# US 2021/0268024 A1 · Notch receptors with hinge domain

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- Number: US 2021/0268024 A1
- Document ID: US20210268024A1
- Status: Published application
- Kind: A1
- Application number: 17/217,618
- Filed: 2021-03-30
- Published: 2021-09-02
- Earliest claimed: 2019-09-24
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Kole T. ROYBAL; Raymond Liu; Iowis Zhu
- Web page: https://patentgrep.com/patent/US20210268024A1

## CPC

- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/42: Cancer antigens
- A61K 40/4211: {CD19 or B4}
- A61K 40/4215: {Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30}
- A61K 40/4254: {Adhesion molecules, e.g. NRCAM, EpCAM or cadherins}
- A61P 35/00: Antineoplastic agents
- C07K 14/4702: {Regulators; Modulating activity}
- C07K 14/70517: {CD8}
- C07K 14/70521: {CD28, CD152}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 16/18: against material from animals or humans
- C07K 16/2803: {against the immunoglobulin superfamily}
- C07K 16/2878: {against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95}
- C07K 16/30: from tumour cells
- C07K 16/32: against translation products of oncogenes
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 2039/505: {comprising antibodies}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- C07K 2317/53: Hinge
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/09: containing a nuclear localisation signal
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K 2319/50: containing protease site
- C07K 2319/70: containing domain for protein-protein interaction
- C07K 2319/71: containing domain for transcriptional activaation, e.g. VP16
- C07K 2319/715: containing a domain for ligand dependent transcriptional activation, e.g. containing a steroid receptor domain
- C07K 2319/74: containing a fusion for binding to a cell surface receptor
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present disclosure generally relates to, inter alia, a new class of receptors engineered to modulate transcriptional regulation in a ligand-dependent manner. Particularly, the new receptors, even though derived from Notch, do not require the Notch negative regulatory regions previously believed to be essential for the functioning of the receptors. In addition, the new receptors described herein incorporate an extracellular oligomerization domain to promote oligomer formation of the chimeric receptors. 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 such as cancers.

## Claims

### Claim 1

A chimeric polypeptide comprising, from N-terminus to C-terminus:
a) an extracellular ligand-binding domain having a binding affinity for a selected ligand;
b) a hinge domain capable of promoting oligomer formation of the chimeric polypeptide via intermolecular disulfide bonding;
c) a transmembrane domain comprising one or more ligand-inducible proteolytic cleavage sites; and
d) an intracellular domain comprising 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,
and wherein the chimeric polypeptide does not comprise a LIN-12-Notch repeat (LNR) and a heterodimerization domain (HD) of a Notch receptor.

### Claim 2 (depends on 1)

The chimeric polypeptide of claim 1, wherein the transmembrane domain further comprises a stop-transfer-sequence.

### Claim 3 (depends on 1)

The chimeric polypeptide of claim 1, wherein the extracellular domain comprises an antigen-binding moiety capable of binding to a ligand on the surface of a cell.

### Claim 4 (depends on 3)

The chimeric polypeptide of claim 3, wherein the cell is a human cell.

### Claim 5 (depends on 3)

The chimeric polypeptide of claim 3, wherein the cell is a tumor cell.

### Claim 6 (depends on 1)

The chimeric polypeptide of claim 1, wherein the ligand comprises a protein or a carbohydrate.

### Claim 7 (depends on 6)

The chimeric polypeptide of claim 6, wherein the ligand is selected from the group consisting of CD1, CD1a, CD1b, CD1c, CD1d, CD1e, 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, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD181 (CXCR1), CD182 (CXCR2), CD183 (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), enhanced green fluorescent protein (EGFP), and signal regulatory protein α (SIRPα).

### Claim 8 (depends on 1)

The chimeric polypeptide of claim 1, wherein the ligand is selected from cell surface receptors, adhesion proteins, integrins, mucins, lectins, tumor associated antigens, and tumor-specific antigens.

### Claim 9 (depends on 3)

The chimeric polypeptide of claim 3, wherein 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.

### Claim 10 (depends on 1)

The chimeric polypeptide of claim 1, wherein the ligand is a tumor-associated antigen selected from the group consisting of CD19, 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-11Rα, KIT (CD117), MUC1, NCAM, PAP, PDGFR-β, PRSS21, PSCA, PSMA, ROR1, SIRPα, SSEA-4, TAG72, TEM1/CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and Axl.

### Claim 11 (depends on 1)

The chimeric polypeptide of claim 1, wherein the one or more ligand-inducible proteolytic cleavage sites comprises a gamma secretase cleavage site.

### Claim 12 (depends on 1)

The chimeric polypeptide of claim 1, wherein the intracellular domain comprises a nuclear localization sequence and a transcriptional regulator sequence selected from the group consisting of Gal4-VP16, Gal4-VP64, tetR-VP64, ZFHD1-VP64, Gal4-KRAB, and HAP1-VP16.

### Claim 13 (depends on 1)

The chimeric polypeptide of claim 1, further comprising an additional proteolytic cleavage site, a signal sequence, a detectable label, a tumor-specific cleavage site, a disease-specific cleavage site, and combinations thereof.

### Claim 14 (depends on 1)

The chimeric polypeptide of claim 1, wherein the hinge domain is derived from a CD8α hinge domain, a CD28 hinge domain, a CD152 hinge domain, a PD-1 hinge domain, a CTLA4 hinge domain, an OX40 hinge domain, an IgG1 hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, and an IgG4 hinge domain, or a functional variant of any thereof.

### Claim 15 (depends on 1)

A recombinant nucleic acid comprising a nucleotide sequence encoding a chimeric polypeptide according to claim 1.

### Claim 16 (depends on 1)

A recombinant cell comprising a chimeric polypeptide according to claim 1.

### Claim 17 (depends on 16)

The recombinant cell of claim 16, wherein the chimeric polypeptide is expressed from a recombinant nucleic acid that has been transduced into the cell.

### Claim 18 (depends on 16)

The recombinant cell of claim 16, wherein the eukaryotic cell is a mammalian cell.

### Claim 19 (depends on 18)

The recombinant cell of claim 18, wherein the mammalian cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell.

### Claim 20 (depends on 19)

The recombinant cell of claim 19, wherein the recombinant cell is an immune cell selected from the group consisting of 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, and other T cells.

### Claim 21 (depends on 20)

The recombinant cell of claim 20, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

### Claim 22 (depends on 16)

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 according to claim 16, wherein the recombinant cell treats the health condition in the individual.

### Claim 23 (depends on 22)

The method of claim 22, further comprising contacting the recombinant cell with the 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.

### Claim 24 (depends on 23)

The method of claim 23, wherein the activity of the recombinant 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.

### Claim 25 (depends on 23)

The method of claim 23, wherein the released transcriptional regulator modulates expression of a gene product in the recombinant cell.

### Claim 26 (depends on 25)

The method of claim 25, wherein the gene product of the recombinant 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, an immuno-inhibitor, and an inhibiting immuno-receptor.

### Claim 27 (depends on 23)

The method of claim 23, wherein the released transcriptional regulator modulates differentiation of the cell, and wherein the cell is an immune cell, a stem cell, a progenitor cell, or a precursor cell.

### Claim 28 (depends on 22)

The method of claim 22, wherein the administered recombinant cell modulates an activity of a target cell in the individual.

### Claim 29 (depends on 28)

The method of claim 28, wherein the target cell is a cancer cell.

### Claim 30 (depends on 29)

The method of claim 29, wherein the cancer 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 cell, a sarcoma cell, a stomach cancer cell, a carcinoma cell, a mesothelioma cell, or a sarcoma cell.