# US 12,595,492 B2 · Rapamycin resistant cells

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- Number: US 12,595,492 B2
- Document ID: US12595492B2
- Status: Granted
- Kind: B2
- Application number: 18/631,675
- Filed: 2024-04-10
- Published: 2024-09-05
- Granted: 2026-04-07
- Earliest claimed: 2018-04-27
- Assignees: Seattle Children's Hospital
- Applicants: Seattle Children's Hospital
- Inventors: Andrew M. Scharenberg; David J. Rawlings; Karen Sommer; Samuel West; Yuchi Chiang Honaker; Ryo Takeuchi
- Web page: https://patentgrep.com/patent/US12595492B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/22: Immunosuppressive or immunotolerising
- A61K 40/416: {Antigens related to auto-immune diseases; Preparations to induce self-tolerance}
- A61K 40/418: {Antigens related to induction of tolerance to non-self}
- C07K 14/4705: {stimulating, promoting or activating activity}
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 14/715: for cytokines; for lymphokines; for interferons
- C07K 14/7155: {for interleukins [IL]}
- C12N 5/0636: {T lymphocytes}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/86: Viral vectors
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 2239/31: characterized by the route of administration
- C07K 2317/53: Hinge
- C07K 2319/00: Fusion polypeptide
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
- C12N 2740/16043: viral genome or elements thereof as genetic vector
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- 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)

## Abstract

The present application relates to, inter alia, compositions including proteins for expression in host cells to render them resistant to rapamycin. The application further relates to methods of using the proteins, cells, and compositions disclosed therein for modulating cell signaling and for selective expansion of cells.

## Claims

### Claim 1

A cell prepared by introducing into a cell:
(i) a first polynucleotide encoding a soluble FK506-binding protein (FKBP)-rapamycin-binding (FRB) domain polypeptide;
(ii) a second polynucleotide encoding a first chemically induced signaling complex (CISC) component, wherein the first CISC component comprises a first extracellular binding domain comprising an FKBP domain, a first transmembrane domain, and a first cytoplasmic signaling domain; and
(iii) a third polynucleotide encoding a second CISC component, wherein the second CISC component comprises a second extracellular binding domain comprising an FRB domain, a second transmembrane domain, and a second cytoplasmic signaling domain; and
wherein the first CISC component and the second CISC component dimerize in the presence of a ligand to create a signaling-competent CISC.

### Claim 2 (depends on 1)

The cell of claim 1, further comprising a DNA endonuclease or a fourth polynucleotide encoding the DNA endonuclease.

### Claim 3 (depends on 2)

The cell of claim 2, further comprising a guide RNA (gRNA) or a fifth polynucleotide encoding the gRNA, wherein the gRNA comprises a spacer sequence that is complementary to a target sequence in a target genomic locus.

### Claim 4

A cell comprising:
(i) a first polynucleotide encoding a soluble FK506-binding protein (FKBP)-rapamycin-binding (FRB) domain polypeptide; or the soluble FRB domain polypeptide;
(ii) a second polynucleotide encoding a first chemically induced signaling complex (CISC) component, wherein the first CISC component comprises a first extracellular binding domain comprising an FKBP domain, a first transmembrane domain, and a first cytoplasmic signaling domain; or the first CISC component; and
(iii) a third polynucleotide encoding a second CISC component, wherein the second CISC component comprises a second extracellular binding domain comprising an FRB domain, a second transmembrane domain, and a second cytoplasmic signaling domain; or the second CISC component; and
wherein the first CISC component and the second CISC component dimerize in the presence of a ligand to create a signaling-competent CISC.

### Claim 5 (depends on 4)

The cell of claim 4, wherein the soluble FRB domain polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1.

### Claim 6 (depends on 5)

The cell of claim 5, wherein the soluble FRB domain polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 2.

### Claim 7 (depends on 4)

The cell of claim 4, wherein the ligand is rapamycin or a rapalog.

### Claim 8 (depends on 7)

The cell of claim 7, wherein the rapalog is selected from the group consisting of everolimus, CCI-779, C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-iRap, AP21967, sodium mycophenolic acid, benidipine hydrochloride, AP1903, AP23573, a metabolite of rapamycin, and an IMID-class drug.

### Claim 9 (depends on 4)

The cell of claim 4, wherein:
(a) the first cytoplasmic signaling domain comprises an IL-2 receptor subunit gamma (IL-2Rγ) cytoplasmic domain and the second cytoplasmic signaling domain comprises an IL-2 receptor subunit beta (IL-2Rβ) cytoplasmic domain; or
(b) the second cytoplasmic signaling domain comprises the IL-2Rγ cytoplasmic domain and the first cytoplasmic signaling domain comprises the IL-2Rβ cytoplasmic domain.

### Claim 10 (depends on 9)

The cell of claim 9, wherein the IL-2Rβ cytoplasmic domain comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 5.

### Claim 11 (depends on 9)

The cell of claim 9, wherein the second cytoplasmic signaling domain comprises the IL-2Rβ cytoplasmic domain, wherein the second CISC component comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 7.

### Claim 12 (depends on 4)

The cell of claim 4, wherein the FKBP domain of the first extracellular binding domain comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 6.

### Claim 13 (depends on 4)

The cell of claim 4, wherein the FRB domain of the second extracellular binding domain comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 1.

### Claim 14 (depends on 4)

The cell of claim 4, wherein the first CISC component comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 12.

### Claim 15 (depends on 4)

The cell of claim 4, wherein the second CISC component comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 13.

### Claim 16 (depends on 4)

The cell of claim 4, wherein
(i) the first CISC component comprises, in N-to-C-terminal order:
(a) the FKBP domain;
(b) an IL-2 receptor subunit gamma (IL-2Rγ) transmembrane domain; and
(c) an IL-2Rγ cytoplasmic domain; and
(ii) the second CISC component comprises, in N-to-C-terminal order:
(a) the FRB domain;
(b) an IL-2 receptor subunit beta (IL-2Rβ) transmembrane domain; and
(c) an IL-2Rβ cytoplasmic domain.

### Claim 17 (depends on 16)

The cell of claim 16, wherein the first CISC component comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 12, and the second CISC component comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 13.

### Claim 18 (depends on 4)

The cell of claim 4, wherein the cell is a T cell, B cell or a natural killer (NK) cell.

### Claim 19 (depends on 4)

The cell of claim 4, wherein the cell is a hematopoietic stem cell.

### Claim 20 (depends on 4)

The cell of claim 4, wherein the cell is a precursor T cell or a T regulatory (Treg) cell.

### Claim 21 (depends on 20)

The cell of claim 20, wherein 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.

### Claim 22 (depends on 20)

The cell of claim 20, wherein 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.

### Claim 23

A method for preparing a cell, comprising introducing into a cell:
(i) a first polynucleotide encoding a soluble FK506-binding protein (FKBP)-rapamycin-binding (FRB) domain polypeptide or the soluble FRB domain polypeptide;
(ii) a second polynucleotide encoding a first chemically induced signaling complex (CISC) component, wherein the first CISC component comprises a first extracellular binding domain comprising an FKBP domain, a first transmembrane domain, and a first cytoplasmic signaling domain; and
(iii) a third polynucleotide encoding a second CISC component, wherein the second CISC component comprises a second extracellular binding domain comprising an FRB domain, a second transmembrane domain, and a second cytoplasmic signaling domain; and
wherein the first CISC component and the second CISC component dimerize in the presence of a ligand to create a signaling-competent CISC.

### Claim 24 (depends on 23)

The method of claim 23, wherein the soluble FRB domain polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1.

### Claim 25 (depends on 23)

The method of claim 23, further comprising contacting the cell with the ligand.

### Claim 26 (depends on 25)

The method of claim 25, wherein the ligand is rapamycin or a rapalog.

### Claim 27 (depends on 26)

The method of claim 26, wherein the rapalog is selected from the group consisting of everolimus, CCI-779, C20-methallylrapamycin, C16-(S)-3-methylindolerapamycin, C16-iRap, AP21967, sodium mycophenolic acid, benidipine hydrochloride, AP1903, AP23573, a metabolite of rapamycin, and an IMID-class drug.

### Claim 28 (depends on 23)

The method of claim 23, wherein:
(a) the first cytoplasmic signaling domain comprises an IL-2 receptor subunit gamma (IL-2Rγ) cytoplasmic domain and the second cytoplasmic signaling domain comprises an IL-2 receptor subunit beta (IL-2Rβ) cytoplasmic domain; or (b) the second cytoplasmic signaling domain comprises the IL-2Rγ cytoplasmic domain and the first cytoplasmic signaling domain comprises the IL-2Rβ cytoplasmic domain.

### Claim 29 (depends on 28)

The method of claim 28, wherein the IL-2Rβ cytoplasmic domain comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 5.

### Claim 30 (depends on 28)

The method of claim 28, wherein the second cytoplasmic signaling domain comprises the IL-2Rβ cytoplasmic domain, wherein the second CISC component comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 7.

### Claim 31 (depends on 23)

The method of claim 23, wherein: (i) the FKBP domain of the first extracellular binding domain comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 6; or (ii) the FRB domain of the second extracellular binding domain comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 1.

### Claim 32 (depends on 23)

The method of claim 23, wherein: (i) the first CISC component comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 12; or (ii) the second CISC component comprises an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 13.