# US 10,308,944 B2 · Polypeptide expression systems

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- Number: US 10,308,944 B2
- Document ID: US10308944B2
- Status: Granted
- Kind: B2
- Application number: 14/790,818
- Filed: 2015-07-02
- Published: 2016-01-07
- Granted: 2019-06-04
- Earliest claimed: 2014-07-03
- Assignees: Genentech, Inc.
- Applicants: Genentech, Inc.
- Inventors: Isidro Hotzel; Yonglei Shang
- Web page: https://patentgrep.com/patent/US10308944B2

## CPC

- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C07K 16/005: {constructed by phage libraries}
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C07K 2317/10: characterized by their source of isolation or production
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/55: Fab or Fab'
- C07K 2317/60: characterized by non-natural combinations of immunoglobulin fragments
- C07K 2317/62: comprising only variable region components
- C07K 2317/64: comprising a combination of variable region and constant region components
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The present invention relates to polypeptide expression systems and methods of using the same.

## Claims

### Claim 1

A polypeptide expression system comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein:
(a) the first nucleic acid molecule comprises a first expression cassette comprising the following components: (i) a first eukaryotic promoter (P1Euk1), (ii) a prokaryotic promoter (P1Prok1), (iii) a first polypeptide-encoding sequence (PES11), (iv) a first 5′ splice site (5′ss11), (v) a hybridizing sequence (HS1), wherein the components are operably linked to each other in a 5′-to-3′ direction as P1Euk1-P1Prok1-PES11-5′ss11-HS1, wherein the HS1 is a gene encoding all or a portion of a pill coat protein of bacteriophage M13, and wherein the HS1 comprises a mutated polyadenylation site; and
(b) the second nucleic acid molecule comprises the following components: (i) a eukaryotic promoter (P2Euk), (ii) a hybridizing sequence capable of hybridizing to HS1 (HS2), (iii) a 3′ splice site (3′ss2), (iv) a second polypeptide-encoding sequence (PES2), and (v) a polyadenylation site (pA2), wherein the components are operably linked to each other in a 5′-to-3′ direction as P2Euk-HS2-3′ss2-PES2-pA2.

### Claim 2 (depends on 1)

The polypeptide expression system of claim 1, wherein the P1Euk1 is a cytomegalovirus (CMV) promoter or a simian virus 40 (SV40) promoter.

### Claim 3 (depends on 1)

The polypeptide expression system of claim 1, wherein the P2Euk is a CMV promoter or an SV40 promoter.

### Claim 4 (depends on 1)

The polypeptide expression system of claim 1, wherein the first expression cassette further comprises a first nucleic acid sequence encoding a eukaryotic signal sequence (ESS11), wherein the ESS11 is positioned between the P1Euk1 and the PES11.

### Claim 5 (depends on 4)

The polypeptide expression system of claim 4, wherein the ESS11 is a variable heavy chain (VH) signal sequence.

### Claim 6 (depends on 1)

The polypeptide expression system of claim 1, wherein the first expression cassette further comprises an excisable prokaryotic promoter module (ePPM1) comprising the following components: (i) a 5′ splice site (5′ss12), (ii) the prokaryotic promoter (P1Prok1, and (iii) a 3′ splice site (3′ss11), wherein the components are operably linked to each other in a 5′-to-3′ direction as 5′ss12-P1Prok1-3′ss11, and wherein the ePPM1 is positioned between the P1Euk1 and the PES11.

### Claim 7 (depends on 6)

The polypeptide expression system of claim 6, wherein the P1Prok1 is a selected from the group consisting of a PhoA promoter, a Tac promoter, a Lac, and a Tphac promoter.

### Claim 8 (depends on 6)

The polypeptide expression system of claim 6, wherein the ePPM1, further comprises a first nucleic acid sequence encoding a prokaryotic signal sequence (PSS11).

### Claim 9 (depends on 8)

The polypeptide expression system of claim 8, wherein the PSS11 is a heat-stable enterotoxin II (stII) signal sequence.

### Claim 10 (depends on 8)

The polypeptide expression system of claim 8, further comprising a polypyrimidine tract positioned between the PSS11 and the 3′ss11 (PPT11).

### Claim 11 (depends on 1)

The polypeptide expression system of claim 1, wherein the PES11 does not comprise a cryptic 5′ splice site.

### Claim 12 (depends on 1)

The polypeptide expression system of claim 1, wherein the first nucleic acid molecule further comprises a second expression cassette comprising (i) a second eukaryotic promoter (P1Euk2), (ii) a second polypeptide-encoding sequence (PES12), and (iii) a polyadenylation site (pA1), wherein the components are operably linked to each other in a 5′-to-3′ direction as P1Euk2-PES12-pA1.

### Claim 13 (depends on 12)

The polypeptide expression system of claim 12, wherein the P1Euk2 is a CMV promoter or an SV40 promoter.

### Claim 14 (depends on 12)

The polypeptide expression system of claim 12, wherein the second expression cassette further comprises a nucleic acid sequence encoding a eukaryotic signal sequence (ESS12).

### Claim 15 (depends on 14)

The polypeptide expression system of claim 14, wherein the ESS12 is the murine binding immunoglobulin protein (mBiP) signal sequence.

### Claim 16 (depends on 12)

The polypeptide expression system of claim 12, wherein the second expression cassette further comprises an excisable prokaryotic promoter module (ePPM2) comprising the following components: (i) a 5′ splice site (5′ss13), (ii) a prokaryotic promoter (P1Prok2), and (iii) a 3′ splice site (3′ss12), wherein the components are operably linked to each other in a 5′-to-3′ direction as 5′ss13-P1Prok2-3′ss12, and wherein the ePPM2 is positioned between the P1Euk2 and the PES12.

### Claim 17 (depends on 16)

The polypeptide expression system of claim 16, wherein the P1Prok2 is a selected from the group consisting of a PhoA promoter, a Tac promoter, and a Lac promoter.

### Claim 18 (depends on 16)

The polypeptide expression system of claim 16, wherein the ePPM2 further comprises a nucleic acid sequence encoding a prokaryotic signal sequence (PSS12).

### Claim 19 (depends on 18)

The polypeptide expression system of claim 18, wherein the PSS12 is a heat-stable enterotoxin II (stII) signal sequence.

### Claim 20 (depends on 18)

The polypeptide expression system of claim 18, further comprising a polypyrimidine tract positioned between the PSS12 and the 3′ss12 (PPT12).

### Claim 21 (depends on 12)

The polypeptide expression system of claim 12, wherein the second expression cassette is positioned 5′ to the first expression cassette.

### Claim 22 (depends on 12)

The polypeptide expression system of claim 12, wherein the PES12 encodes all or a portion of an antibody.

### Claim 23 (depends on 1)

The polypeptide expression system of claim 1, further comprising an intronic splice enhancer (ISE) positioned between the 5′ss11 and the HS1 (ISE1).

### Claim 24 (depends on 23)

The polypeptide expression system of claim 23, wherein the ISE1 comprises a G-run comprising three or more consecutive guanine residues.

### Claim 25 (depends on 23)

The polypeptide expression system of claim 23, further comprising a polypyrimidine tract positioned between the HS2 and the 3′ss2 (PPT2).

### Claim 26 (depends on 25)

The polypeptide expression system of claim 25, further comprising an ISE positioned between the HS2 and the 3′ss2 (ISE2).

### Claim 27 (depends on 26)

The polypeptide expression system of claim 26, wherein the ISE2 comprises a G-run comprising three or more consecutive guanine residues.

### Claim 28 (depends on 1)

The polypeptide expression system of claim 1, wherein the PES11 encodes all or a portion of an antibody.

### Claim 29 (depends on 28)

The polypeptide expression system of claim 28, wherein the PES11 encodes a polypeptide comprising a VH domain.

### Claim 30 (depends on 29)

The polypeptide expression system of claim 29, wherein the polypeptide comprising the VH domain further comprises a CH1 domain.

### Claim 31 (depends on 28)

The polypeptide expression system of claim 28, wherein the PES2 encodes all or a portion of an antibody.

### Claim 32 (depends on 1)

The polypeptide expression system of claim 1, wherein the mutated polyadenylation site comprises one or more silent mutations.

### Claim 33 (depends on 1)

A vector set comprising a first vector and a second vector, wherein the first and second vectors comprise the first and second nucleic acid molecules, respectively, of the polypeptide expression system of claim 1.

### Claim 34

A nucleic acid molecule comprising a set of splice sites consisting of a first 5′ splice site (5′ss11), a second 5′ splice site (5′ss12), and a 3′ splice site (3′ss11), the nucleic acid comprising a first expression cassette, the first expression cassette comprising the following components:
(a) a first eukaryotic promoter (P1Euk1);
(b) a first excisable prokaryotic promoter module (ePPM1) comprising the following components:
(i) the 5′ss12;
(ii) a prokaryotic promoter (P1Prok1); and
(iii) the 3′ss11,
wherein the components of the ePPM1 are operably linked to each other in a 5′-to-3′ direction as 5′ss12-P1Prok1-3′ss11;
(c) a first polypeptide-encoding sequence (PES11);
(d) the 5′ss11; and
(e) a utility peptide-encoding sequence (UPES), wherein the UPES encodes all or a portion of a pill coat protein of bacteriophage M13, and wherein the UPES comprises a mutated polyadenylation site,
wherein the components of the first expression cassette are operably linked to each other in a 5′-to-3′ direction as P1Euk1-ePPM1-PES11-5′ss11-UPES.

### Claim 35 (depends on 34)

The nucleic acid molecule of claim 34, wherein the first expression cassette further comprises a first nucleic acid sequence encoding a eukaryotic signal sequence (ESS11), wherein the ESS11 is positioned between the P1Euk1 and the ePPM1.

### Claim 36 (depends on 34)

The nucleic acid molecule of claim 34, wherein the ePPM1 further comprises a first nucleic acid sequence encoding a prokaryotic signal sequence (PSS11), wherein the PSS11 is positioned between the P1Prok1 and the 3′ss11.

### Claim 37 (depends on 34)

A vector comprising the nucleic acid molecule of claim 34.

### Claim 38 (depends on 33, 37)

A host cell comprising the vector of claim 37 or the vector set of claim 33.

### Claim 39 (depends on 38)

The host cell of claim 38, wherein the host cell is a prokaryotic cell.

### Claim 40 (depends on 38)

The host cell of claim 38, wherein the host cell is a eukaryotic cell.

### Claim 41 (depends on 37)

A method for producing a polypeptide comprising the amino acid sequence encoded by the PES11, the method comprising culturing a host cell that comprises the vector of claim 37 in a culture medium.

### Claim 42 (depends on 41)

The method of claim 41, wherein the method further comprises recovering the polypeptide from the host cell or the culture medium.

### Claim 43 (depends on 33)

A method for producing a polypeptide comprising the amino acid sequence encoded by the PES1 and the amino acid sequence encoded by the PES2, the method comprising culturing a host cell that comprises the vector set of claim 33 in a culture medium.

### Claim 44 (depends on 43)

The method of claim 43, wherein the method further comprises recovering the polypeptide from the host cell or the culture medium.

### Claim 45

A nucleic acid molecule comprising a set of splice sites consisting of a first 5′ splice site (5′ss11), a second 5′ splice site (5′ss12), a third 5′ splice site (5′ss13), a first 3′ splice site (3′ss11), and a second 3′ splice site (3′ss12), wherein the nucleic acid molecule comprises:
(a) a first expression cassette, the first expression cassette comprising the following components:
(i) a first eukaryotic promoter (P1Euk1);
(ii) a first excisable prokaryotic promoter module (ePPM1) comprising the 5′ss12, a prokaryotic promoter (P1Prok1), and the 3′ss11, wherein the components of the ePPM1 are operably linked to each other in a 5′-to-3′ direction as 5′ss12-P1Prok1-3′ss11;
(iii) a first polypeptide-encoding sequence (PES11);
(iv) the 5′ss11; and
(v) a utility peptide-encoding sequence (UPES), wherein the UPES encodes all or a portion of a pill coat protein of bacteriophage M13, and wherein the UPES comprises a mutated polyadenylation site, and
(b) a second expression cassette, the second expression cassette comprising the following components:
(i) a second eukaryotic promoter (P1Euk2);
(ii) a second excisable prokaryotic promoter module (ePPM2) comprising the 5′ss13, a prokaryotic promoter (P1Prok2), and the 3′ss12, wherein the components of the ePPM2 are operatively linked to each other in a 5′-to-3′ direction as 5′ss13-P1Prok2-3′ss12;
(iii) a second polypeptide-encoding sequence (PES12); and
(iv) a polyadenylation site (pA1),
wherein the components are operably linked to each other in a 5′-to-3′ direction as P1Euk2-ePPM2-PES12-pA1-P1Euk1-ePPM1-PES11-5′ss11-UPES.

### Claim 46 (depends on 45)

The nucleic acid molecule of claim 45, wherein the second expression cassette further comprises a second nucleic acid sequence encoding a eukaryotic signal sequence (ESS12), wherein the ESS12 is positioned between the P1Euk2 and the PES12.

### Claim 47 (depends on 45)

The nucleic acid molecule of claim 45, wherein the ePPM2 further comprises a nucleic acid sequence encoding a prokaryotic signal sequence (PSS12), wherein the PSS12 is positioned between the P1Prok2 and the 3′ss12.