAGAGTGGGGGAGAGCAGGCTGGCAGCTCTCCAGTCAGGAGGCATAGTTTTTACTGAACAATCAAAGCACTTGGACTCTTGCTCTTTCTACTCTGAACTAATAAATCTGTTGCCAAGCTGG

[0047] In some embodiments, the nucleic acid sequence encoding Rel-A(p65) described herein is at least 80% identical to the sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In some embodiments, the nucleic acid sequence encoding Rel-A(p65) is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or SEQ ID NO: 23. In some embodiments, a nucleic acid encoding Rel-A(p65) described herein may differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides from the sequence of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23.

[0048] "Linkers" are short amino acid sequences that are naturally produced to separate multiple domains in a single protein, and are generally flexible, rigid, and Linkers can be classified into three groups: cleavable and non-cleavable. Chen, X., et al., 2013, Adv. Drug Deliv. Rev., 65, 1357-1369. Linkers can be natural or synthetic. To achieve this, many linkers are used, including "flexible linkers." Flexible linkers are rich in glycine. Klein et al., Protein Engineering, Design & Selection, Vol. 27, No. 10, pp. 325-330, 2014; Priyanka et al., Protein Sci., 2013 Feb; 22(2):153-167.

[0049] In some embodiments, the linker is a synthetic linker. The synthetic linker may have a length of about 10 to about 200 amino acids, for example, 10 to 25 amino acids, 25 to 50 amino acids, 50 to 75 amino acids, 75 to 100 amino acids, 100 to 125 amino acids, 125 to 150 amino acids, 150 to 175 amino acids, or 175 to 200 amino acids. The synthetic linker may have a length of 10 to 30 amino acids, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. The synthetic linker may have a length of 30 to 50 amino acids, for example, 30 to 35 amino acids, 35 to 40 amino acids, 40 to 45 amino acids, or 45 to 50 amino acids.

[0050] In some embodiments, the linker is a flexible linker. In some embodiments, the linker is rich in glycine (Gly or G) residues. In some embodiments, the linker is rich in glycine (Gly or G) residues. The linker is rich in serine (Ser or S) residues. In some embodiments, the linker is rich in glycine and serine residues. In some embodiments, the linker has one or more glycine-serine residue pairs (GS), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GS pairs. In some embodiments, the linker has one or more Gly-Gly-Gly-Ser (GGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Gly-Gly-Ser (GGGGS) sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGGGS sequences. In some embodiments, the linker has one or more Gly-Gly-Ser-Gly (GGSG) sequences, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more GGSG sequences. In some embodiments, the linker is GSAAAGGSGGSGGS (SEQ ID NO: 3). In some embodiments, the linker is GGGSGGGS (SEQ ID NO: 4).

[0051] "Native or naturally occurring Notch" is meant to encompass all known forms of the Notch receptor. In humans, four forms of Notch are known. Joanna Pancewicz: BMC Cancer 11(1):502, November 2011. The human Notch family includes four receptors and five ligands.

[0052] In some embodiments, the chimeric Notch receptor polypeptide comprises all or a portion of human Notch1, Notch2, Notch3, or Notch4. In some embodiments, the chimeric Notch receptor polypeptide comprises all or a portion of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. In some embodiments, the "portion" of Notch comprises the three NLR domains, the transmembrane domain, and a short cytosolic fragment containing the native nuclear localization sequence (NLS) of Notch.

[0053] Human neurogenic locus notch homolog protein 1 preprotein NP_060087.3 (SEQ ID NO: 24) RRQHGQLWFPEGFKVSEASKKKRREPLGEDSVGLKPLKNASDGALMDDNQNEWGDEDLETKKFRFEEPVVLPDLDDQTDHRQWTQQHLDAADLRMSAMAPTPPQGEVDADCMDVNVRGPDGFTPLMIASCSGGGLETGNSEEEEDAPAVISDFIYQGASLHNQTDRTGETALHLAARYSRSDAAKRLLEASADANIQD NMGRTPLHAAVSADAQGVFQILIRNRATDLDARMHDGTTPLILAARLAVEGMLEDLINSHADVNAVDDLGKSALHWAAAVNNVDAAVVLLKNGANKDMQNNREETPLFLAAREGSYETAKVLLDHFANRDITDHMDRLPRDIAQERMHHDIVRLLDEYNLVRSPQLHGAPLGGTPTLSPPLCSPNGYLGSLKPGVQGKK VRKPSSKGLACGSKEAKDLKARRKKSQDGKGCLLDSGMLSPVDSLESPHGYLSDVASPPLLPSPFQQSPSVPLNHLPGMPDTHLGIGHLNVAAKPEMAALGGGGRLAFETGPPRLSHLPVASGTSTVLGSSSSGGALNFTVGGSTSLNGQCEWLSRLQSGMVPNQYNPLRGSVAPGPLSTQAPSLQHGMVGPLHSSLAA SALSQMMSYQGLPSTRLATQPHLVQTQQVQPQNLQMQQQNLQPANIQQQQSLQPPPPPPQPHLGVSSAASGHLGRSFLSGEPSQADVQPLGPSSLAVHT ILPQESPALPTSLPSSLVPPVTAAQFLTPPSQHSYSPVDNTPSHQLQVPEHPFLTPSPESPDQWSSSSPHSNVSDWSEGVSSPPTSMQSQIARIPEAFK Human neurogenic locus notch homolog protein 2 isoform 1 preprotein NP_077719.2 (SEQ ID NO: 25) Human neurogenic locus notch homolog protein 2 isoform 2 precursor NP_001186930.1 (SEQ ID NO: 26) MPALRPALLWALLALWLCCAAPAHALQCRDGYEPCVNEGMCVTYHNGTGYCKCPEGFLGEYCQHRDPCEKNRCQNGGTCVAQAMLGKATCRCASGFTGEDCQYSTSHPCFVSRPCLNGGTCHMLSRDTYECTCQVGFTGKECQWTDACLSHPCANGSTCTTVANQFSCKCLTGFTGQKCETDVNECDIPGHCQHGGTCLN LPGSYQCQCPQGFTGQYCDSLYVPCAPSPCVNGGTCRQTGDFTFECNCLPGFEGSTCERNIDDCPNHRCQNGGVCVDGVNTYNCRCPPQWTGQFCTEDVDECLLQPNACQNGGTCANRNGGYGCVCVNGWSGDDCSENIDDCAFASCTPGSTCIDRVASFSCMCPEGKAGLLCHLDDACISNPCHKGALCDTNPLNGQYI CTCPQGYKGADCTEDVDECAMANSNPCEHAGKCVNTDGAFHCECLKGYAGPRCEMDINECHSDPCQNDATCLDKIGGFTCLCMPGFKGVHCELEINECQSNPCV NNGQCVDKVNRFQCLCPPGFTGPVCQIDIDDCSSTPCLNGAKCIDHPNGYECQCATGFTGVLCEENIDNCDPDPCHHGQCQDGIDSYTCICNPGYMGAICSDQI DECYSPCLNDGRCIDLVNGYQCNCQPGTSGVNCEINFDDCASNPCIHGICMDGINRYSCVCSPGFTGQRCNIDIDECASNPCRKGATCINGVNGFRCICPEGP HHPSCYSQVNECLSNPCIHGNCTGGLSGYKCLCDAGWVGINCEVDKNECLSNPCQNGGTCDNLVNGYRCTCKKGFKGYNCQVNIDECASNPCLNQGTCFDDISGY TCHCVLPYTGKNCQTVLAPCSPNPCENAAVCKESPNFESYTCLCAPGWQGQRCTIDIDECISKPCMNHGLCHNTQGSYMCECPPGFSGMDCEEDIDDCLANPCQNGGSCMDGVNTFSCLCLPGFTGDKCQTDMNECLSEPCKNGGTCSDYVNSYTCKCQAGFDGVHCENNINECTESSCFNGGTCVDGINSFSCLCPVGFTGSFCLHEI NECSSHPCLNEGTCVDGLGTYRCSCPLGYTGKNCQTLVNLCSRSPCKNKGTCVQKKAESQCLCPSGGAAYCDVPNVSDIAASRRGVLVEHLCQHSGVCINAG NTHYCQCPLGYTGSYCEEQLDECASNPCQHGATCSDFIGGYRCECVPGYQGVNCEYEVDECQNQPCQNGGTCIDLVNHFKCSCPPGTRGMKSSLSIFHPGHCLKL Human neurogenic locus notch homolog protein 3 precursor NP_000426.2 (SEQ ID NO: 27) Human neurogenic locus notch homolog protein 4 preprotein NP_004548.3 (SEQ ID NO: 28) MQPPSLLLLLLLLLCVSVVRPRGLLCGSFPEPCANGGTCLSLSLGQGTCQCAPGFLGETCQFPDPCQNAQLCQNGGSCQALLPAPLGLPSSPSPLTPSFLCTCLPGFTGERCQAKLEDPCPPSFCSKRGRCHIQASGRPQCSCMPGWTGEQCQLRDFCSANPCVNGGVCLATYPQIQCHCPPGFEGHACERDVNECFQ DPGPCPKGTSCHNTLGSFQCLCPVGQEGPRCELRAGPCPPRGCSNGGTCQLMPEKDSTFHLCLCPPGFIGPDCEVNPDNCVSHQCQNGGTCQDGLDTYTCLCPETWTGWDCSEDVDECETQGPPHCRNGGTCQNSAGSFHCVCVSGWGGTSCEENLDDCIAATCAPGSTCIDRVGSFSCLCPPGRTGLLCHLEDMCLSQP

[0054] In some embodiments, the Notch core of the chimeric Notch receptor polypeptide comprises a portion of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. In some embodiments, the chimeric Notch receptor polypeptide comprises 50 to 1000 amino acids of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. In some embodiments, the chimeric Notch receptor polypeptide comprises 50 to 900 amino acids, 100 to 800 amino acids, 200 to 700 amino acids, 300 to 600 amino acids, or 400 to 500 amino acids of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. In some embodiments, the chimeric Notch receptor polypeptide comprises amino acids 1374 to 1734 of SEQ ID NO:27.

[0055] In some embodiments, the amino acid sequences of Notch described herein are at least 80% identical to the corresponding amino acid sequence of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. In some embodiments, the amino acid sequences of Notch are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acid sequence of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. In some embodiments, the amino acid sequence of Notch described herein may differ from the amino acid sequence of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28 by 1 to 50 amino acids, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids.

[0056] In some embodiments, the mRNA sequence of a Notch described herein is SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33.

[0057] Human notch2 (NOTCH2), transcript variant 1, mRNA NM_024408.3 (SEQ ID NO: 30) TAATAAAAGGGAAGGTAAGATGGATAATCACTTTCTCATTTGGGTTCTGAATTGGAGACTCAGTTTTTATGAGACACATC Human notch2 (NOTCH2), transcript variant 2, mRNA NM_001200001.1 (SEQ ID NO: 31) Human notch3 (NOTCH3), mRNA NM_000435.2 (SEQ ID NO: 32) CGGGAGTAGGAGCAGGAGGAGGCCCGACCCCTCGCGGCCGTAGGTTTTCTGCAGGCATGCGCGGGCCTCGGCCCAACCCTGCGATAATGCGAGGAAGATACGGAGTGGCTGCCGGGCGCGGAGGCAGGGTCTCAACGGATGACTGGCCCTGTGATTGGGTGGCCCTGGGAGCTTGCGGTTCTGCCTCCAACATTCCGATCCCGCCTCCTTGCCTTACTCCGTCCCCGGAGCGGGGATCACCTCAACTTGACTGTGGTCCCCCAGCCCTCCAAGAAATGCCCATAAACCAAGGAGGAGAGGGTAAAAAATAGAAGAATACATGGTAGGGAGGAATTCCAAAAATGATTACCCATTAAAAGGCAGGCTGGAAGGCCTTCCTGGTTTTAAGATGGATCCCCCAAAATGAAGGGTTGTGAGTTTAGTTTCTCTCCTAAAATGAATGTATGCCCACCAGAGCAGACATCTTCCACGTGGAGAAGCTGCAGCTCTGGAAAGAGGGTTTAAGATGCTAGGATGAGGCAGGCCCAGTCCTCCTCCAGAAAATAAGACAGGCCACAGGAGGGCAGAGTGGAGTGGAAATACCCCTAAGTTGGAACCAAGAATTGCAGGCATATGGGATGTAAGATGTTCTTTCCTATATATGGTTTCCAAAGGGTGCCCCTATGATCCATTGTCCCCACTGCCCACAAATGGCTGACAAATATTTATTGGGCACCTACTATGTGCCAGGCACTGTGTAGGTGCTGAAAAGTGGCCAAGGGCCACCCCCGCTGATGACTCCTTGCATTCCCTCCCCTCACAACAAAGAACTCCACTGTGGGGATGAAGCGCTTCTTCTAGCCACTGCTATCGCTATTTAAGAACCCTAAATCTGTCACCCATAATAAAGCTGATTTGAAGTGTTAAAAAAAAAAAAAAAAAA

[0058] In some embodiments, the nucleic acid sequences encoding Notch described herein are at least 80% identical to the sequence of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33. In some embodiments, the nucleic acid sequences encoding Notch are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33. In some embodiments, the Notch nucleic acid sequences described herein may differ from the sequences of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides.

[0059] A "chimeric Notch receptor polypeptide" of the present disclosure comprises: a) an extracellular domain comprising a first member of a specific binding pair; b) a Notch receptor polypeptide, wherein the Notch receptor polypeptide is between 50 and 1000 amino acids in length 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, wherein 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.

[0060] The term "antigen-binding domain" refers to a domain that specifically binds to a target antigen. In some instances, an antigen-binding domain can be formed from amino acids present in a single-chain polypeptide. In other instances, an antigen-binding domain can be formed from amino acids present in a first single-chain polypeptide and amino acids present in one or more additional single-chain polypeptides (e.g., a second single-chain polypeptide). Non-limiting examples of antigen-binding domains are described herein and include, but are not limited to, scFvs or LBDs (ligand-binding domains) of growth factors. Additional examples of antigen-binding domains are known in the art.

[0061] As used herein, the term "antigen" generally refers to the binding partner that is specifically recognized by the antigen-binding domains described herein. Exemplary antigens include polypeptides and peptide fragments thereof, small molecules, lipids, carbohydrates, and nucleic acids. The antigen-binding domains of the present invention may be specifically bound by any of various classes of molecules, including, but not limited to, any of the antigen-binding domains. Non-limiting examples of antigen(s) that can be specifically bound by any of the antigen-binding domains are described herein. Additional examples of antigen(s) that can be specifically bound by any of the antigen-binding domains are known in the art.

[0062] 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, diabodies, 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. The terms also encompass Fab', Fv, F(ab')2, and / or other antibody fragments that retain specific binding to an antigen, as well as monoclonal antibodies. 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.

[0063] As used herein, the term "humanized immunoglobulin" refers to an immunoglobulin comprising portions of immunoglobulins of different origins, at least some of which contain amino acid sequences of human origin. For example, a humanized antibody can contain portions derived from an immunoglobulin of non-human origin, such as a mouse, with the required specificity and immunoglobulin sequences of human origin (e.g., a chimeric immunoglobulin), chemically coupled by conventional techniques (e.g., synthetically) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portions of a chimeric antibody can be expressed to produce a contiguous polypeptide chain). Another example of a humanized immunoglobulin is an immunoglobulin comprising 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 modifications). Chimeric or CDR-grafted single-chain antibodies are also encompassed by the term humanized immunoglobulin. See, for example, U.S. Patent No. 4,816,567 to Cabilly et al., U.S. Patent No. 4,816,397 to Boss et al., WO 86 / 01533 to Neuberger, MS et al., and U.S. Patent No. 5,225,539 to Winter. Also, regarding single chain antibodies, see U.S. Patent No. 4,946,778 to Ladner et al., U.S. Patent No. 5,946,778 to Huston, See, e.g., US Pat. No. 5,476,786, and Bird, RE et al., Science, 242:423-426 (1988).

[0064] The term "nanobody" (Nb) refers to the smallest antigen-binding fragment or single variable domain (V) derived from a naturally occurring heavy chain antibody. HH) They are derived from the heavy chain-only antibodies found in camelids. In the "camelidae" family, immunoglobulins that lack light polypeptide chains are found. "Camelidae" refers to the Old World camelids (Camelus bactrianus and Camelus dromedarius) and the New World camelids (e.g., Llama paco) Alpacas (Llama paccos), Llama glama, Llama guana Guanaco (Llama guanicoe) and Llama vicugna (Llama vicugna) )). Single variable domain heavy chain antibodies are referred to herein as nanobodies or V HH They are called antibodies.

[0065] An "antibody fragment" includes a portion of an intact antibody, such as 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., Trends Biotechnol. 21:484, 2003), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0066] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-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 L The six CDRs define 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 antigen, albeit with lower affinity than the entire binding site.

[0067] 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 used 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 that held the hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0068] 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.

[0069] "Single-chain Fv" or "sFv" or "scFv" antibody fragments include the V of the antibody. H Domain and V L In some embodiments, an Fv polypeptide comprises V domains, and these domains are present in a single polypeptide chain. H Domains and V L The 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 eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0070] The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which fragments contain a light-chain variable domain (V L ) connected to the heavy chain variable domain (V H ) including (V H -V L Diabodies are described in European Patent No. 404,097, International Publication No. WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993.

[0071] The terms "polypeptide," "peptide," and "protein," used interchangeably herein, refer to polypeptides that contain both genetically encoded and non-genetically encoded amino acids. "Amino acid" refers to polymeric forms of amino acids of any length, which may include amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term also includes fusion proteins, with or without an N-terminal methionine residue, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences; immunologically tagged proteins; and the like.

[0072] An "isolated" polypeptide is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the polypeptide is more than 90%, more than 95%, or more than 98% purified.

[0073] The terms "chimeric antigen receptor" and "CAR," used interchangeably herein, refer to an artificial multi-module molecule that generally, but not exclusively, contains an extracellular domain (e.g., a ligand / antigen binding domain), a transmembrane domain, and one or more intracellular signaling domains, and is capable of inducing or inhibiting immune cell activation. The term CAR is not particularly limited to CAR molecules, but also includes CAR mutants, i.e., CAR mutants are described, for example, in PCT Application No. US2014 / 016527, Fedorov et al., Sci Transl.Med.5(215):215ra172, 2013, Glienke et al., Front.Pharmacol.6:21, 2015, Kakarla & Gottschalk, Cancer J.20(2):151-155, 2014, Riddell et al., Cancer J.20(2):141-144, 2014, Pegram et al., Cancer J.20(2):127-33, 2014, Cheadle et al., Immunol Rev. 257(1):91-106, 2014, Barrett et al., Ann.Rev. Med. 65:333-347, 2014, Sadelain et al., Cancer Disov. 3(4):388-98, 2013, and Cartellieri et al., J. Biomed. Biotechnol. 956304, 2010, the entire disclosures of which are incorporated herein by reference. vinegar.

[0074] In the present invention, transcription of a nucleotide sequence is activated by a transcription activator fusion protein (e.g., human RelA protein) composed of an HNF1 DNA-binding domain (e.g., human HNF1 DNA-binding domain), which binds with high selectivity to a selected DNA sequence, fused to a variety of polypeptides responsible for the ligand-dependent activity of the transactivator and its transcriptional activity. The fusion proteins of the present invention are useful for regulating the transcription level of any target gene linked to a selected HNF1 DNA-binding site. The fusion proteins can be used to specifically activate transcription from genes controlled by HNF1-responsive promoters in tissues lacking endogenous HNF1 and vHNF1 proteins. The fusion proteins of the present invention are composed primarily of human elements. A fully human protein reduces the risk of immune recognition of the transactivator. Repressors are also provided in a similar manner.

[0075] U.S. Patent No. 6,277,999 describes various chimeric Notch receptors, how to construct them, and methods for their use. In the examples below detailing how chimeric Notch receptors can be humanized to make them less immunogenic, the chimeric Notch receptors shown in U.S. Patent No. 6,277,999 may be used, for example, in cells of the monocyte / macrophage lineage.

[0076] Certain abbreviations are used throughout to describe the domains of the four human Notch proteins: NEC: extracellular subunit, NTM: transmembrane subunit, EGF: epidermal growth factor, HD: heterodimerization domain, ICN: intracellular domain, LNR: cysteine-rich LNR repeat, TM: transmembrane domain, RAM: RAM domain, NLS: nuclear localization signal, ANK: ankyrin repeat domain, NCR: cysteine-responsive region, TAD: transactivation domain, PEST: region rich in proline (P), glutamine (E), serine (S), and threonine (T) residues.

[0077] method In addition to gene therapy uses, ligand-dependent transcription factors incorporating the humanized DBD of the present invention can be used to regulate the expression of genes contained in recombinant viral vectors that may interfere with viral propagation in packaging cell lines during the production process. These recombinant viruses may be derivatives of adenoviruses, retroviruses, lentiviruses, herpesviruses, adeno-associated viruses, and other viruses familiar to those skilled in the art. Another application would be to provide large-scale production of a toxic protein of interest in vitro using cultured cells that do not contain endogenous HNF1 / vHNF1 and that have been modified to contain a nucleic acid encoding a transactivator bearing a DBD of the present invention in a form suitable for expression of the transactivator in the cells, and a gene encoding the protein of interest operably linked to, for example, an HNF1-dependent promoter.

[0078] To induce or repress transcription in vivo, the ligand can be administered (e.g., by injection) to the body or tissue of interest. The body to be treated can be that of an animal, particularly a mammal, and the mammal can be a human or a non-human such as a rabbit, guinea pig, rat, mouse or other rodent, cat, dog, pig, sheep, goat, cow or horse, or an avian such as a chicken. Suitable routes of administration include oral, intraperitoneal, intramuscular, or intravenous (iv).

[0079] One convenient way of producing a polypeptide or fusion protein according to the invention is to express a nucleic acid encoding the polypeptide or fusion protein by using the nucleic acid in an expression system. Thus, the present invention also provides, in various aspects, nucleic acids encoding the transcriptional activators or transcriptional repressors of the invention, which may be used to produce the encoded protein.

[0080] Generally, nucleic acids, whether encoding a protein or component according to the invention, are provided in isolated and / or purified form, or as isolates that are free or substantially free of naturally associated materials, such as free or substantially free of nucleic acids flanking genes in the human genome, possibly except for one or more regulatory sequence(s) for expression. Nucleic acids may be wholly or partially synthetic and may comprise genomic DNA, cDNA, or RNA. Where a nucleic acid according to the invention comprises RNA, reference to the sequence shown should be interpreted as including reference to the RNA equivalent in which T is substituted for U.

[0081] Nucleic acid sequences encoding the polypeptides or fusion proteins according to the present invention can be easily prepared by those skilled in the art using the information and references contained herein and techniques known in the art (Sambrook, et al., A Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989-2016), and Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, (1994-2016)). Techniques include (i) the use of polymerase chain reaction (PCR) to amplify a sample of such nucleic acid, for example, from a genomic source; (ii) chemical synthesis; or (iii) the preparation of cDNA sequences. DNA encoding a portion of a full-length coding sequence (e.g., a DNA-binding domain, or, in some cases, a regulatory domain) can be generated and used by any suitable method known to those of skill in the art, including taking the encoding DNA, identifying suitable restriction enzyme recognition sites on either side of the portion to be expressed, and excising the portion from the DNA. The portion can then be operably linked to a suitable promoter in a standard, commercially available expression system. Another recombinant approach is to amplify the relevant portion of DNA with suitable PCR primers. Modifications of the relevant sequence can be made, for example, using site-directed mutagenesis, to result in expression of an altered peptide or to a nucleic acid used to express the nucleic acid. The codon preference of the host cell in which the gene is to be expressed can be taken into consideration.

[0082] To obtain expression of a nucleic acid sequence, the sequence may be incorporated into a vector having one or more control sequences operably linked to the nucleic acid to control its expression. The vector may also include other sequences, such as a promoter or enhancer, that drive expression of the inserted nucleic acid, a nucleic acid sequence that causes a polypeptide or peptide to be produced as a fusion, and / or a nucleic acid encoding a secretion signal that causes a polypeptide produced in a host cell to be secreted from the cell. The polypeptide can then be obtained by transforming the vector into a host cell in which the vector functions, culturing the host cell so that the polypeptide is produced, and recovering the polypeptide from the host cell or the surrounding medium. E. coli, yeast, and Prokaryotic and eukaryotic cells are used for this purpose in the art, including eukaryotic cell lines such as COS or CHO cells.

[0083] Thus, the present invention also encompasses methods of making the disclosed polypeptides or fusion proteins, which methods involve expression from a nucleic acid encoding the product (generally a nucleic acid according to the invention). This can be conveniently achieved by growing host cells containing such a vector in culture under appropriate conditions that cause or allow expression of the polypeptide. Polypeptides can also be expressed in in vitro systems.

[0084] Systems for cloning and expressing polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of heterologous polypeptides include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells, and many others. A common, preferred bacterial host is E. coli.

[0085] Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as appropriate. Vectors may be plasmids, viruses, such as phage or phagemid, as appropriate. For further details, see, for example, Molecular cloning: a Laboratory Manual: 4th edition, Green and Sambrook et al., See, for example, "Current Protocols in Molecular Biology," Ausubel et al., Eds., John Wiley & Sons, 2016. Many known techniques and protocols for manipulating nucleic acids, for example, in preparing nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells and gene expression, and analyzing proteins, are described in detail in "Current Protocols in Molecular Biology," Ausubel et al., Eds., John Wiley & Sons, 2016.

[0086] For use in mammalian cells, the recombinant expression vector's control functions can be provided by viral genetic material. Exemplary promoters include those derived from polyoma, adenovirus 2, cytomegalovirus, and SV40.

[0087] The regulatory sequences of the recombinant expression vectors used in the present invention can direct the expression of a polypeptide or fusion protein preferentially in a particular cell type, i.e., tissue-specific regulatory elements can be used. In one embodiment, the recombinant expression vectors of the present invention are plasmids. Alternatively, the recombinant expression vectors of the present invention can be viruses or parts of viruses that allow expression of nucleic acids introduced into the viral nucleic acid. For example, replication-defective retroviruses, adenoviruses, and adeno-associated viruses can be used. Protocols for producing recombinant retroviruses and infecting cells in vitro or in vivo with such viruses can be found in Ausubel, et al. (supra). Adenoviruses The genomes of viruses such as HIV-1 encode transactivator or repressor proteins. The virus can be engineered to be activated and expressed but inactivated with respect to its ability to replicate in a normal lytic viral life cycle.

[0088] Accordingly, a further aspect of the present invention provides a host cell comprising a heterologous nucleic acid as disclosed herein.

[0089] Furthermore, recombinant expression vectors can be designed to effect homologous recombination between a nucleic acid encoding a transactivator or repressor and a target gene in a host cell, and such homologous recombination vectors can be used to generate homologously recombinant animals that express the fusion proteins of the invention.

[0090] Examples of mammalian cell lines that can be used include CHO dhfr cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980), 293 cells (Graham et al., J. Gen. Virol. 36:59, 1977), and myeloma cells such as SP2 or NS0 (Meth. Enzymol. 73(B):3-46, 2016). In addition to cell lines, the present invention is applicable to normal cells, such as cells modified for gene therapy purposes or embryonic cells modified to create transgenic or homologously recombinant animals. Examples of cell types of particular interest for gene therapy purposes include hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, muscle cells, neural cells, and skin and airway epithelia. Furthermore, in the case of transgenic or homologously recombinant animals, embryonic stem cells and fertilized oocytes may be modified to contain nucleic acids encoding transactivator or repressor fusion proteins. [Example]

[0091] The following examples are included to demonstrate preferred embodiments of the invention. It will be understood by those of skill in the art that the techniques disclosed in the examples below are techniques discovered by the inventors to work well in the practice of the invention and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0092] All four human Notch proteins (Notch1-4) were tested for the ability of their core LNR, HD, and transmembrane domains to selectively release the GAL4-VP16 transcription factor fused to its C-terminal intracellular portion in response to an N-terminal extracellular CD19 ScFv fusion binding to its cognate antigen. Human Notch2 and Notch3 released functional amounts of transcription factor upon antigen binding. Human Notch1 released small amounts of transcription factor in response to antigen binding, whereas human Notch4 did not release detectable amounts of transcription factor. Human Notch3 showed the highest functional release of transcription factor in response to antigen binding and was used in most designs.

[0093] We further modified the minimal LIN12-HD transmembrane "core" Notch2 and Notch3 domains to include an extra short (approximately 60 aa) intracellular domain containing the native Notch nuclear localization sequence (NLS) to improve nuclear import upon self-cleavage and release of the transcription factor domain.

[0094] To minimize the immunogenicity of chimeric Notch receptors, a series of synthetic humanized transcription factors were designed and constructed from (1) a minimized human DNA-binding domain (DBD) and (2) a minimized potent transactivation domain (TAD). The reason for creating non-naturally occurring, yet humanized, chimeras is to eliminate undesired endogenous cofactor interactions between the chimeric Notch receptor-releasing humanized transcription factors and the natural binding partners with which the full-length human transcription factors may interact. This is to improve the robustness and predictability of transcriptional responses induced by chimeric antigen receptors in cellular applications that utilize humanized antigen receptors.

[0095] A comprehensive screen of human transcription factors was performed to find naturally occurring DNA-binding domains that fulfilled several criteria: (1) the DNA-binding domain belonged to a transcription factor that is not generally naturally expressed in the target host cell type. In this embodiment, we sought a DNA-binding domain that is not present in any hematopoietic lineage, particularly lymphoid and T-cell lineages, and (2) the DNA-binding domain bound to its target DNA sequence with high affinity, with a dissociation constant of 10 nM or less.

[0096] The DNA-binding domains were first tested for their ability to bind to the multisite synthetic promoter by expressing the DNA-binding domain fused to the natural transactivation domain and verifying that they could upregulate GFP driven by the synthetic multisite promoter, thereby validating the correct pairing of the designed cognate promoter and DNA-binding domain.

[0097] The validated DNA-binding domains were then tested as fusions to synNotch along with a potent transactivation domain and assayed for their ability to upregulate a cognate multisite promoter driving GFP upon stimulation with external antigen and release into the nucleus.

[0098] Examples of human DNA-binding domains tested with this strategy include human CRX (Furukawa, Takahisa, Eric M. Morrow, and Constance L. Cepko. "Crx, a novel otx-like homeobox gene, shows photoreceptor-specific expression and regulates photoreceptor differentiation." Cell 91.4 (1997):531-541, / / doi.org / 10.1016 / S0092-8674(00)80439-0), PO U1F1(Jacobson, Eric M., et al. "Structure of Pit-1 POU Domain Bound to DNA as a Dimer: Unexpected Arrangement and Flexibility." Gerald, and Giuseppe Cibelli (Nature biotechnology 33.5 (2015):555-562. doi:10.1038 / nbt.3128) and ZNF528(Najafabadi, Hamed S., et al."C2H2 zinc finger proteins greatly expand the human regulatory lexicon." 33.5(2015):555-562, doi:10.1038 / nbt.3128). All DNA-binding domains were able to induce strong GFP expression under the control of their cognate promoters when expressed as soluble transcription factors. However, when expressed and released from chimeric Notch fusion constructs, only the HNF1A and EGR1 DNA-binding domains were able to induce detectable GFP expression under their cognate promoters. Because only a small fraction of the expressed chimeric Notch protein undergoes self-cleavage in response to antigen-binding stimulation, the effective concentration of the released, nuclear-translocated transcription factor is much lower than that of directly expressed transcription factors. Therefore, to be functional, chimeric Notch-released transcription factors must exhibit very strong binding to their cognate promoters.

[0099] The human transactivation domains were expressed as fusions to the Gal4 DNA-binding domain and chimeric Notch designs were screened for activity by measuring the relative levels of GFP expression under the control of the cognate Gal4 multisite promoter, which were also compared with the GFP expression levels induced by the nonhuman VP64 transactivation domain.

[0100] Examples of human transactivation domains that have been screened using this method include RelA(p65) (Wang, Weixin, et al. "The nuclear factor-κB RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells." Clinical Cancer Research 5.1 (1999):119-127), YAP (Lian, Ian, et al. "The role of YAP transcription coactivator in regulating stem cell self-renewal and differentiation." Genes & development 24.11 (2010):1106-1118, doi:10.1101 / gad.1903310), and WWTR1(TAZ) (Hong, Jeong-Ho, et al. "TAZ, a transcriptional modulator of mesenchymal stem cell differentiation." Science 309.5737 (2005):1074-1078). doi:10.1126 / science.1110955), CREB3(LZIP)(Omori, Yoshihiro, et al. "CREB-H: a novel mammalian transcription factor belonging to the CREB / ATF family and functioning via the box-B element with a liver-specific expression." Nucleic acids research 29.10 (2001):2154-2162, doi: / / doi.org / 10.1093 / nar / 29.10.2154), and MyoD (Weintraub, Harold, and Robert Davis. "The myoD gene family: nodal point during specification of the muscle cell lineage." Science 251.4995 (1991):761, doi:10.1126 / science.1846704). Of these, the transactivation domains of RelA (p65), WWTR1 (TAZ), and CREB3 (LZIP) showed activity in chimeric Notch. The activity of the transactivation domain of RelA (p65) was determined to be the most potent in inducing GFP expression.

[0101] Combining the best performing human Notch domain, the best performing DNA binding domain and the best performing transactivation domain results in the design of Notch3-HNF1a-p65 for a chimeric humanized Notch receptor.

[0102] There are many uses for humanized chimeric Notch receptors, such as for delivering CARs or t-cell receptors to treat diseases.

[0103] References to nucleotide or protein sequences below generally refer to sequences in the National Center for Biotechnology Information (NCBI) (ncbi.nlm.niv.gov). All nucleotide sequences are 5' to 3'.

[0104] Example 1. Construction of a chimeric Notch using Notch3, the DNA binding domain of HNF1 alpha, and the p65 transactivation domain The following sequences were amplified by annealing with a short 3' reverse complement oligo and using Phusion polymerase (Thermo Scientific™ Phusion™ High Fidelity DNA Polymerase). Double-stranded synthetic DNA fragments (IDT gBlock) or single-stranded long oligonucleotides (IDT Ultra) were double-stranded by second-strand synthesis using IDT ELISA (catalog number F534S). I ordered it as Lamar.

[0105] Four synthetic dsDNA pieces were ordered from Integrated DNA Technologies (IDT), including: 1. Human CD8a signal peptide 1 to 22 (NP_001139345 amino acids 1 to 22 (MALPVTALLLPLALLLHAARPS) (SEQ ID NO: 1)), Myc tag (EQKLISEEDL) (SEQ ID NO: 2), anti-human B cell (CD19) antibody, clone FMC63. 2. Human Notch3 core (gi|134244285|NP_000426.2 amino acids 1374 to 1734). 3. GS flexible linker (GSAAAGGSGGSGGS) (SEQ ID NO: 3), human HNF1 alpha (gi|807201167|NP_001293108.1 amino acids 1-283), GS flexible linker (GGGSGGGS) (SEQ ID NO: 4). 4. Human Rel-A (p65) (gi|223468676|NP_068810.3 amino acids 1 to 551) and stop codon.

[0106] These were designed to incorporate 20 nt of homology with the 5' and 3' flanking fragments for in vitro recombination using the In-fusion cloning system (Clontech). All fragments were cloned into MluI / NotI-cleaved vectors of the self-inactivating lentiviral vector pHR-SIN:SFFV (Addgene; catalog no. 79121). The skeleton was assembled by in-fusion.

[0107] The second reporter construct was constructed by assembling three synthetic dsDNA fragments: 1. A tetrad-repeat palindromic DNA binding sequence for the HNF1a DNA binding domain dimer, immediately followed by a minimal CMV promoter. atcgatGTTAATaATTAACatatatGTTAATcATTAACtataGTTAATtATTAACcgctatGTTAATgATTAACactagttaggcgtgtacggtgggaggcc tatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagagacaccgggaccgatccagc (SEQ ID NO: 34) 2. The Kozak sequence (GCCGCCACC) (SEQ ID NO: 35) and the coding sequence for EGFP. 3.EF1α promoter sequence 4. Kozak sequence (GCCGCCACC) (SEQ ID NO: 35) and coding sequence for mCherry.

[0108] These fragments were designed to incorporate an additional 20–25 nt of homology with the 5′ and 3′ flanking fragments for in vitro recombination using the In-Fusion Cloning System (Clontech). All fragments were assembled by in-fusion reaction into the MluI / NotI-cleaved vector backbone of the self-inactivating lentiviral vector pHR-SIN:SFFV.

[0109] The lentiviral construct was then transfected into the virus packaging plasmid pCMVdR8.91 and pCMVdR8.92 using the transfection reagent FuGENE HD (Roche). pMD2.G was co-transfected into 293T cells. Amphotropic VSV-G pseudotyped lentiviral particles were collected from the supernatant 48 hours later.

[0110] Viral particles derived from both the synnotch and reporter constructs were used to simultaneously transduce Jurkat cells or primary CD4+ / CD8+ pan-T cells derived from human donors. A detailed description of the lentiviral protocol can be found in Morsut et al. Cell. 2016 Feb 11;164(4):780-91.

[0111] Transduced Jurkat cells were tested for expression 2 days after transduction, and transduced human primary pan-T cells were tested for expression 7 days after transduction. Expression of the synnotch construct was tested by labeling the expressed cell surface Myc tag marker with an alexa-647 conjugated anti-myc antibody (Cell Signaling Technology, Myc tag (9B11) mouse mAb (Alexa Fluor™ 647 conjugate; catalog number 2233).

[0112] Expression of the cognate reporter construct for synnotch was tested by observing constitutive mCherry expression generated from the reporter vector. For SEQ ID NO: 1, double-positive cells were selected.

[0113] Cells expressing both the synnotch construct and its reporter were isolated using magnetic beads coated with anti-Myc tag antibody (obtained from Thermofisher Scientific, catalogue number 1011111). Synnotch activity was assayed by stimulating cells for 24 hours with either anti-HA tag antibody-coated magnetic beads (obtained from Pierce™, anti-HA magnetic beads, catalog number 88842) or anti-HA tag antibody-coated magnetic beads (anti-HA magnetic beads, catalog number 88836) as a negative control. The mean fluorescence intensity of reporter EGFP expression in response to antibody binding stimulation was measured in stimulated cells relative to the mean fluorescence intensity of negative control stimulated cells.

[0114] Cells expressing both the synnotch construct and its reporter were further assayed for synnotch activity by stimulating the cells for 24 hours by co-incubation with the Raji cell line (American Type Culture Collection (ATCC) CCL-86™ (Raji)), which expresses high levels of the CD19 antigen, and with a cell line negative for cell surface CD19. The mean fluorescence intensity of 9EGFP expression of the co-transfected reporter in response to cell-bound antigen stimulation was measured in stimulated cells relative to the mean fluorescence intensity of negative control stimulated cells.

[0115] Example 2. Construction of chimeric Notch using Notch3, EGR1 DNA binding domain and p65 transactivation domain The construction of the vector was similar to the construct in Example 1, except that the synthetic DNA fragment containing the DNA binding domain of human HNF1a was replaced with the following containing the human EGR1 DNA binding domain: GS flexible linker (GSAAAGGSGGSGGS) (SEQ ID NO: 3), human EGR1 (genbank NP_001955 amino acids 333 to 423), GS flexible linker (GGGSGGGS) (SEQ ID NO: 4)

[0116] The reporter construct contained four cognate binding sites, a five-repeat DNA binding sequence for the EGR1 DNA-binding domain dimer, immediately followed by a minimal CMV promoter: acccggggggacagcagagatccagtttatcgatGCGTGGGCGataGCGGGGGCGtatGCGTGGGCGattGCGGGGGCGttaGCGTGGGCGactagttaggcgtgtacggt gggaggcctataaagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagc (SEQ ID NO: 34)

[0117] Example 3. Construction of the above example using the WWTR1 (TAZ) transactivation domain The construction of the vector was identical to that of Examples 1 and 2, except that the synthetic DNA fragment containing the transactivation domain of human RelA (p65) was replaced with the following containing the transactivation domain of human WWTR1: Human WWTR1 (TAZ) (Genpept NP_056287.1 amino acids 165-395) and a stop codon.

[0118] Example 4. Construction of the above example using the CREB3 (LZIP) transactivation domain The construction of the vector was identical to that of Examples 1 and 2, except that the synthetic DNA fragment containing the transactivation domain of human RelA (p65) was replaced with the following containing the transactivation domain of human CREB3 (LZIP): Human CREB3 (LZIP) (Genpept NP_006359.3 amino acids 1-95) and a stop codon.

[0119] Example 5. Construction of the above example using human Notch2 domain The construction of the vector was identical to the example above, except that the synthetic DNA fragment containing the minimized human notch3 lin12-HD-NLS domain was replaced with the following fragment containing the minimized LIN12-HD-NLS domain of human notch2: human Notch2 core (gi|24041035|NP_077719.2) amino acids 1413 to 1780.

[0120] Example 6. Transduction of monocyte-derived macrophages with chimeric Notch constructed from Notch3, the DNA-binding domain of HNF1 alpha, and the p65 transactivation domain Both mouse Notch1 and human Notch3 proteins were tested for their ability to selectively release Gal4-VP64 (in the case of mouse Notch) or HNF1a-p65 (in the case of human Notch), transcription factors with the core LNR, HD, and transmembrane domains fused C-terminally to the intracellular portion of the protein, in response to binding of the N-terminal extracellular CD19 scFv fusion portion of each protein to its cognate antigen in human monocyte-derived macrophages. Human Notch chimeric proteins were constructed as described herein. Mouse Notch chimeric proteins were constructed as described in U.S. Patent No. 5,629,999.

[0121] The transfection reagent FuGENE HD (Roche) was used to transfect the virus package. The lentiviral constructs were co-transfected into 293T cells with the cloning plasmids pCMV-dR8.91 and pMD2.G and the pVpx plasmid. Amphotropic VSV-G pseudotyped lentiviral particles in the supernatant were collected 48 hours later. Various dilutions of the viral supernatant were used to infect Jurkat cells, and VCN was determined using ddPCR at 7 days postinfection.

[0122] Human macrophages were derived from monocytes isolated from freshly isolated (within 8 hours) healthy adult blood (AllCells Inc.). CD14+ monocytic cells were isolated using RosetteSep negative control. Sex selection (STEMCELL Technologies, RosetteSep™ human monocyte enrichment cocktail) CD14+ cells were enriched from blood using a 24-well plate containing 3x10 CD14+ cells in 1 mL of medium. CD14+ cells were differentiated into macrophages as previously described (Hrecka et al., Nature 2011). Briefly, 24-well plates were seeded with 3x10 CD14+ cells in 1 mL of medium. 5The cells were plated at a density of 100 cells / mL. The culture medium consisted of Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 μg / mL penicillin G, 100 μg / mL streptomycin, and 10 ng / mL macrophage colony-stimulating factor (M-CSF, Miltenyi Biotec) from days 0 to 2 and 20 ng / mL from day 2 onwards.

[0123] Viral particles derived from both synNotch and reporter constructs were used to simultaneously transduce monocyte-derived macrophage cells from human donors 4 days after isolation. Cells were transduced with either human Notch3, HNF1a DNA-binding domain, and p65 transactivation domain (hNotch3 / HNF1a / p65) or mouse Notch1, Gal4 DNA-binding domain, and VP64 transactivation domain (mNotch1 / Gal4 / VP64) at a range of multiplicities of infection (MOI) (0.1-1). Further description of the lentiviral protocol can be found in Morsut L, et al. Cell. 2016 Feb 11;164(4):780-91.

[0124] Transduced human primary bone marrow cells were tested for expression 7 days after transduction by flow cytometry. PE-Cy7 anti-CD14+ antibody (BD Biosciences, PE-Cy™ 7 Mouse Anti-Human CD14 Antibody (Clone M5E2(RUO)), Cat. No. 55 Expression of the synNotch constructs in bone marrow cells was tested by labeling the bone marrow cells with a cell surface-expressed Myc tag marker containing Myc-Tag (9B11) mouse mAb (AlexaFluor™ 647 conjugated; catalog no. 2233), and an alexa-647-conjugated anti-my antibody (Cell Signaling Technology, Myc-Tag (9B11) mouse mAb (AlexaFluor™ 647 conjugated; catalog no. 2233)).

[0125] Expression of the synNotch cognate reporter construct was tested by measuring constitutive mCherry expression resulting from the reporter vector by flow cytometry.

[0126] Cells were assayed for synNotch activity by stimulating them for 24 hours by co-culturing them with the Daudi cell line (American Type Culture Collection (ATCC) CCL-213™ cells (Daudi cells)), which expresses high levels of CD19 antigen, and a cell line negative for cell surface CD19.

[0127] The fluorescence intensity of EGFP expression of the co-transduced reporter in response to cell-bound antigen stimulation was measured in these CD14+ monocyte-derived macrophages when stimulated with antigen-positive CD19+ cells relative to the fluorescence intensity of negative control stimulated cells.

[0128] Overall, in monocyte-derived macrophages, the chimeric humanized Notch receptor, human Notch3-HNF1a-p65, induced unregulated expression of the reporter construct. Notch, DNA-binding domain, and transactivation domain components of the protein were functional in macrophages. The chimeric mouse Notch receptor, Notch1-Gal4-VP64, did not induce selective expression of GFP in response to an N-terminal extracellular CD19 scFv fusion binding to its cognate antigen, compared with a negative control lacking any CD19 expression. See Figures 2, 3A, 3B, 4, 5A, and 5B.

Claims

1. 1. A nucleic acid comprising a nucleotide sequence encoding a chimeric Notch polypeptide, the nucleic acid comprising, covalently linked from N-terminus to C-terminus, a) an extracellular domain comprising a binding factor that specifically binds to an antigen; b) said receptor polypeptide comprising one or more proteolytic cleavage sites; and c) an intracellular domain comprising a transcriptional regulator, wherein binding of the binding factor to the antigen induces cleavage of the Notch receptor polypeptide at the one or more proteolytic cleavage sites to release the intracellular domain and the transcriptional regulator, and the transcriptional regulator comprises a DNA-binding domain of human origin and a transactivation domain of human origin.

2. The nucleic acid of claim 1 , wherein the binding agent comprises an antibody.

3. The nucleic acid of claim 2, wherein the antibody is selected from the group consisting of an scFv, a bispecific antibody, a nanobody, or a bite.

4. The nucleic acid of claim 3 , wherein the transcriptional regulatory factor is a transcriptional activator.

5. The nucleic acid of claim 3 , wherein the transcriptional regulatory factor is a transcriptional repressor.

6. The nucleic acid of claim 1 , wherein the transcriptional regulator is from the HNF transcriptional regulator family.

7. The nucleic acid of claim 6 , wherein the transcriptional regulator is HNF1 alpha or HNF1 beta.

8. 8. The nucleic acid of claim 7, wherein the transactivation domain is selected from the group consisting of RelA (p65), YAP, WWTR1 (TAZ), CREB3 (LZIP), and MyoD.

9. A recombinant vector comprising the nucleic acid of claim 1.

10. A recombinant vector comprising the nucleic acid of claim 8.

11. A host cell transformed with the nucleic acid of claim 1.

12. The host cell of claim 11, which is a macrophage.

13. The host cell of claim 12 , wherein the macrophage is derived from a monocyte.

14. A method for producing a chimeric Notch polypeptide comprising a transcriptional regulator, the transcriptional regulator comprising a DNA binding domain of human origin, the method comprising culturing a host cell described in claim 11.

15. A chimeric Notch polypeptide comprising a humanized transcriptional regulator.

16. 16. The chimeric Notch polypeptide of claim 15, wherein the humanized transcriptional regulator is derived from the HNF transcriptional regulator family.

17. 17. The chimeric Notch polypeptide of claim 16, wherein the humanized transcriptional regulator comprises HNF1 alpha or HNF1 beta.

18. A method of treating a disease in a patient in need thereof, comprising treating the patient with a chimeric Notch polypeptide comprising a humanized transcriptional regulator.

19. 20. The method of treating a disease in a patient according to claim 18, comprising a humanized transcriptional regulator selected from the HNF1 transcriptional regulator family.

20. 20. The method of treating a disease according to claim 19, wherein the disease is cancer.

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