# US 11,897,932 B2 · Receptors for ligand-dependent transcriptional regulation

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- Number: US 11,897,932 B2
- Document ID: US11897932B2
- Status: Granted
- Kind: B2
- Application number: 17/217,635
- Filed: 2021-03-30
- Published: 2021-08-12
- Granted: 2024-02-13
- 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/US11897932B2

## CPC

- C07K 14/4705: {stimulating, promoting or activating activity}
- 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 40/4202: {Receptors, cell surface antigens or cell surface determinants}
- C07K 14/70517: {CD8}
- C07K 16/18: against material from animals or humans
- C07K 16/2803: {against the immunoglobulin superfamily}
- C12N 5/0636: {T lymphocytes}
- C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/09: containing a nuclear localisation signal
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K 2319/41: containing a Myc-tag
- C07K 2319/50: containing protease site
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure generally relates to, among other things, 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. 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.

## Claims

### Claim 1

A chimeric polypeptide comprising, from N-terminus to C-terminus:
a) an extracellular ligand-binding domain comprising a single chain variable fragment (scFv) having a binding affinity for a selected ligand;
b) a linking polypeptide comprising the sequence of SEQ ID NO: 19;
c) a transmembrane domain from a human Notch receptor 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 the ligand-inducible proteolytic cleavage site disposed between the transcriptional regulator and the linking polypeptide,
and wherein the chimeric polypeptide does not comprise a LIN-12-Notch repeat (LNR) nor 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 scFv is 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 1)

The chimeric polypeptide of claim 1, wherein the ligand is selected from the group consisting of CD19, CD59, CD66, CD73, CD80 (B7.1), CD86 (B7.2), CD94, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD246, CD273 (PD-L2), CD274 (PD-L1), CD295, CD340 (HER2), FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placental-like 2 (ALPPL2), 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 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, 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-11Ra, KIT (CD117), MUC1, NCAM, PAP, PDGFR-beta, PRSS21, PSCA, PSMA, ROR1, SSEA-4, TAG72, TEM1/CD248, TEM7R, TSHR, VEGFR2, BCMA (CD269), ALPPL2, ALPI, citrullinated vimentin, cMet, and Axl.

### Claim 10 (depends on 1)

The chimeric polypeptide of claim 1, wherein the ligand-inducible proteolytic cleavage site is a gamma-secretase cleavage site.

### Claim 11 (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 Ga14-VP16, Ga14-VP64, tetR-VP64, ZFHD1-VP64, Ga14-KRAB, and HAP1-VP16.

### Claim 12 (depends on 1)

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

### Claim 13 (depends on 1)

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

### Claim 14 (depends on 1)

The chimeric polypeptide of claim 1, wherein the transmembrane domain is from human Notch 1 receptor.

### Claim 15 (depends on 1)

The chimeric polypeptide of claim 1, wherein the transmembrane domain is from human Notch 2 receptor.

### Claim 16 (depends on 1)

The chimeric polypeptide of claim 1, wherein the transmembrane domain is from human Notch 3 receptor.

### Claim 17 (depends on 1)

The chimeric polypeptide of claim 1, wherein the transmembrane domain is from human Notch 4 receptor.