# US 11,028,410 B2 · Hybrid promoter and uses thereof

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- Number: US 11,028,410 B2
- Document ID: US11028410B2
- Status: Granted
- Kind: B2
- Application number: 16/072,180
- Filed: 2017-01-26
- Published: 2019-02-21
- Granted: 2021-06-08
- Earliest claimed: 2016-01-27
- Assignees: Just-Evotec Biologics, Inc.
- Applicants: Just-Evotec Biologics, Inc.
- Inventors: Jeffrey T. McGrew; Pauline S. Smidt; E-Ching Ong
- Web page: https://patentgrep.com/patent/US11028410B2

## CPC

- C12N 15/86: Viral vectors
- C12N 9/2471: {Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase}
- C12N 15/85: for animal cells
- C12Y 302/01023: Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
- C12N 2710/16122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2710/16143: viral genome or elements thereof as genetic vector
- C12N 2830/003: tet inducible
- C12N 2830/15: chimeric enhancer/promoter combination
- C12N 2830/42: being an intron or intervening sequence for splicing and/or stability of RNA
- C12N 2840/105: enhancing translation
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

A hybrid promoter for recombinant expression of proteins of interest is disclosed that combines a mCMV enhancer sequence with a rat EF-1alpha intron sequence. Also disclosed are an expression cassette containing the hybrid promoter and a recombinant expression vector containing the expression cassette. A mammalian host cell, which comprises the recombinant expression vector is also disclosed, as is a method of producing a protein of interest that employs the mammalian host cell, optionally involving tetracycline-inducible expression of the protein.

## Claims

### Claim 1

A hybrid promoter, comprising:
(i) a murine cytomegalovirus (mCMV) enhancer sequence, comprising a mCMV enhancer element (mCMV-E) and a CMV promoter (CMV-P) sequence at its 3′ end, operably linked 5′ to a rat EF-1alpha intron sequence;
(ii) an intervening first leader sequence operably linked, 3′ to the CMV promoter sequence of the mCMV enhancer sequence, and 5′ to the rat EF-1alpha intron sequence; and
(iii) a second leader sequence operably linked 3′ to the rat EF-1alpha intron sequence.

### Claim 2 (depends on 1)

The hybrid promoter of claim 1, wherein the CMV promoter sequence at the 3′ end of the mCMV enhancer sequence comprises a segment having the nucleotide sequence of SEQ ID NO:24 or SEQ ID NO:26.

### Claim 3 (depends on 1)

The hybrid promoter of claim 1, wherein the rat EF-1alpha intron sequence comprises the nucleotide sequence of SEQ ID NO:4.

### Claim 4 (depends on 1)

The hybrid promoter of claim 1, wherein the mCMV enhancer sequence comprises the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:33; and the rat EF-1alpha intron sequence comprises the nucleotide sequence of SEQ ID NO:4.

### Claim 5 (depends on 4)

The hybrid promoter of claim 4, wherein the mCMV enhancer sequence comprises the nucleotide sequence of SEQ ID NO:2.

### Claim 6 (depends on 1)

The hybrid promoter of claim 1, wherein the first leader sequence comprises the nucleotide sequence of SEQ ID NO:3.

### Claim 7 (depends on 1)

The hybrid promoter of claim 1, wherein the second leader sequence comprises the nucleotide sequence of SEQ ID NO:5.

### Claim 8 (depends on 1)

The hybrid promoter of claim 1, further comprising one or more TetO sequences inserted within the CMV promoter sequence.

### Claim 9 (depends on 1)

The hybrid promoter of claim 1, comprising a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:30, SEQ ID NO:31, or SEQ ID NO:32.

### Claim 10 (depends on 1)

The hybrid promoter of claim 1, comprising the nucleotide sequence of SEQ ID NO:1.

### Claim 11 (depends on 1)

The hybrid promoter of claim 1, comprising the nucleotide sequence of SEQ ID NO:30.

### Claim 12 (depends on 1)

The hybrid promoter of claim 1, comprising the nucleotide sequence of SEQ ID NO:31.

### Claim 13 (depends on 1)

The hybrid promoter of claim 1, comprising the nucleotide sequence of SEQ ID NO:32.

### Claim 14 (depends on 1, 8)

An expression cassette, comprising:
(a) the hybrid promoter of claim 1 or claim 8, operably linked 5′ to an open reading frame encoding an exogenous protein of interest; and
(b) a polyadenylation site operably linked 3′ to the open reading frame.

### Claim 15 (depends on 14)

A recombinant expression vector comprising the expression cassette of claim 14.

### Claim 16 (depends on 15)

A mammalian host cell comprising the recombinant expression vector of claim 15.

### Claim 17 (depends on 16)

The mammalian host cell of claim 16, further being capable of expressing TetR.

### Claim 18 (depends on 16)

The mammalian host cell of claim 16, being derived from a Chinese Hamster Ovary (CHO) cell.

### Claim 19 (depends on 18)

The mammalian host cell of claim 18, selected from the group consisting of a CHO-K1 cell, a DXB11 cell, and a DG44 cell.

### Claim 20 (depends on 16)

A method of producing a protein of interest, comprising:
(a) culturing the mammalian host cell of claim 16, in an aqueous medium under physiological conditions permitting expression of the protein of interest; and
(b) recovering the protein of interest from the medium.

### Claim 21 (depends on 8)

A method of producing a protein of interest, comprising:
(a) culturing a mammalian host cell that comprises an expression vector comprising the hybrid promoter of claim 8, in an aqueous medium under physiological conditions, wherein the mammalian host cell is capable of expressing TetR, whereby, in the absence of tetracycline in the medium, the expression of the protein of interest is repressed;
(b) adding tetracycline to the aqueous medium in an amount sufficient to bind TetR in the host cell, whereby expression of the protein of interest by the host cell is derepressed; and
(c) recovering the protein of interest from the medium.

### Claim 22 (depends on 20)

The method of producing a protein of interest of claim 20, wherein the aqueous medium is serum-free.

### Claim 23 (depends on 20)

The method of producing a protein of interest of claim 20, wherein culturing the mammalian host cell is in a suspension in liquid aqueous medium.

### Claim 24 (depends on 20)

The method of producing a protein of interest of claim 20, wherein culturing the mammalian host cell is in an adhesion layer on a solid or semisolid substrate.

### Claim 25 (depends on 17)

The mammalian host cell of claim 17, being derived from a Chinese Hamster Ovary (CHO) cell.

### Claim 26 (depends on 25)

The mammalian host cell of claim 25, selected from the group consisting of a CHO-K1 cell, a DXB11 cell, and a DG44 cell.

### Claim 27 (depends on 21)

The method of producing a protein of interest of claim 21, wherein the aqueous medium is serum-free.

### Claim 28 (depends on 21)

The method of producing a protein of interest of claim 21, wherein culturing the mammalian host cell is in a suspension in liquid aqueous medium.

### Claim 29 (depends on 21)

The method of producing a protein of interest of claim 21, wherein culturing the mammalian host cell is in an adhesion layer on a solid or semisolid substrate.