WO2022204324A1 - Hybrid receptors with multiple transcriptional regulators - Google Patents

Hybrid receptors with multiple transcriptional regulators Download PDF

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Publication number
WO2022204324A1
WO2022204324A1 PCT/US2022/021605 US2022021605W WO2022204324A1 WO 2022204324 A1 WO2022204324 A1 WO 2022204324A1 US 2022021605 W US2022021605 W US 2022021605W WO 2022204324 A1 WO2022204324 A1 WO 2022204324A1
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Prior art keywords
cell
chimeric receptor
domain
receptor
cells
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PCT/US2022/021605
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English (en)
French (fr)
Inventor
Axel Hyrenius WITTSTEN
Kole. T. ROYBAL
Julia Garcia
Iowis ZHU
Raymond Liu
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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Priority to AU2022246086A priority Critical patent/AU2022246086A1/en
Priority to CN202280032802.3A priority patent/CN117597140A/zh
Priority to US18/551,931 priority patent/US20240165163A1/en
Priority to EP22776599.7A priority patent/EP4313111A4/en
Priority to JP2023558163A priority patent/JP2024511423A/ja
Priority to CA3213189A priority patent/CA3213189A1/en
Publication of WO2022204324A1 publication Critical patent/WO2022204324A1/en
Anticipated expiration legal-status Critical
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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Definitions

  • the present disclosure relates generally to the field of immunology, and particularly relates to hybrid chimeric antigen receptors designed to combine fast time-scale intracellular signal transduction and long time-scale transcription regulation.
  • the disclosure also provides compositions and methods useful for producing such receptors, nucleic acids encoding same, host cells genetically modified with the nucleic acids, as well as methods for modulating an activity of a cell and/or for the treatment of various health conditions or diseases, such as cancers.
  • Notch receptors are transmembrane proteins that mediate cell-cell contact signaling and play a central role in development and other aspects of cell-to-cell communication. Notch receptors are involved in and are required for a variety of cellular functions during development, and are important for the function of a vast number of cell-types across species.
  • a number of existing first-generation synthetic derivatives of Notch receptors which are often referred to as “SynNotch” have been developed recently by replacing the extracellular ligand-binding domain, which in wild-type Notch contains multiple EGF-like repeats, with an antibody derivative, and replacing the cytoplasmic domain with a transcription activator of choice, but still relying on the Notch NRR (L. Morsut et al., Cell (2016) 164:780-91) and the standard two-step proteolysis.
  • the NRR spans approximately 160 amino acids, making this domain alone about three times the size of some mature proteins, such as insulin or epidermal growth factor (EGF).
  • first-generation SynNotch and the second-generation SynNotch receptors in contrast to chimeric antigen receptor (CARs), do not elicit membrane proximal signaling via kinase cascades.
  • the receptors instead, translate ligand-binding to release of a receptor-tethered transcription factor that shuttles to the nucleus to regulate a user-defined transcriptional circuit.
  • these receptors lack the ability to initiate fast time-scale signaling that regulates cellular processes such as metabolic reprogramming, proliferation, growth factor production, or cytotoxicity.
  • the present disclosure provides, among other things, a new class of hybrid SynNotch receptors that incorporate intracellular signaling domains (e.g. stimulation domains and co stimulation domains of a CAR, for example, co-stimulation domains from 4-1BB, CD28, and a cytoplasmic tail of CD3zeta, etc.) that can initiate activation of T cells concomitant with custom transcriptional regulation.
  • intracellular signaling domains e.g. stimulation domains and co stimulation domains of a CAR, for example, co-stimulation domains from 4-1BB, CD28, and a cytoplasmic tail of CD3zeta, etc.
  • a chimeric receptor comprising, from N- terminus to C-terminus: a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) a linking polypeptide; c) a transmembrane domain (TMD) comprising one or more ligand-inducible proteolytic cleavage sites; and d) an intracellular domain (ICD).
  • the ICD comprises, in any order: (i) an intracellular signaling domain (SD) comprising at least one costimulatory domain derived from a signaling molecule and an activation domain, and (ii) a transcriptional regulator.
  • binding of the selected ligand to the extracellular ligand-binding domain induces cleavage at a ligand-inducible proteolytic cleavage site disposed between the ICD and the linking polypeptide. In other embodiments, binding of the selected ligand to the extracellular ligand-binding domain induces proximal signaling cascades through the intracellular SD.
  • the chimeric receptor does not comprise a LIN-12-Notch repeat (LNR) and/or a heterodimerization domain (HD) of a Notch receptor.
  • the extracellular domain comprises an antigen-binding moiety capable of binding to a ligand on the surface of a cell.
  • the cell is a pathogenic cell.
  • the cell is a human cell.
  • the human cell is a tumor cell.
  • the human cell is a terminally differentiated cell.
  • the ligand comprises a protein or a carbohydrate. In certain embodiments, the ligand is selected from the group consisting of CD1, CD la, CD lb, CDlc,
  • CD 134, CD 140 (PDGFR4), CD152, CD154, CD158, CD178, CD181 (CXCR1), CD182 (CXCR2), CD 183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD- L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placental-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), blue fluorescent protein (BFP) enhanced green fluorescent protein (EGFP), and signal regulatory protein a (SIRPa).
  • BCMA B-cell maturation antigen
  • BFP green fluorescent protein
  • BFP blue fluorescent protein
  • EGFP signal regulatory protein a
  • SIRPa signal regulatory protein a
  • the ligand is selected from cell surface receptors, adhesion proteins, integrins, mucins, lectins, tumor-associated antigens, and tumor-specific antigens. In some embodiments, the ligand is a tumor-associated antigen or a tumor-specific antigen. In some embodiments, the extracellular ligand-binding domain comprises the ligand-binding portion of a receptor.
  • the antigen-binding moiety is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, a minibody, an F(ab')2 fragment, an F(ab)v fragment, a single chain variable fragment (scFv), a single domain antibody (sdAb), and a functional fragment thereof.
  • the antigen-binding moiety comprises an scFv.
  • the antigen-binding moiety specifically binds to a tumor- associated antigen selected from the group consisting of CD 19, B7H3 (CD276), BCMA (CD269), ALPPL2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2/neu), IGLL1, IL-1 IRa, KIT (CD117), MUC1, NCAM, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1/CD248, TEM7R, TSHR, V
  • the tumor-associated antigen is CD 19, BCMA,
  • the linking polypeptide of the chimeric receptor provided herein comprises a hinge domain.
  • the hinge domain is capable of promoting oligomer formation of the chimeric polypeptide via intermolecular disulfide bonding.
  • the hinge domain is derived from a CD8a hinge domain, a CD28 hinge domain, a CD 152 hinge domain, a PD-1 hinge domain, a CTLA4 hinge domain, an 0X40 hinge domain, an IgGl hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, and an IgG4 hinge domain, or a functional variant of any thereof.
  • the linking polypeptide is derived from the group selected from: a CD8a hinge domain or a functional variant thereof, a CD28 hinge domain or a functional variant thereof, 0X40 hinge domain or a functional variant thereof, and an IgG4 hinge domain or a functional variant thereof.
  • the linking polypeptide is derived from a CD8a hinge domain or a functional variant thereof. In other embodiments, the linking polypeptide is derived from an CD28 hinge domain or a functional variant thereof. In some specific embodiments, the linking polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3.
  • the one or more ligand-inducible proteolytic cleavage sites comprises a g secretase cleavage site.
  • the TMD comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 4.
  • the chimeric receptor of the present disclosure further comprises a stop-transfer-sequence (STS) positioned between the TMD and the ICD.
  • STS stop-transfer-sequence
  • the stop-transfer- sequence comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5.
  • the signaling molecule comprises a class 1 or a class 3 human membrane protein.
  • the signaling molecule is selected from the group consisting of CD28, ICOS, CTLA4, PD1, PD1H, BTLA, B71, B7H1, CD226, CRT AM, TIGIT, CD96, TIMl, TIM2, TIM3, TIM4, CD2, SLAM, 2B4, Lyl08, CD84, Ly9, CRACC, BTN1, BTN2, BTN3, LAIRl, LAG3, CD160, 4-1BB, 0X40, CD27, GITR, CD30, TNFR1, TNFR2, HVEM, LT_R, DR3, DCR3, FAS, CD40, RANK, OPG, TRAILRl, TACI, BAFFR, BCMA, TWEAKR, EDAR, XEDAR, RELT, DR6, TROY, NGFR, CD22, SIGLEC-3, SIGLEC
  • the signaling molecule is selected from the list consisting of 0X40, ICOS, 4-1BB, CTLA4, CD28, CD30, CD2, CD27, and CD226.
  • the activation domain comprises one or more immunoreceptor tyrosine-based activation motifs (IT AMs).
  • IT AMs immunoreceptor tyrosine-based activation motifs
  • the one or more IT AMs are derived from O ⁇ 3z, CD3o, CD3/, and CD3e.
  • the one or more ITAMs have at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to a O ⁇ 3z ITAM.
  • the transcriptional regulator comprises a transcriptional activator or a transcriptional repressor.
  • the transcriptional regulator further comprises a nuclear localization sequence (NLS) derived from a protein selected from the group consisting of Gal4, tetR, ZFHD1, and HAPl, and wherein the transcriptional regulator comprises a transactivation domain derived from a protein selected from the group consisting of VP64, VP65, KRAB, and VP 16.
  • NLS nuclear localization sequence
  • the chimeric receptor provided herein comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOS: 15-31 and 34-45. [0023] In certain embodiments, the chimeric receptor provided herein further comprises a signal sequence, a detectable label, a tumor-specific cleavage site, a disease-specific cleavage site, or a combination thereof.
  • the present disclosure also includes a recombinant nucleic acid comprising a nucleotide sequence encoding the chimeric receptor described herein.
  • the nucleotide sequence is incorporated into an expression cassette or an expression vector.
  • the expression vector is a viral vector.
  • the viral vector is a lentiviral vector, an adeno virus vector, an adeno-associated virus vector, or a retroviral vector.
  • the present disclosure includes a recombinant cell comprising the chimeric receptor and/or the recombinant nucleic acid described herein.
  • the recombinant cell is a eukaryotic cell.
  • the eukaryotic cell is a mammalian cell.
  • the mammalian cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell.
  • the immune cell is a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, a cytotoxic T cell, or other T cell.
  • the recombinant cell of the present disclosure comprises: a) a first chimeric receptor and a second chimeric receptor described herein; and/or b) a first nucleic acid and a second nucleic acid described herein.
  • the first chimeric receptor and the second chimeric receptor do not have the same sequence.
  • the first nucleic acid or the second nucleic acid do not have the same sequence.
  • the first chimeric receptor modulates the expression and/or activity of the second chimeric receptor.
  • the recombinant cell of the present disclosure further comprises an expression cassette encoding a protein operably linked to a promoter, wherein expression of the protein is modulated by the transcriptional regulator.
  • the protein is heterologous to the cell.
  • the promoter is a yeast GAL4 promoter.
  • the protein is a cytokine, a cytotoxin, a chemokine, an immunomodulator, a pro- apoptotic factor, an anti-apoptotic factor, a hormone, a differentiation factor, a de-differentiation factor, an immune cell receptor (e.g., a TCR or CAR), or a reporter.
  • a method for making the recombinant cell described herein comprising: a) providing a cell capable of protein expression; and b) contacting the provided cell with a recombinant nucleic acid described herein into the provided cell.
  • the cell is obtained by leukapheresis performed on a sample obtained from a subject, and the cell is contacted ex vivo.
  • the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle.
  • compositions comprising a pharmaceutically acceptable carrier, and one or more of the following: a) the recombinant nucleic acid described herein; and b) the recombinant cell described herein.
  • the composition comprises a recombinant nucleic acid described herein and a pharmaceutically acceptable carrier.
  • the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle.
  • the system comprises one or more of the following: a) a chimeric receptor described herein; b) a recombinant nucleic acid described herein; c) a recombinant cell described herein; and d) a pharmaceutical composition described herein.
  • the present disclosure also provided a method for modulating an activity of a cell, comprising: a) providing a recombinant cell described herein; and b) contacting the recombinant cell with a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain induces cleavage of a ligand-inducible proteolytic cleavage site and releases the transcriptional regulator, wherein the released transcriptional regulator modulates an activity of the recombinant cell.
  • the contacting is carried out in vivo, ex vivo, or in vitro.
  • the activity of the cell to be modulated is selected from the group consisting of: expression of a selected gene, proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of a molecule, cellular adhesion, and cytolytic activity.
  • the released transcriptional regulator modulates expression of a gene product of the cell.
  • the released transcriptional regulator modulates expression of a heterologous gene product.
  • the gene product of the cell is selected from the group consisting of chemokine, a chemokine receptor, a chimeric antigen receptor, a cytokine, a cytokine receptor, a differentiation factor, a growth factor, a growth factor receptor, a hormone, a metabolic enzyme, a pathogen-derived protein, a proliferation inducer, a receptor, an RNA guided nuclease, a site-specific nuclease, a T cell receptor, a toxin, a toxin derived protein, a transcriptional regulator, a transcriptional activator, a transcriptional repressor, a translational regulator, a translational activator, a translational repressor, an activating immuno-receptor, an antibody, an apoptosis inhibitor, an apoptosis inducer, an engineered T cell receptor, an immuno- activator,
  • the released transcriptional regulator modulates differentiation of the cell, and wherein the cell is an immune cell, a stem cell, a progenitor cell, or a precursor cell.
  • the method includes a method for inhibiting an activity of a target cell in an individual, the method comprising administering to the individual an effective number of the recombinant cells described herein.
  • the recombinant cells inhibit an activity of the target cell in the individual.
  • the target cell is a pathogenic cell.
  • the pathogenic cell is a cancer cell.
  • the target cell is an acute myeloma leukemia cell, an anaplastic lymphoma cell, an astrocytoma cell, a B-cell cancer cell, a breast cancer cell, a colon cancer cell, an ependymoma cell, an esophageal cancer cell, a glioblastoma cell, a glioma cell, a leiomyosarcoma cell, a liposarcoma cell, a liver cancer cell, a lung cancer cell, a mantle cell lymphoma cell, a melanoma cell, a neuroblastoma cell, a non-small cell lung cancer cell, an oligodendroglioma cell, an ovarian cancer cell, a pancreatic cancer cell, a peripheral T-Cell lymphoma cell, a renal cancer
  • the present disclosure provides a method for the treatment of a health condition in an individual in need thereof, the method comprising administering to the individual a first therapy comprising an effective number of the recombinant cell described herein, wherein the recombinant cell treats the health condition in the individual.
  • the method for the treatment of a health condition in an individual in need thereof further comprises administering to the individual a second therapy.
  • the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, and toxin therapy.
  • the first therapy and the second therapy are administered together in the same composition or in separate compositions. In some embodiments, the first therapy and the second therapy are administered at the same time. In other embodiments, the first therapy and the second therapy are administered sequentially. In certain embodiments, the first therapy is administered before the second therapy. In other embodiments, the first therapy is administered after the second therapy. In yet other embodiments, the first therapy and the second therapy are administered in rotation.
  • the present disclosure also provides the use of one or more of the following for the treatment of a health condition: a) a chimeric receptor described herein; b) a recombinant nucleic acid described herein; c) a recombinant cell described herein; and d) a composition described herein.
  • the present disclosure relates to the use of any of the forgoing for the manufacture of a medicament for the treatment of a health condition.
  • the health condition is cancer.
  • the cancer is a solid tumor, a soft tissue tumor, or a metastatic lesion.
  • FIGS 1 A- 1C illustrate the design of exemplary hybrid SynNotch CARs in accordance with some embodiments of the disclosure and predicted function of hybrid SynNotch CAR circuits.
  • FIG. 1 A is a diagram of all possible intracellular domain configurations.
  • FIG. IB is a detailed diagram of Hybrid SynNotch CAR domains.
  • FIG. 1C shows hypothesized short term proximal and long term transcriptional signaling induced by Hybrid SynNotch CARs.
  • FIGS 2A-2D schematically summarize the results from experiments performed to illustrate the expression of various exemplary hybrid SynNotch CARs and circuit induction.
  • Primary human T-cells were activated with anti-CD3/anti-CD28 Dynabeads (Gibco) and transduced with two lentiviral constructs expressing either a receptor or a transcriptional reporter construct.
  • Hybrid SynNotch CAR with 4 IBB costimulatory domains (FIG. 2A) or CD28 costimulatory domains (FIG. 2C) were sorted on Day 5 post initial T-cell stimulation, purifying the receptor and reporter dual positive population.
  • T-cells expressing anti-CD 19 receptors with 4 IBB costimulatory domains (FIG. 2B) or CD28 costimulatory domains (FIG. 2D) and the BFP reporter were co cultured with K562 cells (blue), or CD 19+ K562 cells (red) for 48 hours. Transcriptional activation of the inducible BFP reporter gene was subsequently measured using a Fortessa X-50 (BD).
  • FIGS 3A-3B illustrate the hybrid SynNotch CAR activation marker expression.
  • T-cells expressing anti-CD 19 receptors with 4 IBB costimulatory domains (FIG. 3 A) or CD28 costimulatory domains (FIG. 3B) and the BFP reporter were produced as described in FIG. 2.
  • Transduced cells were co-cultured with K562 cells (gray), or CD 19+ K562 cells (blue or red) for 48 hours.
  • Expression of activation markers CD25, CD39, CD69 and PD-1 were subsequently measured using a Fortessa X-50 (BD).
  • FIG 4 schematically summarizes the results from experiments performed to illustrate the proliferation of exemplary hybrid SynNotch CARs.
  • T-cells expressing anti-CD 19 receptors with 4 IBB costimulatory domains or CD28 costimulatory domains and the BFP reporter were produced as described in FIG. 2.
  • Transduced cells were stained with Cell Trace Far Red (CTFR), then co-cultured with K562 cells (gray), or CD 19+ K562 cells (blue or red) for 5 days. Dilution of CTFR dye was subsequently measured using a Fortessa X-50 (BD).
  • CTFR Cell Trace Far Red
  • FIGS 5A-5B summarize the results from experiments performed to illustrate the cytokine secretion by exemplary hybrid SynNotch CARs.
  • Transduced cells were co cultured with K562 cells (gray), or CD 19+ K562 cells (blue or red). After 48 hours, Brefeldin A, Monesin and a second bolus of K562 cells (either with or without CD 19+ expression) was added to the co-cultures. Co-cultures were incubated for an additional 6 hours, then transduced cells were assessed using a Fortessa X50 (BD) for intracellular expression of the cytokines Granzyme B, IFNy, IL-2 and TNFa. Note that data was not collected for the Hybrid SynNotch CAR with ICD CD28 - CD3C - Gal4VP64.
  • BD Fortessa X50
  • FIGS 6A-6B summarize the results from experiments performed to illustrate the target killing by exemplary hybrid SynNotch CARs.
  • T-cells expressing anti-CD19 receptors with 41BB costimulatory domains (FIG. 6A) or CD28 costimulatory domains (FIG. 6B) and the BFP reporter were produced as described in FIG. 2.
  • A549 cells expressing the CD 19 ligand and the nuclear stain mkate2 were allowed to adhere to a 96 well flat bottom plate for 24 hours, then transduced T cells were added at a 1:1 ratio. The plate was incubated in an Incucyte, which captured plate images and fluorescence every 2 hours for 5 days. Imaging software was used to calculate the number of A549 CD19+ mkate2+ cells in culture at each timepoint. For each experimental group, the A549 cell count was normalized to that of the Hinge Notch experimental group.
  • FIGS 7A-7C illustrate the in vivo efficacy of the hybrid SynNotch CARs.
  • FIG. 7A is a description of experimental timeline.
  • NOD.Cg- Prkdc sad Il2rgtm i wJ1/SzJ (NSG) mice were dosed intravenously with 0.5 xl06 Nalm6-Luc-GFP tumor cells.
  • Bulk CD3+ T-cells were co transduced with the anti-CD19 Hybrid SynNotch CAR with ICD CD3z - Gal4VP64 - CD28 and the BFP reporter as described in FIG. 2.
  • 0.5 xl06 transduced CD3+ T cells were dosed to animals via retro-orbital injection 4 days post tumor injection.
  • FIG. 7B shows tumor burden as measured via bioluminescence imaging of luciferase secreting tumor cells using an IVIS Spectrum.
  • FIG. 7C shows a survival curve.
  • FIGS 8A-8D illustrate that minimized 4-1BB variants improve NF-kB signaling and reduce noise.
  • FIG. 8 A is a description of 4 IBB variants, depicting the amino acids deleted to create the “no STS” and “trunc” 41BB costimulatory domains.
  • FIG. 8B shows T cells were co transduced to express the anti-CD 19 Hybrid SynNotch CAR with 4 IBB variants and the BFP reporter as described in FIG. 2.
  • FIG. 8C shows circuit induction was assessed as described in FIG. 2.
  • FIG. 8D shows a Jurkat cell line transduced to express an mCherry reporter under a common promoter for NfKB.
  • This NfKB reporter cell line was then transduced with anti-CD19 Hybrid SynNotch CARs, and co-cultured with K562 cells expressing CD19. mCherry expression was assessed as a proxy for NfKB activity at 24, 48 and 72 hours post co-culture via flow cytometry.
  • FIGs. 9A-9D shows further iterations on minimized 4 IBB variants.
  • FIG. 9A is a description of 4 IBB variants, depicting the amino acids deleted to create the “min” 4 IBB costimulatory domains, and amino acid regions duplicated to create the “trunc41BBtrunc41BB” costimulatory domain.
  • FIG. 9B shows T cells were co-transduced to express the anti-CD 19 Hybrid SynNotch CAR with 4 IBB variants and the BFP reporter as described in FIG 2.
  • FIG. 9C shows circuit induction was assessed as described in FIG 2.
  • FIG. 9D shows a Jurkat cell line was transduced to express an mCherry reporter under a common promoter for NfKB.
  • This NfKB reporter cell line was then transduced with anti-CD 19 Hybrid SynNotch CARs, and co-cultured with K562 cells expressing CD19. mCherry expression was assessed as a proxy for NfKB activity at 24, 48 and 72 hours post co-culture via flow cytometry.
  • FIGs. 10A-10B show Trunc41BB Hybrid SynNotch CAR in vivo efficacy.
  • FIG. 10A shows a description of experimental timeline.
  • NOD.Cg- Prkdc sad Il2rgtm i wJ1/SzJ (NSG) mice were dosed subcutaneously with 4xl0 6 CD19 ligand expressing M28 tumor cells.
  • Bulk CD3+ T- cells were co-transduced with the anti-CD 19 Hybrid SynNotch CAR with ICDs as indicated in 10B and the BFP reporter as described in FIG 2.
  • 6 xlO 6 transduced CD3+ T cells were dosed to animals via retro-orbital injection 7 days post tumor injection.
  • FIG. 10B shows tumor volume assessed via caliper measurements weekly.
  • FIGs. 11 A-l 1C show minimized CD28 variants reduce noise.
  • FIG. 11 A is a description of CD28 variants, depicting the amino acids deleted to create the “no STS” and “trunc” CD28 costimulatory domains.
  • FIG. 1 IB shows T cells co-transduced to express the anti-CD 19 Hybrid SynNotch CAR with CD28 variants and the BFP reporter as described in FIG. 2.
  • FIG. 11C shows circuit induction assessed as described in FIG. 2.
  • FIGs. 12A-12C show further iterations on minimized CD28 variants.
  • FIG. 12A is a description of CD28 variants, depicting the amino acids deleted to create the “CD28ATPRRP,” “truncCD28ATPRRP” and “fullytruncCD28” costimulatory domains.
  • FIG. 12B shows T cells co-transduced to express the anti-CD 19 Hybrid SynNotch CAR with CD28 variants and the BFP reporter as described in FIG. 2.
  • FIG. 12C shows circuit induction assessed as described in FIG.
  • FIGs. 13A-13C show “Third Generation” variants.
  • FIG. 13A is a description of “third generation” variants, which include one of the CD28 signaling motifs appended to the C terminus of atrunc41BB costimulatory domain.
  • FIG. 13B shows T cells co-transduced to express the anti-CD 19 Hybrid SynNotch CAR with third generation variants and the BFP reporter as described in FIG. 2.
  • FIG. 13C shows circuit induction assessed as described in FIG.
  • FIGs. 14A-14B show Trunc41BB Hybrid SynNotch CAR In Vivo Efficacy.
  • FIG. 14A is a description of experimental timeline.
  • NOD.Cg- Prkdc sad Il2rgtm ⁇ w ⁇ Sz ⁇ (NSG) mice were dosed subcutaneously with 4xl0 6 CD19 ligand expressing M28 tumor cells.
  • Bulk CD3+ T-cells were co-transduced with the anti-CD 19 Hybrid SynNotch CAR with ICDs as indicated in 14B and the BFP reporter as described in FIG 2.
  • 3 xlO 6 transduced CD3+ T cells were dosed to animals via retro-orbital injection 7 days post tumor injection.
  • FIG. 14B show tumor volume assessed via caliper measurements weekly.
  • FIGs. 15A-15B shows BCMA and ALPPL2 Targeted Hybrid SynNotch CAR Expression and Circuit Induction.
  • T-cells expressing anti-BCMA and anti-ALPPL2 receptors with 4 IBB costimulatory domains and the BFP reporter were produced as described in FIG 2 (FIG. 15 A).
  • Transduced cells were co-cultured with K562 cells (blue), or antigen positive (either BCMA or ALPPL2) K562 cells (red) for 48 hours (FIG. 15B).
  • FIG. 16 shows ALPPL2 Targeted Hybrid SynNotch CAR In Vivo Efficacy.
  • NOD.Cg- Prkdc sad Il2rgtm ⁇ VSzS (NSG) mice were dosed subcutaneously with 4xl0 6 M28 tumor cells as described in FIG. 14.
  • Bulk CD3+ T-cells were co-transduced with anti-ALPPL2 CAR or the anti-ALPPL2 Hybrid SynNotch CAR with ICDs as indicated in figure and the BFP reporter as described in FIG. 2.
  • 3 xlO 6 transduced CD3+ T cells were dosed to animals via retro-orbital injection 7 days post tumor injection. Tumor volume was assessed via caliper measurements weekly.
  • the present disclosure relates generally to a new class of chimeric receptors designed to combine fast time-scale intracellular signal transduction and long time-scale transcription regulation.
  • some embodiments of the disclosure provides exemplary chimeric receptors (referred to herein as “hybrid SynNotch CARs”) that incorporate (i) costimulatory domains and stimulatory domains of a CAR, for example, the cytoplasmic tail of the CD3zeta chain, and a transcriptional regulator.
  • the architecture of the cytoplasmic tail of these new receptors costimulatory domain, CD3zeta, transcription factor
  • costimulatory domain, CD3zeta, transcription factor can be configured in multiple ways.
  • the present disclosure also identifies hybrid receptor architectures that reliably induce proximal T-cell receptor costimulatory signals and gene regulation in a target cell type, such as, primary human T cells.
  • the new hybrid SynNotch CARs provided herein can simultaneously stimulate (i) fast time-scale (e.g., from seconds to minutes) proximal signaling and (ii) long-time scale transcriptional regulation that usually takes hours to induce to sufficient levels to observe cellular state changes.
  • Hybrid SynNotch CARs exhibit antigen independent induction of the inducible transcriptional reporter element.
  • the present disclosure demonstrates that the spatial configuration of the intracellular domains influences receptor behavior in both the presence and absence of ligand. Additionally, the present disclosure exemplifies that, upon engagement with the target antigen, Hybrid SynNotch CARs functionally induce signaling through their intracellular signaling domains (e.g., 4-1BB or CD28, and CD3zeta), leading to expression of activation markers.
  • the present disclosures demonstrates that, upon engagement with antigen, the Hybrid SynNotch CARs provided herein functionally induce short term signaling cascades through their intracellular signaling domains (e.g., 4-1BB or CD28, and CD3zeta), leading to proliferation of the T cells.
  • the present disclosures demonstrates that, the signaling induced by the costimulatory domains and activation domain (e.g., CD3zeta) of the Hybrid SynNotch CARs of the present disclosure differs in type or mechanism, strength, intensity, or length of time to the CARs.
  • the present disclosures demonstrates that the Hybrid SynNotch CAR T cells disclosed herein can kill target cells at similar rates as the CAR T cells.
  • the present disclosure provides that the Hybrid SynNotch CARs induce T cell activation and cytotoxic programs that are sufficient to cause target cell killing over a period of longer time (such as multiple days).
  • the present disclosure demonstrates that the Hybrid SynNotch CAR T cells are effective in controlling and clearing tumor burden in vivo.
  • the present disclosure further provides, among others, that modification of the costimulatory domain (e.g., the 4- IBB costimulatory domain) can optimize the Hybrid SynNotch CARs described herein with antigen-independent activity, resulting in improved designs that are capable of both antigen dependent transcriptional circuit induction and T cell signaling.
  • modification of the costimulatory domain e.g., the 4- IBB costimulatory domain
  • administration refers to the delivery of a composition or formulation by an administration route including, but not limited to, intravenous, intra-arterial, intracerebral, intrathecal, intramuscular, intraperitoneal, subcutaneous, intramuscular, and combinations thereof.
  • administration includes, but is not limited to, administration by a medical professional and self-administration
  • heterologous refers to a polypeptide sequence or domain which is not native to a flanking sequence, e.g., wherein the heterologous sequence is not found in nature coupled to the polypeptide sequences occurring at one or both ends.
  • a protein or polypeptide derived from an origin or source refers to an origin or source, and may include naturally occurring, recombinant, unpurified or purified polypeptide that is obtained from, is obtained based on a source or original protein or polypeptide.
  • a protein or polypeptide derived from an original protein or polypeptide may include the original protein or polypeptide, in part or in whole, and may be a fragment or variant of the original protein or polypeptide.
  • the polypeptide sequence or domain that is derived from a source or origin can be genetically or chemically modified.
  • host cell and “recombinant cell” are used interchangeably herein. It is understood that such terms, as well as “cell”, “cell culture”, “cell line”, refer not only to the particular subject cell or cell line but also to the progeny or potential progeny of such a cell or cell line, without regard to the number of transfers. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g., deliberate or inadvertent mutations) or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the originally cell or cell line.
  • operably linked denotes a physical or functional linkage between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion.
  • percent identity refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection.
  • sequences are then said to be “substantially identical.”
  • This definition also refers to, or may be applied to, the complement of a test sequence.
  • This definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • Sequence identity can be calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res.
  • Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof.
  • a “therapeutically effective amount” of an agent is an amount sufficient to provide a therapeutic benefit in the treatment or management of a health condition, such as a disease (e.g., a cancer), or to delay or minimize one or more symptoms associated with the cancer.
  • a therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer.
  • the term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the cancer, or enhances therapeutic efficacy of another therapeutic agent.
  • an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.”
  • a “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).
  • the exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.
  • a “subject” or an “individual” includes animals, such as human (e.g., human individuals) and non-human animals.
  • a “subject” or “individual” is an individual under the care of a physician.
  • the subject can be a human individual or an individual who has, is at risk of having, or is suspected of having a disease of interest (e.g., cancer) and/or one or more symptoms of the disease.
  • the subject can also be an individual who is diagnosed with a risk of the condition of interest at the time of diagnosis or later.
  • non-human animals includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non mammals, such as non-human primates, e.g., sheep, dogs, cows, chickens, amphibians, reptiles, and the like.
  • the chimeric receptors disclosed herein provide signals having a range of characteristics, from low to high ligand-induced transduction and (independently) low to moderate non-induced signal transduction. This range of activities is a new feature that can be exploited to enhance and tune the actions of engineered cells. Further, as described in greater detail below, a number of the receptor variants disclosed herein exhibit improved expression compared to existing SynNotch receptors.
  • Notch receptors are large transmembrane proteins that normally communicate signals upon binding to surface-bound ligands expressed on adjacent cells. Notch signals rely on cell- to- cell communication, e.g., communication between two contacting cells, in which one contacting cell is a “receiver” cell and the other contacting cell is a “sender” cell. Notch receptors expressed in a receiver cell recognize their ligands (the delta/serrate/lag, or “DSL” family of proteins) expressed on a sending cell. The engagement of notch and delta on these contacting cells leads to a two-step proteolysis of the notch receptor, which ultimately causes the release of the intracellular portion of the receptor (“ICD”) from the membrane into the cytoplasm.
  • ICD intracellular portion of the receptor
  • Notch has a matrix metalloprotease cleavage site (denoted “S2”), which, when the receptor is not activated is protected from cleavage by the Notch negative regulatory region (“NRR”).
  • NRR consists of three LIN-12-Notch repeat (“LNR”) modules and a heterodimerization domain (“HD”). It is believed that this proteolysis is regulated by the force exerted by the sending cell: the DSL ligand pulls on the Notch receptor, which changes the conformation of the NRR and exposes the metalloprotease site. This is cleaved by a constitutively active protease (such as ADAM10), which releases the extracellular binding portion and negative regulatory region of the receptor.
  • ADAM10 constitutively active protease
  • Notch signals are transduced by a process called regulated intramembrane proteolysis.
  • Notch receptors are normally maintained in a resting, proteolytically resistant conformation on the cell surface, but ligand binding initiates a proteolytic cascade that releases the intracellular domain of the receptor (ICD) from the membrane.
  • the critical, regulated cleavage step is effected by ADAM metalloproteases and occurs at a site called S2 immediately external to the plasma membrane.
  • This truncated receptor, dubbed NEXT for Notch extracellular truncation
  • NEXT for Notch extracellular truncation
  • the ICD After g-secretase cleavage, the ICD ultimately enters the nucleus, where it nucleates assembly of a transcriptional activation complex that contains a DNA-binding transcription factor, and a transcriptional coactivator of the Mastermind family. This complex then engages one or more additional coactivator proteins such as p300 to recruit the basal transcription machinery and activate the expression of downstream target genes.
  • Notch receptors have a modular domain organization.
  • the ectodomains of Notch receptors consist of a series of N-terminal epidermal growth factor (EGF)-like repeats that are responsible for ligand binding.
  • EGF epidermal growth factor
  • O-linked glycosylation of these EGF repeats including modification by O-fucose, Fringe, and Rumi glycosyltransferases, also modulates the activity of Notch receptors in response to different ligand subtypes in flies and mammals.
  • the EGF repeats are followed by three LIN-12/Notch repeat (LNR) modules, which are unique to Notch receptors, and are widely reported to participate in preventing premature receptor activation.
  • LNR LIN-12/Notch repeat
  • the heterodimerization (HD) domain of Notchl is divided by furin cleavage, so that its N-terminal part terminates the extracellular subunit, and its C-terminal half constitutes the beginning of the transmembrane subunit. Following the extracellular region, the receptor has a transmembrane segment and an intracellular domain (ICD), which includes a transcriptional regulator.
  • ICD intracellular domain
  • the present disclosure provides, among other things, a new class of chimeric receptors designed to combine fast time-scale intracellular signal transduction and long time-scale transcription regulation.
  • some embodiments of the disclosure provides new hybrid SynNotch receptor architectures that incorporate signaling domains (e.g. co-stimulation,
  • the new receptors provided herein have linear amino acid signaling motif to mediate signaling in T cells added into the cytoplasmic tail of SynNotch receptors. As demonstrated in the Examples and figures, these new receptors can stimulate fast time-scale (e.g., from seconds to a minute) proximal signaling as well as long-time scale transcriptional regulation that takes hours to induce to sufficient levels to observe cellular state changes.
  • the present disclosure provides chimeric receptor comprising, from N- terminus to C-terminus,: a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; b) a linking polypeptide; c) a transmembrane domain comprising one or more ligand-inducible proteolytic cleavage sites; and d) an intracellular domain comprising, in any order: (i) an intracellular signaling domain (SD) comprising (1) at least one costimulatory domain derived from a signaling molecule and (2) an activation domain, and (ii) a transcriptional regulator.
  • SD intracellular signaling domain
  • the binding of the selected ligand to the extracellular ligand binding domain induces cleavage at a ligand-inducible proteolytic cleavage site disposed between the intracellular domain and the linking polypeptide.
  • the binding of the selected ligand to the extracellular ligand-binding domain can also induce proximal signaling cascades through the intracellular SD.
  • the proximal signaling cascades refer to fast time-scale signaling. For instance, the signaling cascades can be induced in seconds to minutes. Alternatively, the signaling cascades can last for seconds to minutes. In some embodiments, such proximal signaling cascades are induced through T-cell receptor costimulatory signals.
  • the chimeric receptor provided herein does not comprise a LIN-12-Notch repeat (LNR) and/or a heterodimerization domain (HD) of a Notch receptor.
  • the linking polypeptide is capable of promoting oligomer formation of the chimeric receptor via intermolecular disulfide bonding.
  • the ECD of the chimeric receptors (e.g., hybrid SynNotch CARs) disclosed herein has a binding affinity for one or more target ligands.
  • the target ligand can be expressed on the surface of a cell, or is otherwise anchored, immobilized, or restrained so that it can exert a mechanical force on the chimeric receptor.
  • the cell can be a pathogenic cell or a human cell.
  • the human cell can be a tumor cell.
  • the human cell can be a terminally differentiated cell.
  • binding of the ECD of a chimeric receptor provided herein to a cell-surface ligand does not necessarily remove the target ligand from the target cell surface, but instead enacts a mechanical pulling force on the chimeric receptor.
  • an otherwise soluble ligand may be targeted if it is bound to a surface, or to a molecule in the extracellular matrix.
  • the target ligand is a cell-surface ligand.
  • suitable ligand types include cell surface receptors; adhesion proteins; carbohydrates, lipids, glycolipids, lipoproteins, and lipopolysaccharides that are surface-bound; integrins; mucins; and lectins.
  • the ligand is a protein. In some embodiments, the ligand is a carbohydrate. [0085] In some embodiments, the ligand is a cluster of differentiation (CD) marker.
  • the CD marker is selected from the group consisting of CD1, CD la, CD lb, CDlc, CD Id, CDle, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95,
  • the extracellular domain includes the ligand-binding portion of a receptor.
  • the extracellular domain includes an antigen-binding moiety that binds to one or more target antigens.
  • the antigen-binding moiety includes one or more antigen-binding determinants of an antibody or a functional antigen-binding fragment thereof.
  • the term “functional fragment thereof’ or “functional variant thereof’ refers to a molecule having quantitative and/or qualitative biological activity in common with the wild-type molecule from which the fragment or variant was derived.
  • a functional fragment or a functional variant of an antibody is one which retains essentially the same ability to bind to the same epitope as the antibody from which the functional fragment or functional variant was derived.
  • an antibody capable of binding to an epitope of a cell surface receptor may be truncated at the N-terminus and/or C-terminus, and the retention of its epitope binding activity assessed using assays known to those of skill in the art.
  • the antigen binding moiety is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, or a minibody, an F(ab’)2 fragment, an F(ab) fragment, a single chain variable fragment (scFv), and a single domain antibody (sdAb), or a functional fragment thereof.
  • the antigen-binding moiety includes an scFv.
  • the antigen-binding moiety can include naturally-occurring amino acid sequences or can be engineered, designed, or modified to provide desired and/or improved properties such as, e.g., binding affinity.
  • binding affinity of an antigen-binding moiety e.g., an antibody
  • a target antigen e.g., CD19 antigen
  • binding affinity is measured by an anti gen/ antibody dissociation rate.
  • binding affinity is measured by a competition radioimmunoassay.
  • binding affinity is measured by ELISA.
  • antibody affinity is measured by flow cytometry.
  • An antibody that “selectively binds” an antigen is an antigen-binding moiety that does not significantly bind other antigens but binds the antigen with high affinity, e.g., with an equilibrium constant (KD) of 100 nM or less, such as 60 nM or less, for example, 30 nM or less, such as, 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or 200 pM or less, or 100 pM or less.
  • KD equilibrium constant
  • a skilled artisan can select an ECD based on the desired localization or function of a cell that is genetically modified to express a chimeric receptor or hybrid SynNotch CAR of the present disclosure.
  • a chimeric receptor or hybrid SynNotch CAR with an ECD including an antibody specific for a HER2 antigen can target cells to HER2-expressing breast cancer cells.
  • the ECD of the disclosed hybrid SynNotch CARs is capable of binding a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).
  • TAAs include a molecule, such as e.g., protein, present on tumor cells and on normal cells, or on many normal cells, but at much lower concentration than on tumor cells.
  • TSAs generally include a molecule, such as e.g., protein which is present on tumor cells but absent from normal cells.
  • the antigen-binding moiety is specific for an epitope present in an antigen that is expressed by a tumor cell, i.e., a tumor-associated antigen.
  • the tumor-associated antigen can be an antigen associated with, e.g., a breast cancer cell, a B cell lymphoma, a pancreatic cancer, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell, a non-Hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a myelogenous leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma
  • a tumor-associated antigen may also be expressed by a non-cancerous cell.
  • the antigen-binding domain is specific for an epitope present in a tissue- specific antigen. In some embodiments, the antigen-binding domain is specific for an epitope present in a disease-associated antigen.
  • Non-limiting examples of suitable target antigens include CD 19, B7H3 (CD276), BCMA (CD269), alkaline phosphatase, placental-like 2 (ALPPL2), green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), signal regulatory protein a (SIRPa), CD 123, CD171, CD 179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2/neu), IGLL1, IL-1 IRa, KIT (CD 117), MUC1, NCAM, PAP, PDGFR-b, PRSS21, PSCA, PSMA, ROR1, S SEA
  • the target antigen is selected from CD 19, B7H3 (CD276), BCMA (CD269), ALPPL2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2/neu), IGLL1, IL-1 IRa, KIT (CD117), MUC1, NCAM, PAP, PDGFR- b, PRSS21, PSCA, PSMA, ROR1, SSEA-4, TAG72, TEM1/CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, Axl, G
  • suitable antigens include PAP (prostatic acid phosphatase), prostate stem cell antigen (PSCA), prostein, NKG2D, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAPl (six-transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, an abnormal p53 protein, integrin b3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), Ral-B, GPC2, CD276 (B7H3), or IL-13Ra.
  • the antigen is Her2.
  • the antigen is ALPPL2. In some embodiments, the antigen is BCMA. In some embodiments, the antigen binding moiety of the ECD is specific for a reporter protein, such as BFP, GFP, and eGFP. Non limiting examples of such antigen binding moiety include a LaG17 anti-GFP nanobody. In some embodiments, the antigen-binding moiety of the ECD includes an anti-BCMA fully-humanized VH domain (FHVH). In some embodiments, the antigen is signal regulatory protein a (SIRPa).
  • SIRPa signal regulatory protein a
  • Additional antigens suitable for targeting by the chimeric receptors disclosed herein include, but are not limited to GPC2, human epidermal growth factor receptor 2 (Her2/neu), CD276 (B7H3), IL-13Ral, IL-13Ra2, a-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA).
  • GPC2 human epidermal growth factor receptor 2
  • CD276 B7H3
  • IL-13Ral IL-13Ral
  • IL-13Ra2 a-fetoprotein
  • CEA carcinoembryonic antigen
  • CA-125 cancer antigen-125
  • CA19-9 calretinin
  • MUC-1 epithelial membrane protein
  • EMA epithelial membrane protein
  • ETA epithelial tumor antigen
  • target antigens include, but are not limited to, tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD123, CD93, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), ALK, DLK1, FAP, NY-ESO, WT1, HMB-45 antigen, protein melan- A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1.
  • MAGE melanoma-associated antigen
  • CD34 CD45
  • CD123 CD93
  • CD99 chromogranin
  • GFAP glial fibrillary acidic protein
  • Additional antigens suitable for targeting by the chimeric receptors disclosed herein include, but are not limited to, those associated with an inflammatory disease such as, AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (a subunit of the heteromeric of IL-2 receptor), CD3, CD4, CD5, IFN-a, IFN-g, IgE, IgE 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 a.4, integrin a4b7, LFA-1 (CDlla), myostatin, OX-40, scleroscin, SOST, TGF l, TNF-a, and VEGF-A.
  • an inflammatory disease such as, AOC3
  • antigens suitable for targeting by the chimeric receptors and hybrid SynNotch CARs disclosed herein include, but are not limited to the pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD20, CD5, CD7, CD3, TRBCl, TRBC2, BCMA, CD38, CD123, CD93, CD34, CD la, SLAMF7/CS1, FLT3, CD33, CD123, TALLA-1, CSPG4, DLL3, Kappa light chain, Lamba light chain, CD 16/ FcyRIII, CD64, FITC, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), GD3, EGFRvIII (epidermal growth factor variant III), EGFR and isovariants thereof, TEM-8, sperm protein 17 (Spl7), mesothelin.
  • pyruvate kinase isoenzyme type M2 tumor M2-PK
  • CD20 CD5, CD7,
  • suitable antigens include PAP (prostatic acid phosphatase), prostate stem cell antigen (PSCA), prostein, NKG2D, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six- transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, an abnormal p53 protein, integrin b3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), and Ral-B.
  • the antigen is GPC2, CD19, Her2/neu, CD276 (B7H3), IL-13Ral, or IL-13Ra2.
  • the antigen is Her2. In some embodiments, the antigen is ALPPL2. In some embodiments, the antigen is BCMA. In some embodiments, the antigen binding moiety of the ECD is specific for a reporter protein, such as GFP and eGFP. Non limiting examples of such antigen binding moiety include a LaG17 anti-GFP nanobody. In some embodiments, the antigen-binding moiety of the ECD includes an anti-BCMA fully-humanized VH domain (FHVH).
  • FHVH fully-humanized VH domain
  • antigens suitable for targeting by the chimeric receptors and hybrid SynNotch CARs disclosed herein include ligands derived from a pathogen.
  • the antigen can be HER2 produced by HER2 -positive breast cancer cells.
  • the antigen can be CD 19 that is expressed on B-cell leukemia.
  • the antigen can be EGFR that is expressed on glioblastoma multiform (GBM) but much less expressed so on healthy CNS tissue.
  • the antigen can be CEA that is associated with cancer in adults, for example colon cancer.
  • the antigen-binding moiety of the ECD is specific for a cell surface target, where non-limiting examples of cell surface targets include CD 19, CD30, Her2, CD22, ENPP3, EGFR, CD20, CD52, CDlla, and a-integrin.
  • the chimeric receptors and hybrid SynNotch CARs disclosed herein include an extracellular domain having an antigen-binding moiety that binds CD 19, CEA, HER2, MUC1, CD20, ALPPL2, BCMA, or EGFR.
  • the chimeric receptors provided herein include an extracellular domain including an antigen-binding moiety that binds CD19.
  • the chimeric receptors provided herein include an extracellular domain including an antigen-binding moiety that binds ALPPL2.
  • the chimeric receptors provided herein include an extracellular domain including an antigen-binding moiety that binds BCMA.
  • the chimeric receptors provided herein include an extracellular domain including an antigen-binding moiety that binds Her2.
  • the chimeric receptors and hybrid SynNotch CARs disclosed herein include an extracellular domain including an antigen-binding moiety that binds CD 19, ALPPL2, BCMA, or Her2.
  • the extracellular domain includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a sequence set forth in SEQ ID NOS: 1, 2, 46, and 47. In some embodiments, the extracellular domain includes an amino acid sequence having at least 90% sequence identity to a sequence set forth in SEQ ID NOS: 1, 2, 46, and 47. In some embodiments, the extracellular domain includes an amino acid sequence having at least 95% sequence identity to a sequence set forth in SEQ ID NOS: 1, 2, 46, and 47.
  • the extracellular domain includes an amino acid sequence having 100% sequence identity to a sequence set forth in SEQ ID NOS: 1, 2, 46, and 47. In some embodiments, the extracellular domain includes an amino acid sequence set forth in SEQ ID NOS: 1, 2, 46, and 47, wherein one, two, three, four, or five of the amino acid residues in any one of the SEQ ID NOS: 1, 2, 46, and 47 is/are substituted by a different amino acid residue.
  • the chimeric receptors of the disclosure include a linking polypeptide sequence disposed between the extracellular binding domain (ECD) and the transmembrane domain (TMD).
  • ECD extracellular binding domain
  • TMD transmembrane domain
  • Existing “SynNotch” receptors comprise a heterologous extracellular ligand-binding domain, a linking polypeptide having substantial sequence identity with a Notch receptor JMD including the NRR, a TMD, and an ICD.
  • the chimeric receptors and hybrid SynNotch CARs comprise a heterologous extracellular ligand-binding domain, a linking polypeptide having substantial sequence identity with a Notch receptor JMD but lacking the NRR (the LIN-12-Notch repeat (LNR) modules, and the heterodimerization domain), a TMD, and an ICD.
  • the linking polypeptide replaces the negative regulatory region (NRR) and heterodimerization (HD) domain of the native Notch.
  • Three to 50 amino acid residues e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
  • the linker polypeptide sequence can be optimized to vary the orientation and/or proximity of the ECD and the TMD relative to one another to achieve a desired activity of the chimeric receptor of the disclosure. All of these sequences can be used as a linking polypeptide for the chimeric receptors of the present disclosure.
  • the linking polypeptide encompassed by the present disclosure can include a polypeptide capable of promoting oligomer formation of the chimeric receptor via intermolecular disulfide bonding, for example, a hinge linker.
  • hinge linkers of the disclosure include an oligomerization domain (e.g., a hinge domain) containing one or more polypeptide motifs that promote oligomer formation of the chimeric receptors via intermolecular disulfide bonding.
  • the hinge domain generally includes a flexible polypeptide connector region disposed between the ECD and the TMD.
  • the hinge domain provides flexibility between the ECD and TMD and also provides sites for intermolecular disulfide bonding between two or more chimeric receptor monomers to form an oligomeric complex.
  • the hinge domain includes motifs that promote dimer formation of the chimeric receptors disclosed herein.
  • the hinge domain includes motifs that promote trimer formation of the chimeric receptors disclosed herein (e.g., a hinge domain derived from 0X40).
  • Hinge polypeptide sequences suitable for the compositions and methods of the disclosure can be naturally-occurring hinge polypeptide sequences (e.g., those from naturally-occurring immunoglobulins) or can be engineered, designed, or modified so as to provide desired and/or improved properties, e.g., modulating transcription.
  • Suitable hinge polypeptide sequences include, but are not limited to, those derived from IgA, IgD, and IgG subclasses, such as IgGl hinge domain, IgG2 hinge domain, IgG3 hinge domain, and IgG4 hinge domain, or a functional variant thereof.
  • the hinge polypeptide sequence contains one or more CXXC motifs.
  • the hinge polypeptide sequence contains one or more CPPC motifs. Additional information in this regard can be found in, for example, a recent review by G. Vidarsson et ah, Frontiers Immunol (2014) 5:520 (doi: 10.3389/fimmu.2014.00520), which is hereby incorporated by reference in its entirety.
  • Hinge polypeptide sequences can also be derived from a CD8a hinge domain, a CD28 hinge domain, a CD 152 hinge domain, a PD-1 hinge domain, a CTLA4 hinge domain, an 0X40 hinge domain, and functional variants thereof.
  • the hinge domain includes a hinge polypeptide sequence derived from a CD8a hinge domain or a functional variant thereof.
  • the hinge domain includes a hinge polypeptide sequence derived from a CD28 hinge domain or a functional variant thereof.
  • the hinge domain includes a hinge polypeptide sequence derived from an 0X40 hinge domain or a functional variant thereof.
  • the hinge domain includes a hinge polypeptide sequence derived from an IgG4 hinge domain or a functional variant thereof.
  • the hinge linker can include about 5 to about 60 amino acids from or overlapping with the selected hinge domain, for example at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 12, at least about 15, at least about 17, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, or , at least about 60 amino acids.
  • the Hinge linker has no more than about 60 amino acids, less than about 55, less than about 50, less than about 45, less than about 40, less than about 35, less than about 32, less than about 30, less than about 29, less than about 28, less than about 27, less than about 26, less than about 25, less than about 24, less than about 23, less than about 22, less than about 21, less than about 20, less than about 18, less than about 16, less than about 14, less than about 12, or less than about 10 amino acids.
  • the linking polypeptide sequence includes a sequence having at least 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to a sequence set forth in SEQ ID NO: 3.
  • the linking polypeptide sequence includes an amino acid sequence having at least 90% sequence identity to a sequence set forth in SEQ ID NO: 3.
  • the linking polypeptide sequence includes an amino acid sequence having at least 95% sequence identity to a sequence set forth in SEQ ID NO: 3.
  • the linking polypeptide sequence includes an amino acid sequence having at least 99% sequence identity to a sequence set forth in SEQ ID NO: 3.
  • the linking polypeptide sequence includes an amino acid sequence identical to a sequence set forth in SEQ ID NO: 3. In some embodiments, the linking polypeptide sequence includes an amino acid sequence set forth in SEQ ID NO: 3, wherein one, two, three, four, or five of the amino acid residues in any one of the SEQ ID NO: 3 is/are substituted by a different amino acid residue.
  • TMD Transmembrane domains
  • the chimeric receptors of the disclosure include a TMD comprising one or more ligand-inducible proteolytic cleavage sites.
  • a Notch receptor e.g., S2 or S3
  • Additional proteolytic cleavage sites suitable for the compositions and methods disclosed herein include, but are not limited to, a metalloproteinase cleavage site for a MMP selected from collagenase-1, -2, and -3 (MMP-1, - 8, and -13), gelatinase A and B (MMP -2 and - 9), stromelysin 1, 2, and 3 (MMP-3, -10, and -11), matrilysin (MMP-7), and membrane metalloproteinases (MTl-MMP and MT2-MMP).
  • MMP-1, -2, and -3 MMP-1, - 8, and -13
  • MMP-2 and - 9 gelatinase A and B
  • MMP-3, -10, and -11 stromelysin 1, 2, and 3
  • MMP-7 matrilysin
  • MTl-MMP and MT2-MMP membrane metalloproteinases
  • the cleavage sequence of MMP - 9 is Pro-X-X-Hy (wherein, X represents an arbitrary residue; Hy, a hydrophobic residue such as Leu, He, Val, Phe, Trp, Tyr, Val, Met, and Pro), e.g., Pro-X-X-Hy-(Ser/Thr), e.g., Pro-Leu/Gln- Gly-Met-Thr-Ser or Pro-Leu/Gln-Gly-Met-Thr.
  • X represents an arbitrary residue
  • Hy a hydrophobic residue such as Leu, He, Val, Phe, Trp, Tyr, Val, Met, and Pro
  • Pro-X-X-Hy-(Ser/Thr) e.g., Pro-Leu/Gln- Gly-Met-Thr-Ser or Pro-Leu/Gln-Gly-Met-Thr.
  • a suitable protease cleavage site is a plasminogen activator cleavage site, e.g., a urokinase plasminogen activator (uPA) or a tissue plasminogen activator (tPA) cleavage site.
  • a suitable protease cleavage site is a prolactin cleavage site.
  • Specific examples of cleavage sequences of uPA and tPA include sequences comprising Val-Gly-Arg.
  • protease cleavage site that can be included in a proteolytically cleavable linker is a tobacco etch virus (TEV) protease cleavage site, e.g., Glu-Asn-Leu-Tyr-Thr-Gln-Ser, where the protease cleaves between the glutamine and the serine.
  • TSV tobacco etch virus
  • Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is an enterokinase cleavage site, e.g., Asp-Asp-Asp-Asp-Lys, where cleavage occurs after the lysine residue.
  • protease cleavage site that can be included in a proteolytically cleavable linker is a thrombin cleavage site, e.g., Leu-Val- Pro-Arg.
  • Additional suitable linkers comprising protease cleavage sites include sequences cleavable by the following proteases: a PreScissionTM protease (a fusion protein comprising human rhinovirus 3C protease and glutathione-S-transferase), a thrombin, cathepsin B, Epstein- Barr virus protease, MMP-3 (stromelysin), MMP-7 (matrilysin), MMP-9; thermolysin-like MMP, matrix metalloproteinase 2 (MMP -2), cathepsin L; cathepsin D, matrix metalloproteinase 1 (MMP-1), urokinase-type plasminogen activator, membrane type 1 matrix metall
  • proteases that are not native to the host cell in which the receptor is expressed can be used as a further regulatory mechanism, in which activation of the receptor is reduced until the protease is expressed or otherwise provided.
  • a protease may be tumor-associated or disease-associated (expressed to a significantly higher degree than in normal tissue), and serve as an independent regulatory mechanism.
  • some matrix metalloproteases are highly expressed in certain cancer types.
  • the TMD suitable for the chimeric receptors disclosed herein can be any transmembrane domain of a Type 1 transmembrane receptor including at least one g-secretase cleavage site.
  • a Type 1 transmembrane receptor including at least one g-secretase cleavage site.
  • Detailed description of the structure and function of the g-secretase complex as well as its substrate proteins, including amyloid precursor protein (APP) and Notch, can, for example, be found in a recent review by Zhang et al., Frontiers Cell Neurosci (2014).
  • Non limiting suitable TMDs from Type 1 transmembrane receptors include those from CLSTN1, CLSTN2, APLP1, APLP2, LRP8, APP, BTC, TGBR3, SPN, CD44, CSF1R, CXCL16,
  • TMD includes at least one g-secretase cleavage site.
  • TMDs suitable for the compositions and methods described herein include, but are not limited to, transmembrane domains from Type 1 transmembrane receptors IL1R1, IL1R2, IL6R, INSR, ERN1, ERN2, JAG2, KCNEl, KCNE2, KCNE3, KCNE4, KL, CHL1, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGER, ROBOl, SORCS3, SORCS1, SORL1, SDC1, SDC2, SPN, TYR, TYRP1, DCT, VASN, FLT1, CDH5, PKHD1, NECTINl,
  • the TMD of the chimeric receptors of the disclosure is a TMD derived from the TMD of a member of the calsyntenin family, such as, alcadein alpha and alcadein gamma.
  • the TMD of the chimeric receptors of the disclosure is a TMD known for Notch receptors.
  • the TMD of the chimeric receptors of the disclosure is a TMD derived from a different Notch receptor.
  • the Notchl TMD can be substituted with a Notch2 TMD, Notch3 TMD, Notch4 TMD, or a Notch TMD from a non-human animal such as Danio rerio, Drosophila melanogaster, Xenopus laevis, or Gallus gallus.
  • a non-human animal such as Danio rerio, Drosophila melanogaster, Xenopus laevis, or Gallus gallus.
  • the amino acid substitution(s) within the TMD includes one or more substitutions within a “GV” motif of the TMD.
  • at least one of such substitution(s) comprises a substitution to alanine.
  • one, two, three, four, five, or more of the amino acid residues of the sequence FMYVAAAAFVLLFFVGCGVLL (SEQ ID NO: 4) may be substituted by a different amino acid residue.
  • the amino acid residue at position 18 and/or 19 of the “GV” motif within SEQ ID NO: 4 is substituted by a different amino acid residue.
  • the glycine residue at position 18 of SEQ ID NO: 4 is substituted by a different amino acid residue.
  • the valine residue at position 19 of SEQ ID NO: 4 is substituted by a different amino acid residue.
  • the transmembrane domain comprises an amino acid sequence having a sequence corresponding to SEQ ID NO: 4 with a mutation at the position corresponding to position 18 of SEQ ID NO: 4, such as G18A mutations.
  • the transmembrane domain comprises an amino acid sequence having a sequence corresponding to SEQ ID NO: 4 with a mutation at the position corresponding to position 19 of SEQ ID NO: 4, such as V19A mutations.
  • the TMD can be derived from but longer or shorter than SEQ ID NO: 4.
  • the TMD can be one, two, three, four, or more amino acids longer or shorter than SEQ ID NO: 4.
  • the TMD includes a sequence having at least 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to SEQ ID NO: 4.
  • the chimeric receptors of the disclosure include an STS which comprises a charged, hydrophilic domain located between the TMD and the ICD. Without being bound to any particular theory, this domain disposed between the TMD and the ICD prevents the ICD from entering the plasma membrane.
  • STS which comprises a charged, hydrophilic domain located between the TMD and the ICD.
  • this domain disposed between the TMD and the ICD prevents the ICD from entering the plasma membrane.
  • a single-chain peptide comprising about 1 to about 40 amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) in which most of the residues have charged side chains under physiological conditions can be used as a STS.
  • STS In short STS embodiments (e.g., less than about 6 amino acids), about 5 or 6 of the amino acids will have charged side chains.
  • the STS includes about 1 to 15, about 5 to 20, about 8 to 25, about 10 to 30, about 12 to 35, about 14 to 40, about 5 to 40, about 10 to 35, about 15 to 30, about 20 to 25, about 20 to 40, about 10 to 30, about 4 to 20, or about 5 to 25 amino acid residues. In some embodiments, the STS includes about 4 to 10, about 5 to 12, about 6 to 14, about 7 to 18, about 8 to 20, about 9 to 22, about 10 to 24, or about 11 to 26 amino acid residues. In some embodiments, the STS includes about 4 to 10 residues, such as, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.
  • the STS includes a sequence having at least about 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to the STS domain of a Type 1 receptor.
  • the STS includes an amino acid sequence having at least 90% sequence identity to the STS domain of a Type 1 receptor.
  • the STS includes a sequence having at least 70% sequence identity, such as, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to a STS sequence from Notchl, Notch2, Notch3, Notch4, CSF1R, CXCL16, DAG1, GHR, PTPRF, AGER, KL, NRG1, LRPIB, Jag2, EPCAM, KCNE3, CDH2, NRG2, PTPRK, BTC, EPHA3, IL1R2, or PTPRM.
  • Notchl Notch2, Notch3, Notch4, CSF1R, CXCL16, DAG1, GHR, PTPRF, AGER, KL, NRG1, LRPIB, Jag2, EPCAM, KCNE3, CDH2, NRG2, PTPRK, BTC, EPHA3, IL1R2, or PTPRM.
  • the STS includes a sequence comprising only Lys (K) or Arg (R) in the first 4 residues. In some embodiments, the STS includes one, two, three, four, five, or more basic residues. In some embodiments, the STS includes five, four, three, two, one, or zero aromatic residues or residues with hydrophobic and/or bulky side chains.
  • the STS includes a sequence having at least 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to SKRKRKH (SEQ ID NO: 5).
  • the STS includes an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5.
  • the STS can be derived from but longer or shorter than SEQ ID NO: 5.
  • the STS can be one, two, three, four, or more amino acids longer or shorter than SEQ ID NO: 5.
  • the STS includes a sequence having at least 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to SEQ ID NO: 5.
  • the STS includes an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 5.
  • the STS includes an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 5.
  • the STS includes the amino acid sequence of SEQ ID NO: 5, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO:
  • ICD Intracellular Domain
  • the chimeric receptors of the disclosure includes an intracellular domain (ICD) comprising, in any order: (i) an intracellular signaling domain (SD) comprising at least one costimulatory domain derived from a signaling molecule and an activation domain, and (ii) a transcriptional regulator.
  • ICD intracellular domain
  • SD intracellular signaling domain
  • a transcriptional regulator a transcriptional regulator
  • the ICD of the chimeric receptors of the disclosure can have at least three distinct domains, as depicted in FIGS. 1 A-1B.
  • the three distinct domains can be arranged in specific orders, and can be operably linked to one another via one or more linkers.
  • the three distinct domains are linked via (GS)n linkers.
  • the n can be any number selected from 1 to 100.
  • n can be 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other embodiments, the n can be 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • An exemplary GS linker can have 3 GS repeats and the sequence of GSGSGSGS (SEQ ID NO: 6). One skilled in the art would know how to modify the linker to suit specific uses.
  • the intracellular domain of the chimeric receptors of the disclosure further comprises an intracellular signaling domain.
  • the intracellular signaling domain can have at least two distinct domains: at least one costimulatory domain and an activation domain.
  • the costimulatory domain comprises a sequence that is derived from a signaling molecule.
  • the signaling molecule can be a protein selected from a class 1 or a class 3 human membrane protein.
  • the signaling molecule is selected from CD28, ICOS, CTLA4, PD1, PD1H, BTLA, B71, B7H1, CD226, CRT AM, TIGIT, CD96, TIM1, TIM2, TIM3, TIM4, CD2, SLAM, 2B4, Lyl08, CD84, Ly9, CRACC, BTN1, BTN2, BTN3, LAIR1, LAG3, CD160, 4-1BB, 0X40, CD27, GITR, CD30, TNFR1, TNFR2, HVEM, LT_R, DR3, DCR3, FAS, CD40, RANK, OPG, TRAILR1, TACI, BAFFR, BCMA, TWEAKR, EDAR, XEDAR, RELT, DR
  • the signaling molecule is selected from the list consisting of 0X40, ICOS, 4-1BB, CTLA4, CD28, CD30, CD2, CD27, and CD226, and derivatives, mutants, variants, fragments and combinations thereof.
  • the signaling molecule is selected from the group consisting of 4- 1BB, BAFF-R, BCMA, BTLA, CD2, CD200R, CD244, CD28, CD300a, CD300f, CD40, CD7, CD72, CD96, CRACC, CRT AM, CTLA4, CXADR, DC-SIGN, GITR, HAVCR2, ICOS, ILT2, ILT3, ILT4, KIR2DL1, KIR3DL1, KLRG1, LAG3, LAIRl, NKG2D, NKR-P1A, NTB-A, PD1, Siglec-3, TACI, TIGIT, TLT-1, and TNR8 (CD30), and derivatives, mutants, variants, fragments and combinations thereof.
  • the signaling molecule is CD28 or 4-1BB.
  • the costimulatory domain comprises a sequence that is derived from CD28.
  • the costimulatory domain comprises a sequence that is derived from 4-1BB.
  • the costimulatory domain comprises one of the CD28 signaling motifs appended to the C terminus of a trunc41BB costimulatory domain.
  • the activation domain includes one or more conserved amino acid motifs that serve as substrates for phosphorylation such as, for example, immunoreceptor tyrosine-based activation motifs (IT AMs).
  • IT AMs immunoreceptor tyrosine-based activation motifs
  • the activation domain includes at least 1, at least 2, at least 3, at least 4, or at least 5 specific tyrosine-based motifs selected from IT AM motifs, an ITIM motifs, or related intracellular motifs that serve as a substrate for phosphorylation.
  • the activation domain of the intracellular signaling domain includes at least 1, at least 2, at least 3, at least 4, or at least 5 IT AMs.
  • any activation domain including an IT AM can be suitably used for the construction of the chimeric receptor s as described herein.
  • An IT AM generally includes a conserved protein motif that is often present in the tail portion of signaling molecules expressed in many immune cells. The motif may include two repeats of the amino acid sequence YxxL/I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing the conserved motif YxxL/Ix(6-8)YxxL/I. IT AMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule.
  • the activation domain is derived from O ⁇ 3z, CD3o, CD3/, and CD3e.
  • the IT AMs are derived from O ⁇ 3z, CD3o, CD3/, and CD3e.
  • the ITAM is derived from O ⁇ 3z.
  • the ITAM comprises a sequence that is at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a O ⁇ 3z ITAM.
  • the activation domain comprises at least 1, at least 2, at least 3, at least 4, or at least 5 IT AMs independently selected from the IT AMs derived from O ⁇ 3z, FcRy, and combinations thereof. In some embodiments, the activation domain comprises a O ⁇ 3z ITAM.
  • the intracellular domain of the chimeric receptors of the disclosure further comprises a transcriptional regulator.
  • the transcriptional regulator is a biochemical element that acts to activate or repress the transcription of a promoter-driven DNA sequence.
  • Transcriptional regulators suitable for the compositions and methods of the disclosure can be naturally-occurring transcriptional regulators or can be engineered, designed, or modified so as to provide desired and/or improved properties, e.g., modulating transcription.
  • the transcriptional regulator directly regulates expression of one or more genes involved in differentiation of the cell.
  • the transcriptional regulator indirectly modulates expression of one or more genes involved in differentiation of the cell by modulating the expression of a second transcription factor which in turn modulates expression of one or more genes involved in differentiation of the cell.
  • a transcriptional regulator can be a transcriptional activator or a transcriptional repressor.
  • the transcriptional regulator is a transcriptional repressor.
  • the transcriptional regulator is a transcriptional activator.
  • the transcriptional regulator can further include a nuclear localization signal.
  • the transcriptional regulator comprises a nuclear localization sequence derived from Gal4, tetR, ZFHD1, or HAP1.
  • the transcriptional regulator comprises a transcriptional regulator sequence derived from VP64, VP65, KRAB, or VP 16.
  • the transcriptional regulator is selected from Gal4-VP16, Gal4-VP64, tetR- VP64, ZFHD1-VP64, Gal4-KRAB, and HAP 1 -VP 16.
  • the transcriptional regulator is Gal4-VP64.
  • the ICD includes a sequence having at least 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to one or more of SEQ ID NOs: 7-14, 33, 49-53, and 59-62.
  • the ICD includes an amino acid sequence having at least 90% sequence identity to one or more of SEQ ID NOs: 7-14, 33, 49-53, and 59-62.
  • the ICD includes an amino acid sequence having at least 95% sequence identity to one or more of SEQ ID NOs: 7-14, 33, 49-53, and 59-62.
  • the ICD includes an amino acid sequence having at least 100% sequence identity to one or more of SEQ ID NOs: 7-14, 33, 49-53, and 59-62. In some embodiments, the ICD includes an amino acid sequence of one or more of SEQ ID NOs: 7-14, 33, 49-53, and 59-62, wherein one, two, three, four, or five of the amino acid residues in one or more of SEQ ID NOs: 7-14, 33, 49-53, and 59- 62 is/are substituted by a different amino acid residue.
  • the chimeric receptors provided herein can further include an additional region or domain.
  • the extracellular domains located N-terminally to the TMD can include a membrane localization signal such as a CD8A signal.
  • the chimeric receptors can include a detectable label, such as a myc tag or His tag, and the like.
  • the chimeric receptors provided herein can also include a tumor-specific cleavage site, or a disease-specific cleavage site.
  • the chimeric receptors provided herein can include a combination of these additional regions.
  • the chimeric receptors of the disclosure include: (a) a linking polypeptide including an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3; (b) a transmembrane domain including an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 4; and (c) a stop transfer sequence domain including an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5.
  • the chimeric receptors of the disclosure include: (a) an extracellular ligand-binding domain having at least 80% sequence identity to any one of SEQ ID NOS: 1, 2, 45, and 46; (b) a linking polypeptide including an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3; (c) a transmembrane domain including an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 4; (d) a stop transfer sequence domain including an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5; and (e) an intracellular domain comprising including one or more amino acid sequences having at least 80% sequence identity to one or more of SEQ ID NOs: 7-14, 33, 49-53, and 59- 62.
  • the chimeric receptors of the disclosure include: (a) an extracellular ligand-binding domain having at least 80% sequence identity to any one of SEQ ID NOS: 1, 2, 45, and 46; (b) a linking polypeptide including an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3; (c) a transmembrane domain including an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 4; (d) a stop transfer sequence domain including an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5; and (e) an intracellular domain comprising including three amino acid sequences, each having at least 80% sequence identity to any one of SEQ ID NOs: 7-14, 33, 49-53, and 59-62, linked by a GS linker.
  • the chimeric receptors of the disclosure includes: (a) an extracellular ligand-binding domain having a sequence set forth in SEQ ID NO: 1, 2, 45, and 46; (b) a linking polypeptide including an amino acid sequence having a sequence set forth in SEQ ID NO: 3; (c) a transmembrane domain including an amino acid sequence having a sequence set forth in SEQ ID NO: 4; (d) a stop transfer sequence domain including an amino acid sequence having a sequence set forth in SEQ ID NO: 5; and (e) an intracellular domain comprising including three amino acid sequences, each having a sequence set forth in SEQ ID NOs: 7-14, 33, 49-53, and 59-62, linked by a GS linker.
  • the chimeric receptors of the disclosure include: (a) an extracellular ligand-binding domain having at least 80% sequence identity to any one of SEQ ID NOS: 1, 2, 45, and 46; (b) a linking polypeptide including an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3; (c) a transmembrane domain including an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 4; and (d) an intracellular domain comprising including one or more amino acid sequences having at least 80% sequence identity to one or more of SEQ ID NOs: 7-14, 33, 49-53, and 59-62.
  • the chimeric receptor of the disclosure includes an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to a chimeric receptor disclosed herein.
  • chimeric receptors including an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97,
  • nucleic acid molecules including nucleotide sequences encoding the chimeric receptors and hybrid SynNotch receptors of the disclosure, including expression cassettes, and expression vectors containing these nucleic acid molecules operably linked to heterologous nucleic acid sequences such as, for example, regulatory sequences which facilitate in vivo expression of the receptor in a host cell.
  • Nucleic acid molecules of the present disclosure can be of any length, including for example, between about 1.5 Kb and about 50 Kb, between about 5 Kb and about 40 Kb, between about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.
  • nucleic acid molecule including a nucleotide sequence encoding a chimeric receptor or hybrid SynNotch receptor including, from N-terminus to C-terminus: (a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; (b) a linking sequence; (c) a transmembrane domain including one or more ligand-inducible proteolytic cleavage sites; and (d) an intracellular domain including (i) an intracellular signaling domain (SD) comprising at least one costimulatory domain derived from a signaling molecule and an activation domain, and (ii) a transcriptional regulator, wherein binding of the selected ligand to the extracellular ligand-binding domain induces cleavage at a ligand- inducible proteolytic cleavage site disposed between the transcriptional regulator and the hinge domain.
  • SD intracellular signaling domain
  • a transcriptional regulator wherein binding of the selected ligand to the extracellular ligand-bind
  • the nucleotide sequence is incorporated into an expression cassette or an expression vector.
  • an expression cassette generally includes a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and/or translation of the coding sequences in a recipient cell, in vivo and/or ex vivo.
  • the expression cassette may be inserted into a vector for targeting to a desired host cell and/or into an individual.
  • an expression cassette of the disclosure include a coding sequence for the chimeric receptor as disclosed herein, which is operably linked to expression control elements, such as a promoter, and optionally, any or a combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence.
  • the nucleotide sequence is incorporated into an expression vector.
  • vector generally refers to a recombinant polynucleotide construct designed for transfer between host cells, and that may be used for the purpose of transformation, e.g., the introduction of heterologous DNA into a host cell.
  • the vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • the expression vector can be an integrating vector.
  • the expression vector can be a viral vector.
  • viral vector is widely used to refer either to a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that generally facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer.
  • Viral particles will generally include various viral components and sometimes also host cell components in addition to nucleic acid(s).
  • the term viral vector may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself.
  • Viral vectors and transfer plasmids contain structural and/or functional genetic elements that are primarily derived from a virus.
  • retroviral vector refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus.
  • lentiviral vector refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus, which is a genus of retrovirus.
  • nucleic acid molecules encoding a polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to a chimeric receptor disclosed herein.
  • the nucleic acid molecules encode a polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOS: 15-32 ,34-44, 47-48, 54-58, and 63-68.
  • nucleic acid molecules encoding a polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, provided herein are nucleic acid molecules encoding a polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • nucleic acid molecules encoding a polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 7-14,33, 49-53, and 59-62.
  • the nucleic acid sequences encoding the chimeric receptors can be optimized for expression in the host cell of interest.
  • the G-C content of the sequence can be adjusted to average levels for a given cellular host, as calculated by reference to known genes expressed in the host cell.
  • Methods for codon usage optimization are known in the art. Codon usages within the coding sequence of the chimeric receptor disclosed herein can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a particular host cell.
  • Some embodiments disclosed herein relate to vectors or expression cassettes including a recombinant nucleic acid molecule encoding the chimeric receptors disclosed herein.
  • the expression cassette generally contains coding sequences and sufficient regulatory information to direct proper transcription and/or translation of the coding sequences in a recipient cell, in vivo and/or ex vivo.
  • the expression cassette may be inserted into a vector for targeting to a desired host cell and/or into an individual.
  • An expression cassette can be inserted into a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, as a linear or circular, single- stranded or double-stranded, DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, including a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, i.e., operably linked.
  • nucleic acid molecules can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transformed/transduced with the vector.
  • Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available, or readily prepared by a skilled artisan. See for example, Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W.
  • DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, nucleoporation, hydrodynamic shock, and infection.
  • Viral vectors that can be used in the disclosure include, for example, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors, lentivirus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.).
  • a chimeric receptor as disclosed herein can be produced in a eukaryotic host, such as a mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells).
  • nucleic acid molecules can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide, e.g., antibody.
  • nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphoramidite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids.
  • the nucleic acid molecules can be double-stranded or single-stranded (e.g., either a sense or an antisense strand).
  • the nucleic acid molecules are not limited to sequences that encode polypeptides (e.g., antibodies); some or all of the non-coding sequences that lie upstream or downstream from a coding sequence (e.g., the coding sequence of a chimeric receptor) can also be included.
  • polypeptides e.g., antibodies
  • some or all of the non-coding sequences that lie upstream or downstream from a coding sequence e.g., the coding sequence of a chimeric receptor
  • Those of ordinary skill in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules. They can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or by performance of the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the nucleic acid molecule is a ribonucleic acid (RNA) molecules can be produced, for example, by in vitro transcription.
  • the nucleic acid of the present disclosure can be introduced into a host cell, such as, for example, a human T lymphocyte, to produce a recombinant or engineered cell containing the nucleic acid molecule. Accordingly, some embodiments of the disclosure relate to methods for making a recombinant or engineered cell, including (a) providing a cell capable of protein expression and (b) contacting the provided cell with a recombinant nucleic acid of the disclosure.
  • nucleic acid molecules of the disclosure can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like.
  • the nucleic acid molecules can be delivered by viral or non-viral delivery vehicles known in the art.
  • the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for transient expression. Accordingly, in some embodiments, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell.
  • Stable integration can be achieved using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR/Cas9 genome editing, or DNA-guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases).
  • the nucleic acid molecule is present in the recombinant host cell as a mini-circle expression vector for transient expression.
  • the nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle, or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation.
  • introduction of nucleic acids into cells may be achieved by viral transduction.
  • adeno-associated virus AAV is engineered to deliver nucleic acids to target cells via viral transduction.
  • AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.
  • Lentiviral-derived vector systems are also useful for nucleic acid delivery and gene therapy via viral transduction.
  • Lentiviral vectors offer several attractive properties as gene- delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell -therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.
  • host cells can be genetically engineered (e.g., transduced or transformed or transfected) with, for example, a vector construct of the present application that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the polypeptides of interest.
  • Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.
  • the recombinant cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the cell is in vivo.
  • the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the cell is a non-human primate cell. In some embodiments, the mammalian cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell.
  • the recombinant cell is an immune system cell, e.g., a lymphocyte (e.g., a T cell or NK cell), or a dendritic cell.
  • the immune cell is a B cell, a monocyte, a natural killer (NK) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell (TH), a cytotoxic T cell (TCTL), or other T cell.
  • the immune system cell is a T lymphocyte.
  • the cell is a stem cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments of the cell, the cell is a lymphocyte. In some embodiments, the cell is a precursor T cell or a T regulatory (Treg) cell. In some embodiments, the cell is a CD34+, CD8+, or a CD4+ cell. In some embodiments, the cell is a CD8+ T cytotoxic lymphocyte cell selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells.
  • the cell is a CD4+ T helper lymphocyte cell selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells.
  • the cell can be obtained by leukapheresis performed on a sample obtained from a subject.
  • the subject is a human patient.
  • the recombinant cell further includes a first and a second nucleic acid molecule as disclosed herein, wherein the first nucleic acid molecule and the second nucleic acid molecule do not have the same sequence.
  • the recombinant cell further includes a first and a second chimeric receptor or hybrid SynNotch receptor as disclosed herein, wherein the first chimeric receptor or hybrid SynNotch receptor and the second chimeric receptor or hybrid SynNotch receptor do not have the same sequence.
  • the first chimeric receptor or hybrid SynNotch receptor modulates the expression and/or activity of the second chimeric receptor or hybrid SynNotch receptor.
  • the recombinant cell further includes an expression cassette encoding a protein of interest operably linked to a promoter, wherein expression of the protein of interest is modulated by the chimeric receptor transcriptional regulator.
  • a promoter Any suitable promoter can be used in connection with the present disclosure.
  • the promoter comprises a yeast GAL4 promoter.
  • the protein of interest is heterologous to the recombinant cell.
  • a heterologous protein is one that is not normally found in the cell, e.g., not normally produced by the cell. In principle, there are no particular limitations with regard to suitable proteins whose expression can be modulated by the chimeric receptor transcriptional regulator.
  • Exemplary types of proteins suitable for use with the compositions and methods disclosed herein include cytokines, cytotoxins, chemokines, immunomodulators, pro-apoptotic factors, anti-apoptotic factors, hormones, differentiation factors, dedifferentiation factors, immune cell receptors, or reporters.
  • the immune cell receptor is a T-cell receptor (TCR).
  • the immune cell receptor is a chimeric antigen receptor (CAR).
  • the expression cassette encoding the protein of interest is incorporated into the same nucleic acid molecule that encodes the chimeric receptor of the disclosure.
  • the expression cassette encoding the protein of interest is incorporated into a second expression vector that is separate from the nucleic acid molecule encoding the chimeric receptor of the disclosure.
  • cell cultures including at least one recombinant cell as disclosed herein, and a culture medium.
  • the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.
  • compositions including pharmaceutical compositions.
  • Such compositions generally include the nucleic acids, and/or recombinant cells, and a pharmaceutically acceptable excipient, e.g., carrier.
  • a pharmaceutically acceptable excipient e.g., carrier.
  • Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate.
  • surfactants e.g., sodium dodecyl sulfate.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be generally to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the chimeric receptors and Notch receptors of the disclosure can also be administered by transfection or infection using methods known in the art, including but not limited to the methods described in McCaffrey et al. (Nature 418:6893, 2002), Xia et al. (Nature Biotechnol. 20:1006-10, 2002), or Putnam (Am. J. Health Syst. Pharm. 53:151-60, 1996, erratum at Am. J. Health Syst. Pharm. 53:325, 1996).
  • nucleic acids, recombinant cells, and pharmaceutical compositions can be used to treat patients for relevant health conditions or diseases, such as cancers and chronic infections.
  • nucleic acids, recombinant cells, and pharmaceutical compositions described herein can be incorporated into therapeutic agents for use in methods of treating an individual who has, who is suspected of having, or who may be at high risk for developing one or more autoimmune disorders or diseases associated with checkpoint inhibition.
  • Exemplary autoimmune disorders and diseases can include, without limitation, celiac disease, type 1 diabetes, Graves’ disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.
  • some embodiments of the disclosure relate to methods for inhibiting an activity of a target cell in an individual, the methods include administering to the individual a first therapy including one or more of nucleic acids, recombinant cells, and pharmaceutical compositions as disclosed herein, wherein the first therapy inhibits the target cell.
  • the target cell may be inhibited if its proliferation is reduced, if its pathologic or pathogenic behavior is reduced, if it is destroyed or killed, etc.
  • Inhibition includes a reduction of the measured pathologic or pathogenic behavior of at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.
  • the methods include administering to the individual an effective number of the recombinant cells disclosed herein, wherein the recombinant cells inhibit an activity of the target cells in the individual.
  • the target cells of the disclosed methods can be any cell type in an individual and can be, for example an acute myeloma leukemia cell, an anaplastic lymphoma cell, an astrocytoma cell, a B-cell cancer cell, a breast cancer cell, a colon cancer cell, an ependymoma cell, an esophageal cancer cell, a glioblastoma cell, a glioma cell, a leiomyosarcoma cell, a liposarcoma cell, a liver cancer cell, a lung cancer cell, a mantle cell lymphoma cell, a melanoma cell, a neuroblastoma cell, a non-small cell lung cancer cell, an oligodendroglioma cell, an ovarian cancer cell, a pancreatic cancer cell, a peripheral T-cell lymphoma cell, a renal cancer cell, a sarcoma cell, a stomach cancer cell, a carcinoma
  • the target cell is a pathogenic cell.
  • some embodiments of the disclosure relate to methods for the treatment of a health condition (e.g., disease) in an individual in need thereof, the methods include administering to the individual a first therapy including one or more of the recombinant cells including a chimeric receptor as disclosed herein, and/or pharmaceutical compositions as disclosed herein, wherein the first therapy treats the health condition in the individual.
  • the methods include administering to the individual a first therapy including an effective number of the recombinant cells as disclosed herein, wherein the recombinant cells treat the health condition.
  • some embodiments of the disclosure relate to methods for assisting in the treatment of a health condition (e.g., disease) in an individual in need thereof, the methods including administering to the individual a first therapy including one or more of chimeric receptor s, Hinge-Notch receptors, nucleic acids, recombinant cells, and pharmaceutical compositions as disclosed herein, and a second therapy, wherein the first and second therapies together treat the disease in the individual.
  • the methods include administering to the individual a first therapy including an effective number of the recombinant cells as disclosed herein, wherein the recombinant cells treat the health condition.
  • the methods of the disclosure involve administering an effective amount of the recombinants cells of the disclosure to an individual in need of such treatment.
  • This administering step can be accomplished using any method of implantation delivery in the art.
  • the recombinant cells of the disclosure can be infused directly in the individual’s bloodstream or otherwise administered to the individual.
  • the methods disclosed herein include administering, which term is used interchangeably with the terms “introducing,” implanting,” and “transplanting,” recombinant cells into an individual, by a method or route that results in at least partial localization of the introduced cells at a desired site such that a desired effect(s) is/are produced.
  • the recombinant cells or their differentiated progeny can be administered by any appropriate route that results in delivery to a desired location in the individual where at least a portion of the administered cells or components of the cells remain viable.
  • the period of viability of the cells after administration to an individual can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of the individual, i.e., long-term engraftment.
  • the recombinant cells described herein can be administered to an individual in advance of any symptom of a disease or condition to be treated. Accordingly, in some embodiments the prophylactic administration of a recombinant cell population prevents the occurrence of symptoms of the disease or condition.
  • recombinant cells are provided at (or after) the onset of a symptom or indication of a disease or condition, e.g., upon the onset of disease or condition.
  • an effective amount of recombinant cells as disclosed herein can be at least 10 2 cells, at least 5 c 10 2 cells, at least 10 3 cells, at least 5 c 10 3 cells, at least 10 4 cells, at least 5 c 10 4 cells, at least 10 5 cells, at least 2 c
  • the recombinant cells can be derived from one or more donors or can be obtained from an autologous source. In some embodiments, the recombinant cells are expanded in culture prior to administration to an individual in need thereof.
  • a recombinant cell composition e.g., a composition including a plurality of recombinant cells according to any of the cells described herein
  • a composition including recombinant cells can be administered by any appropriate route that results in effective treatment in the individual, e.g., administration results in delivery to a desired location in the individual where at least a portion of the composition delivered, e.g., at least 1 c 10 4 cells, is delivered to the desired site for a period of time.
  • Modes of administration include injection, infusion, instillation.
  • “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrastemal injection and infusion.
  • the route is intravenous.
  • delivery by injection or infusion is a preferred mode of administration.
  • the recombinant cells are administered systemically, e.g., via infusion or injection.
  • a population of recombinant cells are administered other than directly into a target site, tissue, or organ, such that it enters, the individual’s circulatory system and, thus, is subject to metabolism and other similar biological processes.
  • efficacy of a treatment including any of the compositions provided herein for the treatment of a disease or condition can be determined by a skilled clinician. However, one skilled in the art will appreciate that a treatment is considered effective if any one or all of the signs or symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by decreased hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein.
  • Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.
  • a therapeutically effective amount includes an amount of a therapeutic composition that is sufficient to promote a particular beneficial effect when administered to an individual, such as one who has, is suspected of having, or is at risk for a disease.
  • an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.
  • the individual is a mammal.
  • the mammal is a human.
  • the individual has or is suspected of having a disease associated with inhibition of cell signaling mediated by a cell surface ligand or antigen.
  • the diseases suitable for being treated by the compositions and methods of the disclosure include, but are not limited to, cancers, autoimmune diseases, inflammatory diseases, and infectious diseases.
  • the disease is a cancer or a chronic infection.
  • the engineered CARs may be introduced into T cells using retroviruses, which efficiently and stably integrate a nucleic acid sequence encoding the chimeric antigen receptor into the target cell genome.
  • retroviruses which efficiently and stably integrate a nucleic acid sequence encoding the chimeric antigen receptor into the target cell genome.
  • lentiviral transduction e.g., type I, type II, or type III systems using a suitable Cas protein such Cas3, Cas4, Cas5, Cas5e (or CasD),
  • a recombinant adeno-associated virus (AAV) vector can be used for delivery.
  • Techniques to produce rAAV particles, in which an AAV genome to be packaged that includes the polynucleotide to be delivered, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (e.g., not in) the rAAV genome, and helper virus functions.
  • the AAV rep and cap genes can be from any AAV serotype for which recombinant virus can be derived, and can be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13 and AAV rh.74. Production of pseudotyped rAAV is disclosed in, for example, international patent application publication number WO 01/83692.
  • the CAR-T cells once they have been expanded ex vivo in response to, for example, an autoimmune disease antigen, can be reinfused into the subject in a therapeutically effective amount.
  • CAR T cells administered can be determined by a physician with consideration of individual differences in age, weight, extent of disease and condition of the subject.
  • T cell therapies may be defined by number of total cells per infusion or number of cells per kilogram of body weight, especially for pediatric subjects (e.g., patients). As T cells replicate and expand after transfer, the administered cell dose may not resemble the final steady-state number of cells.
  • a pharmaceutical composition including the CAR T cells of the present disclosure may be administered at a dosage of 104 to 1010 total cells.
  • a pharmaceutical composition including the CAR T cells of the present disclosure may be administered at a dosage of 103 to 108 cells/kg body weight, including all integer values within those ranges.
  • compositions including the CAR T cells of the present disclosure may also be administered multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are known in the art (see, for example, Rosenberg et ah, New Engl J Med, (1988) 319: 1676).
  • the optimal dosage and treatment regimen for a particular subject can be determined by one skilled in the art by monitoring the subject for signs of disease and adjusting the treatment accordingly.
  • compositions embodied herein for the treatment of, for example, an autoimmune or inflammatory disease
  • other cell-based therapies for example, stem cells, antigen presenting cells, pancreatic islets etc.
  • the composition of the present disclosure may be prepared in a manner known in the art and in a manner suitable for parenteral administration to mammals, particularly humans, including a therapeutically effective amount of the composition alone, with one or more pharmaceutically acceptable carriers or diluents.
  • compositions of the present disclosure may also include other supplementary physiologically active agents.
  • compositions include those suitable for parenteral administration, including subcutaneous, intramuscular, intravenous and intradermal administration.
  • the compositions may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. Such methods include preparing the carrier for association with the CAR-T cells. In general, the compositions are prepared by uniformly and intimately bringing into association any active ingredients with liquid carriers.
  • the composition is suitable for parenteral administration. In another embodiment, the composition is suitable for intravenous administration.
  • compositions suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes, which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • any one of the compositions as disclosed herein e.g., the chimeric receptors, recombinant nucleic acids, recombinant cells, cell cultures, and pharmaceutical compositions described herein can be administered to a subject in need thereof as a single therapy (e.g., monotherapy).
  • the chimeric receptors, recombinant nucleic acids, recombinant cells, cell cultures, and pharmaceutical compositions described herein can be administered to the subject in combination with one or more additional therapies, e.g., at least one, two, three, four, or five additional therapies.
  • Suitable therapies to be administered in combination with the compositions of the disclosure include, but are not limited to chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery.
  • Other suitable therapies include therapeutic agents such as chemotherapeutics, anti-cancer agents, and anti-cancer therapies.
  • Administration “in combination with” one or more additional therapies includes simultaneous (concurrent) and consecutive administration in any order.
  • the one or more additional therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery.
  • chemotherapy as used herein encompasses anti-cancer agents.
  • Various classes of anti-cancer agents can be suitably used for the methods disclosed herein.
  • Non-limiting examples of anti cancer agents include: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxin, antibodies (e.g., monoclonal or polyclonal), tyrosine kinase inhibitors (e.g., imatinib mesylate (Gleevec® or Glivec®)), hormone treatments, soluble receptors and other antineoplastics.
  • alkylating agents include: antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxin, antibodies (e.g., monoclonal or polyclonal), tyrosine kinase inhibitors (e.g., imatinib mesylate (Gleevec® or Glivec®)), hormone treatments, soluble receptors and other antineoplastics.
  • the present disclosure also contemplates the combination of the composition of the disclosure with other drugs and/or in addition to other treatment regimens or modalities such as surgery.
  • the composition of the present disclosure is used in combination with known therapeutic agents the combination may be administered either in sequence (either continuously or broken up by periods of no treatment) or concurrently or as an admixture.
  • treatment includes administering to the subject the compositions embodied herein, e.g. autologous T cells transduced or contacted with a CAR embodied herein and one or more anti-inflammatory agents and/or therapeutic agents.
  • the anti-inflammatory agents include one or more antibodies which specifically bind to pro-inflammatory cytokines, e.g., pro-inflammatory cytokines such as IL-1, TNF, IL-6, GM-CSF, and IFN-g.
  • pro-inflammatory cytokines such as IL-1, TNF, IL-6, GM-CSF, and IFN-g.
  • the antibodies are anti-TNFa, anti -IL-6 or combinations thereof.
  • one or more agents, other than antibodies can be administered which decrease pro- inflammatory cytokines, e.g. non-steroidal anti-inflammatory drugs (NSAIDs). Any combination of antibodies and one or more agents can be administered which decrease pro-inflammatory cytokines.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • Treatment in combination is also contemplated to encompass the treatment with either the composition of the disclosure followed by a known treatment, or treatment with a known agent followed by treatment with the composition of the disclosure, for example, as maintenance therapy.
  • autoimmune diseases excessive and prolonged activation of immune cells, such as T and B lymphocytes, and overexpression of the master pro- inflammatory cytokine tumor necrosis factor alpha (TNF), together with other mediators such as interleukin-6 (IL-6), interleukin- 1 (IL-1), and interferon gamma (IFN-g), play a central role in the pathogenesis of autoimmune inflammatory responses in rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Crohn’s disease (CD), and ankylosing spondylitis (AS).
  • RA rheumatoid arthritis
  • IBD inflammatory bowel disease
  • CD Crohn’s disease
  • AS ankylosing spondylitis
  • Non-steroidal anti-inflammatory drugs NSAIDs
  • glucocorticoids disease-modifying anti-rheumatic drugs
  • DMARDs disease-modifying anti-rheumatic drugs
  • Anti-TNF biologies (such as infliximab, adalimumab, etanercept, golimumab, and certolizumab pepol) have markedly improved the outcome of the management of autoimmune inflammatory diseases.
  • Non-steroidal anti-inflammatory drugs have the analgesic, antipyretic, and anti inflammatory effect, frequently used for the treatment of conditions like arthritis and headaches.
  • NSAIDs relieve pain through blocking cyclooxygenase (COX) enzymes. COX promotes the production of prostaglandins, a mediator which causes inflammation and pain.
  • COX cyclooxygenase
  • NSAIDs have different chemical structures, all of them have the similar therapeutic effect, e.g., inhibition of autoimmune inflammatory responses.
  • NSAIDs can be divided into two broad categories: traditional non-selective NSAIDs and selective cyclooxygenase-2 (COX-2) inhibitors (For a review, see, P. Li et ah, Front Pharmacol (2017) 8:460).
  • abatacept a fully humanized fusion protein of extracellular domain of CTLA-4 and Fc fraction of IgGl, has been approved for the RA patients with inadequate response to anti-TNF therapy.
  • the major immunological mechanism of abatacept is selective inhibition of co stimulation pathway (CD80 and CD86) and activation of T cells.
  • Tocilizumab a humanized anti- IL-6 receptor monoclonal antibody was approved for RA patients intolerant to DMARDs and/or anti-TNF biologies.
  • This therapeutic mAh blocks the transmembrane signaling of IL-6 through binding with soluble and membrane forms of IL-6 receptor.
  • Biological drugs targeting IL-1 anakinra
  • Thl immune responses IL-12/IL-23, ustekinumab
  • Thl7 immune responses IL-17, secukinumab
  • CD20 rituximab
  • the methods of the disclosure include administration of a composition disclosed herein to a subject individually as a single therapy (e.g., monotherapy).
  • a composition of the disclosure is administered to a subject as a first therapy in combination with a second therapy.
  • the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery.
  • the first therapy and the second therapy are administered concomitantly.
  • the first therapy is administered at the same time as the second therapy.
  • the first therapy and the second therapy are administered sequentially.
  • the first therapy is administered before the second therapy.
  • the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and/or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in rotation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.
  • the methods include the steps of: (a) providing an effective amount of any of the recombinant cells provided herein, and (b) contacting it with a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain induces cleavage of a ligand- inducible proteolytic cleavage site and releases the intracellular domain comprising the intracellular signaling domain and the transcriptional regulator, wherein the released intracellular signaling domain and the transcriptional regulator modulates an activity of the recombinant cell.
  • a selected ligand binding of the selected ligand to the extracellular ligand-binding domain induces cleavage of a ligand- inducible proteolytic cleavage site and releases the intracellular domain comprising the intracellular signaling domain and the transcriptional regulator, wherein the released intracellular signaling domain and the transcriptional regulator modulates an activity of the recombinant cell.
  • Non-limiting exemplary cellular activities that can be modulated using the methods include, but are not limited to, gene expression, proliferation, apoptosis, non- apoptotic death, differentiation, dedifferentiation, migration, secretion of a gene product, cellular adhesion, and cytolytic activity.
  • the released transcriptional regulator modulates expression of a gene product of the cell. In some embodiments, the released transcriptional regulator modulates expression of a heterologous gene product in the cell.
  • a heterologous gene product is one that is not normally found in the native cell, e.g., not normally produced by the cell.
  • the cell can be genetically modified with a nucleic acid including a nucleotide sequence encoding the heterologous gene product.
  • the heterologous gene product is a secreted gene product. In some embodiments, the heterologous gene product is a cell surface gene product. In some cases, the heterologous gene product is an intracellular gene product. In some embodiments, the released transcriptional regulator simultaneously modulates expression of two or more heterologous gene products in the cell.
  • the heterologous gene product in 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 T-cell receptor (TCR), a chimeric antigen receptor (CAR), a toxin, a toxin-derived protein, a transcriptional regulator, a transcriptional activator, a transcriptional repressor, a translation regulator, a translational activator, a translational repressor, an activating immuno-receptor, an antibody, an apoptosis inhibitor, an apoptosis inducer, an engineered T cell receptor, an immuno-activator, an immuno
  • the released transcriptional regulator modulates differentiation of the cell, and wherein the cell is an immune cell, a stem cell, a progenitor cell, or a precursor cell.
  • the chimeric receptors of the disclosure provide a higher degree of expression than a standard SynNotch receptor, when using identical binding domains and ICDs.
  • the chimeric receptor s or Hinge-Notch receptors of the disclosure can provide expression enhancement of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% higher than a corresponding SynNotch receptor.
  • the chimeric receptors of the disclosure can provide transcriptional regulation that responds to the degree of T cell activation, independent of ligand binding. For example, when expressed in a T cell, some receptors of the disclosure provide a stronger ligand- induced signal when the T-cell is activated as compared to the ligand-induced signal when the T- cell is not activated. This permits additional flexibility in use, for example in cases where it is desired to enhance or suppress a T cell response when activated despite the absence of the chimeric receptor ligand.
  • kits including the chimeric receptor s, Hinge- Notch receptors, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions provided and described herein as well as written instructions for making and using the same.
  • systems and/or kits that include one or more of: an chimeric receptor as described herein, a Hinge-Notch receptor as described herein, a recombinant nucleic acids as described herein, a recombinant cell as described herein, or a pharmaceutical composition as described herein.
  • kits of the disclosure further include one or more syringes (including pre-filled syringes) and/or catheters (including pre-filled syringes) used to administer one any of the provided chimeric receptor s, Hinge-Notch receptors, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to an individual.
  • a kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the other kit components for a desired purpose, e.g., for modulating an activity of a cell, inhibiting a target cancer cell, or treating a health condition (e.g., disease) in an individual in need thereof.
  • any of the above-described systems and kits can further include one or more additional reagents, where such additional reagents can be selected from: dilution buffers; reconstitution solutions, wash buffers, control reagents, control expression vectors, negative control polypeptides, positive control polypeptides, reagents for in vitro production of the chimeric receptor polypeptides.
  • the components of a system or kit can be in separate containers. In some other embodiments, the components of a system or kit can be combined in a single container.
  • a system or kit can further include instructions for using the components of the kit to practice the methods.
  • the instructions for practicing the methods are generally recorded on a suitable recording medium.
  • the instructions can be printed on a substrate, such as paper or plastic, etc.
  • the instructions can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging), etc.
  • the instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided.
  • kits that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.
  • Intracellular domains containing the appropriate costimulatory domain, CD3zeta domain, Gal4-VP64 and GS linkers were synthesized as gene fragments from Twist.
  • Receptors were built by fusing the CD 19, BCMA or ALPPL2 targeting scFv to the corresponding receptor scaffold and intracellular tail. All receptors contain an n-terminal CD8a signal peptide (MALPVTALLLPLALLLHAARP, SEQ ID NO: 69) for membrane targeting and a flag-tag (DYKDDDDK, SEQ ID NO: 70) for easy determination of surface expression with a-flag PE (Biolegend 637310). The receptors were cloned into a modified pHR’SIN:CSW vector containing a PGK promoter for all primary T cell experiments.
  • pHR’SIN:CSW vector was also modified to make the response element plasmids. Five copies of the Gal4 DNA binding domain target sequence
  • GGAGCACTGTCCTCCGAACG, SEQ ID NO: 71 were cloned 5' to a minimal pybTATA promoter. Also included in the response element plasmids was a PGK promoter that constitutively drives mCitrine expression to easily identify transduced T cells. For all inducible BFP vectors, BFP was cloned via a BamHI site in the multiple cloning site 3 ' to the Gal4 response elements. All constructs were cloned via In-fusion cloning (Clontech #ST0345).
  • FIG. 1A shows a diagram of all possible intracellular domain configurations.
  • FIG. IB shows a detailed diagram of hybrid SynNotch CAR domains.
  • FIG. 1C illustrates the principle of short term proximal and long term transcriptional signaling induced by hybrid SynNotch CARs.
  • the components of the hybrid SynNotch CARs comprising a 4-1BB costimulatory domain are described in Table 1 below.
  • the N-JMDs of the hybrid SynNotch CARs tested here included a truncated form of the CD8a hinge that is composed of an N-terminal fragment of the typical CD8a Hinge domain.
  • T cells Primary CD4+ and CD8+ T cells were isolated from anonymous donor blood after apheresis by negative selection (STEMCELL Technologies #15062 & 15063). Blood was obtained from Blood Centers of the Pacific (San Francisco, CA) as approved by the University Institutional Review Board. T cells were cryopreserved in RPMI-1640 (UCSF cell culture core) with 20% human AB serum (Valley Biomedical Inc., #HP1022) and 10% DMSO.
  • T cells were cultured in human T cell medium consisting of X- VIVO 15 (Lonza #04-418Q), 5% Human AB serum and 10 mM neutralized N-acetyl L-Cysteine (Sigma- Aldrich #A9165) supplemented with 30 units/mL IL-2 (NCI BRB Preclinical Repository) for all experiments. In vivo experiments were completed with bulk CD3+ cells isolated in a similar manner.
  • Pantropic VSV-G pseudotyped lentivirus was produced via transfection of Lenti-X 293T cells (Clontech #1113 ID) with a pHR’ SIN:CSW transgene expression vector and the viral packaging plasmids pCMVdR8.91 and pMD2.G using Mirus TransIT-Lenti (Mirus #MIR 6606).
  • Primary T cells were thawed the same day, and after 24 hours in culture, were stimulated with Human T-Activator CD3/CD28 Dynabeads (Life Technologies #1113 ID) at a 1 :3 celkbead ratio. At 48 hours, viral supernatant was harvested and the primary T cells were exposed to the virus for 24 hours.
  • T cells were sorted, and the T cells expanded until day 10-14 when they were rested and could be used in vitro or in vivo assays. T cells were sorted for assays with a Beckton Dickinson (BD) FACs ARIA II. Cancer Cell Lines
  • the cancer cell lines used were K562 myelogenous leukemia cells (ATCC #CCL-243), A549 lung epithelial carcinoma cells (ATCC #CCL-18) and M28 human epithial type mesothelioma cells.
  • K562s, A549s and M28s were lentivirally transduced to stably express human CD 19.
  • CD 19 levels were determined by staining the cells with a-CD19 APC (Biolegend #302212) or BV421 (Biolegend # 302234).
  • A549s were additionally transduced to express the nuclear stain mkate2. All cell lines were sorted for expression of the transgenes.
  • the expression of surface activation markers was used as a measure of short-term activation by either CAR or hybrid SynNotch CAR Signaling.
  • transduced cells were co-cultured with K562s, either with or without CD 19 expression, at a 1 : 1 ratio for 24-72 hours.
  • Co-cultured cells were then centrifuged, washed twice with flow buffer (PBS + 2% FBS), and stained in 50 uL of a master mix of antibodies targeting surface activation markers CD69, PD-1, CD25 and CD39.
  • Cells were washed twice after stain with flow buffer and resuspended in flow buffer with diluted DRAQ7 to assess viability. Stained cells were immediately analyzed on a flow cytometer to assess expression of activation markers.
  • transduced primary human T cells were co-cultured with K562s, either with or without CD 19 expression, at a 1 : 1 ratio for 18-24 hours (overnight), 48 hours (short term) or 96 hours (long term).
  • Overnight co-cultures included Brefeldin A (eBioscience #00-4506-51) and Monesin (VWR #420701-BL) to stop secretion of cytokines.
  • Brefeldin A and Monesin were added to the co-culture and incubated for an additional 6 hours before beginning staining.
  • Co-cultured cells were washed twice with PBS and stained in 50uL of Fixable NEAR IR (Invitrogen #L34975) for 20 minutes at room temperature in the dark. 50uL of a master mix containing fluorescently tagged anti-CD4 or anti-CD8 antibodies was then added to the cells and incubated for 20 minutes at room temperature in the dark. Stained cells were then washed twice with flow buffer (PBS + 2% FBS). Stained cells were then resuspended in lOOuL of IC Fix Buffer (eBioscience #00-8222-49) and incubated for 45 minutes at 4C in the dark. Fixed cells were then washed twice with IX Permeabilization Buffer (eBioscience #00-8333-56).
  • An intracellular cytokine staining master mix was made of fluorescently tagged antibodies targeting intracellular cytokines TNFa, IL-2, IFNy and GranzymeB diluted in IX Permeabilization Buffer. Washed cells were stained in 50uL of this master mix for 30 minutes at 4C in the dark. Stained cells were washed twice with IX Permeabilization Buffer and resuspended in lOOuL of flow buffer. Stained cells were immediately analyzed on a flow cytometer.
  • CD 19+ A549 cells expressing mkate2 were seeded in a flat bottom 96 well plate and incubated overnight to allow adherence.
  • Transduced primary human T cells were centrifuged and resuspended in Jurkat media + 30 U/mL IL-2; Jurkat media (RPMI-1640 medium + 10% FBS + 1% PenStrep + IX Glutamax) as RPMI has less fluorescence than media based on X-VIVO-15.
  • Media was removed from the adherent A549 cells, and transduced human T cells were added to cultures at a 1 : 1 ratio. Images were taken every 2 hours using the Incucyte software over the course of the experiments (see relevant figures for imaging total assay times, which varied between conditions).
  • Transduced human T cells were taken from culture and washed into PBS with diluted CellTrace Far Red (CTFR) (Invitrogen #C34564). Cells were stained for 20 minutes at 37 °C in the dark, then 5x the staining volume of culture media with protein was added, and cells were incubated for an additional 5 minutes at 37 °C in the dark. Stained cells were centrifuged, washed into human T cell media. K562 cells with and without CD 19 expression were washed into human T cell media and added to CTFR stained T cells at a 1:1 ratio. Co-cultures were incubated for 5 days, with a media change occurring halfway through incubation.
  • CTFR CellTrace Far Red
  • Co-cultures were then centrifuged, washed twice with flow buffer (PBS + 2% FBS), and stained in 50 pL of master mix containing fluorescently tagged anti-CD8 antibodies. Cells were washed twice after stain with flow buffer and resuspended in flow buffer with diluted DRAQ7 to assess viability. Stained cells were immediately analyzed on a flow cytometer to assess dilution of CTFR dye.
  • NOD.Cg-Prkdcscid I12rgtmlWjl/SzJ (NSG) (UCSF LARC Breeding Core) mice were dosed with 0.5 x 106 Luciferase expressing Naim 6 cells via tail vein injection. 4 days post tumor injection, hybrid SynNotch CAR or CAR transduced T cells were dosed to tumor bearing animals via retro-orbital injection (see figures details for the number of T cells dosed per experiment). Bioluminescence imaging was performed using an IVIS Spectrum In Vivo Imaging system at regular time points to assess tumor burden. Animals were dosed with 200 pL of 15 mg/mL Luciferin via IP injection, and allowed to ambulate for 12-20 minutes prior to capturing prone and supine images.
  • Image capture time was adjusted based on bioluminescence intensity, and average radiance [p/s/cm 2 /sr] was used as a measurement of tumor burden. Throughout experiment animal drinking water was supplemented with Clavomox (Zoetis #55-101) to prevent bacterial infections.
  • NOD.Cg-Prkdcscid I12rgtmlWjl/SzJ (NSG) mice were dosed with 4 x 106 CD19 ligand expressing M28 cells via subcutaneous injection. 7 days post tumor injection, 3-6 x 106 Hybrid SynNotch CAR or CAR tranduced T cells were dosed to tumor bearing animals via retro- orbital injection. Tumors were measured with calipers twice weekly, and tumor volume was calculated using the following formula: (length x width2)/2. Throughout experiment animal drinking water was supplemented with Clavomox to prevent bacterial infections.
  • Hybrid SynNotch CARs with 4- IBB or CD28 costimulatory domains exhibit antigen independent induction of the inducible transcriptional BFP reporter element (e.g., For 4-1BB Configuration 1 and 4, for CD28 configurations 1, 2, 4, 5, and 6).
  • other intracellular configurations of the Hybrid SynNotch CARs exhibit antigen specific induction of the BFP reporter element, expressing BFP only when in the presence of ligand (e.g., For 4-1BB configurations 2, 3, 5, and 6, for CD28 configuration 3).
  • This data set demonstrates the ability of specific configurations of Hybrid SynNotch CAR circuits that incorporate either the 4- IBB or CD28 co-stimulatory domains to induce transcription in an antigen specific manner. Additionally, this data set indicates that the spatial configuration of the intracellular domains influences receptor behavior in both the presence and absence of ligand.
  • This Example shows the expression of the activation markers of the T cells transduced with the exemplary hybrid SynNotch CARs.
  • T-cells expressing anti-CD19 receptors with 4-1BB costimulatory domains (FIG. 3A) or CD28 costimulatory domains (FIG. 3B) and the BFP reporter were produced as described above.
  • Transduced cells were co-cultured with K562 cells (gray), or CD 19+ K562 cells (light gray) for 48 hours.
  • Expression of activation markers CD25, CD39, CD69 and PD-1 were subsequently measured using a Fortessa X-50 (BD).
  • Hybrid SynNotch CAR T cells express activation markers at a similar mean fluorescence intensity and overall percentage as compared to the CAR control that employs the same co-stimulatory domain. Additionally, the Hybrid SynNotch CAR T cells only express activation markers when in the presence of ligand. Together, this data set indicates that, upon engagement with antigen, Hybrid SynNotch CARs functionally induce signaling through their intracellular signaling domains (4- 1BB or CD28, and CD3zeta), leading to expression of activation markers.
  • This Example shows the proliferation of the T cells transduced with the exemplary hybrid SynNotch CARs.
  • T-cells expressing anti-CD19 receptors with 4-1BB costimulatory domains or CD28 costimulatory domains and the BFP reporter were produced as described above.
  • Transduced cells were stained with Cell Trace Far Red (CTFR), then co-cultured with K562 cells (gray), or CD19+ K562 cells (blue or red) for 5 days. Dilution of CTFR dye was subsequently measured using a Fortessa X-50 (BD).
  • CTFR Cell Trace Far Red
  • Hybrid SynNotch CARs when engaged with ligand, the Hybrid SynNotch CARs induce T cell proliferation at a rate similar to the CAR alone. Additionally, the Hybrid SynNotch CAR T cells specifically proliferate extensively when antigen is present, indicating that the proliferative response of the Hybrid SynNotch CAR T cells is antigen specific. Altogether, this data set indicates that, upon engagement with antigen, Hybrid SynNotch CARs functionally induce short term signaling cascades through their intracellular signaling domains (4- IBB or CD28, and CD3zeta), leading to proliferation of the T cells.
  • This Example shows the cytokine secretion by the T cells transduced with theexemplary hybrid SynNotch CARs.
  • T-cells expressing anti-CD19 receptors with 4-1BB costimulatory domains (FIG. 5A) or CD28 costimulatory domains (FIG. 5B) and the BFP reporter were produced as above.
  • Transduced cells were co-cultured with K562 cells (gray), or CD 19+ K562 cells (blue or red). After 48 hours, Brefeldin A, Monesin and a second bolus of K562 cells (either with or without CD 19+ expression) was added to the co-cultures.
  • This Example shows the cell killing activity of the exemplary hybrid SynNotch CARs.
  • T-cells expressing anti-CD19 receptors with 4-1BB costimulatory domains (FIG. 6A) or CD28 costimulatory domains (FIG. 6B) and the BFP reporter were produced as described above.
  • A549 cells expressing the CD 19 ligand and the nuclear stain mkate2 were allowed to adhere to a 96 well flat bottom plate for 24 hours, then transduced T cells were added at a 1:1 ratio. The plate was incubated in an Incucyte, which captured plate images and fluorescence every 2 hours for 5 days.
  • Imaging software was used to calculate the number of A549 CD 19+ mkate2+ cells in culture at each time point. For each experimental group, the A549 cell count was normalized to that of the Hinge Notch experimental group. As demonstrated, the Hybrid SynNotch CAR T cells kill target cells at similar rates as the CAR T cells. This indicates that, in this in vitro setting, the Hybrid SynNotch CARs induce T cell activation and cytotoxic programs that are sufficient to cause target cell killing over a period of multiple days.
  • This Example shows the in vivo efficacy of the exemplary hybrid SynNotch CARs provided herein.
  • FIG. 7A describes the experimental timeline.
  • NOD.Cg-PrkdcscidI12rgtmlWjl/SzJ (NSG) mice were dosed intravenously with 0.5 xl06 Nalm6-Luc-GFP tumor cells.
  • Bulk CD3+ T-cells were co-transduced with the anti-CD19 hybrid SynNotch CAR with the CD3z - Gal4VP64 - CD28 intracellular domain and the BFP reporter as described above.
  • 0.5 xl06 transduced CD3+ T cells were dosed to animals via retro-orbital injection 4 days post tumor injection.
  • FIG. 7B shows the tumor burden measured via bioluminescence imaging of luciferase secreting tumor cells using an IVIS Spectrum and FIG. 7C shows the survival curve of the experimental mice.
  • This data demonstrates Hybrid SynNotch CAR T cells are effective in clearing Nalm6 tumors in vivo similarly to CAR T cells with a CD28 costimulatory domain.
  • the Nalm6 tumor burden initially grows in both Hybrid SynNotch CAR and CAR T cell treated groups, however the tumor burden is reduced, and eventually cleared in both treated groups at approximately day 10 post tumor injection.
  • FIG. 10A shows a description of experimental timeline.
  • NOD.Cg- PrkdcscidI12rgtmlWjl/SzJ (NSG) mice were dosed subcutaneously with 4x106 CD19 ligand expressing M28 tumor cells.
  • Bulk CD3+ T-cells were co-transduced with the anti-CD19 Hybrid SynNotch CAR with ICDs as indicated in 10B and the BFP reporter as described above.
  • 6 xl06 transduced CD3+ T cells were dosed to animals via retro-orbital injection 7 days post tumor injection.
  • FIG. 10B shows tumor volume assessed via caliper measurements weekly.
  • FIG. 14A shows a description of experimental timeline.
  • NOD.Cg- PrkdcscidI12rgtmlWjl/SzJ (NSG) mice were dosed subcutaneously with 4x106 CD19 expressing M28 tumor cells.
  • Bulk CD3+ T-cells were co-transduced with the anti-CD 19 Hybrid SynNotch CAR with ICDs as indicated in 14B and the BFP reporter as described above.
  • FIG. 14B shows tumor volume assessed via caliper measurements weekly.
  • FIG. 16 shows ALPPL2 Targeted Hybrid SynNotch CAR In Vivo Efficacy.
  • NOD.Cg-PrkdcscidI12rgtmlWjl/SzJ (NSG) mice were dosed subcutaneously with 4x106 M28 tumor cells as described in FIG. 14.
  • Bulk CD3+ T-cells were co-transduced with anti-ALPPL2 CAR or the anti-ALPPL2 Hybrid SynNotch CAR with ICDs as indicated in figure and the BFP reporter as described in FIG. 2.
  • 3 xl06 transduced CD3+ T cells were dosed to animals via retro- orbital injection 7 days post tumor injection. Tumor volume was assessed via caliper measurements weekly.
  • EXAMPLE 9 MINIMIZED 4-1BB OR CD28 VARIANTS IMPROVE NF-KB SIGNALING AND REDUCE NOISE
  • This example shows the improved NF-kB Signaling and reduced noise conferred by a 4- IBB variant and a CD28 variant.
  • FIG. 8A shows the alignment of wildtype 4-1BB and 4-1BB variants, depicting the amino acids deleted to create the “no STS” and “trunc” 4- IBB costimulatory domains.
  • FIG. 9A shows the alignment of wildtype 4-1BB and 4-1BB variants, depicting the amino acids deleted to create the “trunc 41BB”, “min41BB”, and “trunc41BBtrunc41BB” costimulatory domains.
  • 11 A and 12A show the alignment of wildtype CD28 and CD28 variants, depicting the amino acids deleted to create the “no STS”, “trunc”, “CD28ATPRRP”, “truncCD28ATPRRP”, and “fullytruncCD28 ” CD28 costimulatory domains.
  • FIGs. 8B, 9B, 1 IB, and 12B demonstrate that the Hybrid SynNotch CARs with 4-1BB variants or CD28 variants are expressed on the surface of the T cell after induction at similar rates as the Hybrid SynNotch CAR with wild type 4- IBB.
  • FIGs. 8B, 9B, 1 IB, and 12B demonstrate that the Hybrid SynNotch CARs with 4-1BB variants or CD28 variants are expressed on the surface of the T cell after induction at similar rates as the Hybrid SynNotch CAR with wild type 4- IBB.
  • a Jurkat cell line was transduced to express an mCherry reporter under a common promoter for NF-KB. This NF-KB reporter cell line was then transduced with anti-CD19 hybrid SynNotch CARs, and co-cultured with K562 cells expressing CD 19. mCherry expression was assessed as a proxy for NF-KB activity at 24, 48 and 72 hours post co-culture via flow cytometry. FIGs.
  • FIG. 13 A shows the alignment of wildtype 4- IBB and CD28 and variants, depicting the amino acids deleted and/or added to create the “trunc41BB PYAP”, “trunc41BB_YMFM”, “trunc41BB YMFMTPRRP”, and “trunc41BB AAYRS” costimulatory domains.
  • FIG. 13B demonstrates that the Hybrid SynNotch CARs with one of the CD28 signaling motifs appended to the C terminus of a trunc41BB costimulatory domain are expressed on the surface of the T cell after induction at similar levels as the Hybrid SynNotch CAR with wild type 4-1BB.
  • T cells were co-transduced to express the anti-BCMA or anti-ALPPL2 hybrid SynNotch CAR with 4 IBB costimulatory domain variants and the BFP reporter as described above. Circuit induction was assessed as described above.
  • FIG. 15A demonstrates that the anti- BCMA and anti-ALPPLS Hybrid SynNotch CARs are expressed on the surface of the T cell after induction.
  • FIG. 15B demonstrates that the anti-BCMA and anti-ALPPLS Hybrid SynNotch CARs with “trunc41BB” “trunc41BB PYAP” have less antigen independent induction of the transcriptional circuit, while maintaining the antigen-dependent induction of the inducible transcriptional circuit. This data set indicates that Hybrid SynNotch CAR scaffolds can be fused to other antigen targeting scFvs and maintain antigen-dependent transcriptional regulation of the circuit and T cell activation.

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