# US 2014/0155285 A1 · Novel cell line screening method

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- Number: US 2014/0155285 A1
- Document ID: US20140155285A1
- Status: Published application
- Kind: A1
- Application number: 14/092,786
- Filed: 2013-11-27
- Published: 2014-06-05
- Earliest claimed: 2012-11-30
- Assignees: Larix Biosciences LLC
- Applicants: Larix Biosciences LLC
- Inventors: Bo YU; James LARRICK
- Web page: https://patentgrep.com/patent/US20140155285A1

## CPC

- G01N 33/56966: {Animal cells}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/2887: {against CD20}
- C12N 15/1086: {Preparation or screening of expression libraries, e.g. reporter assays}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12Q 1/6897: involving reporter genes operably linked to promoters
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The invention provides a novel cell line development method useful to screen for recombinant protein production. The method utilizes a membrane-anchored reporter or an intracellular reporter residing in the expression vector for a gene of interest to facilitate initial cell selection by FACS or MACS. A switching mechanism can be used to delete the reporter from the chromosome by providing an appropriate DNA recombinase, which turns the selected cells into production cells that secrete the protein of interest without co-expression of the reporter.

## Claims

### Claim 1

A DNA construct comprising (a) promoter; (b) a gene of interest (GOI); and (c) a reporter gene flanked by a DNA recombinase recognition sequence (DRRS).

### Claim 2 (depends on 1)

The DNA construct of claim 1, wherein the DRSS is selected from the group consisting of an FRT sequence (DRRS for flippase or FLP), an LoxP (DRRS for Cre), and an attB or attP (DRRS for φC31 integrase).

### Claim 3 (depends on 1)

The DNA construct of claim 1, further comprising an IRES upstream of the reporter gene.

### Claim 4 (depends on 1)

The DNA construct of claim 1, wherein the reporter gene encodes a membrane anchored reporter (MAR) molecule, wherein the MAR is any molecule that can be detected on the cell surface.

### Claim 5 (depends on 4)

The DNA construct of claim 4, wherein the MAR is selected from the group consisting of membrane-anchored GFP, and a membrane-anchored protein of interest.

### Claim 6 (depends on 1)

The DNA construct of claim 1, wherein the reporter gene encodes an intracellular reporter molecule.

### Claim 7 (depends on 1)

The DNA construct of claim 1, wherein the gene of interest has two or more exons and one or more introns.

### Claim 8 (depends on 7)

The DNA construct of claim 7, wherein an intron of the gene of the interest comprises a splice acceptor sequence followed by a membrane association sequence.

### Claim 9 (depends on 8)

The DNA construct of claim 8, wherein a splice acceptor sequence provides a means for alternative splicing wherein the expressed protein of interest can be membrane associated or secreted.

### Claim 10 (depends on 1)

The DNA construct of claim 1, wherein the gene of interest encodes a recombinant protein.

### Claim 11 (depends on 10)

The DNA construct of claim 10, wherein the gene of interest encodes an antibody.

### Claim 12 (depends on 11)

The DNA construct of claim 11, wherein the gene of the interest encoding said antibody comprises a splice acceptor sequence in the third intron of the antibody gamma constant region coding sequence.

### Claim 13 (depends on 1)

An expression vector for delivering a gene of interest into a host cell, comprising the DNA construct of claim 1.

### Claim 14 (depends on 1)

A cell comprising the DNA construct of claim 1.

### Claim 15 (depends on 14)

A cell culture comprising the cell of claim 14.

### Claim 16 (depends on 15)

The cell culture of claim 15, wherein a site specific DNA recombinase is added to the culture media.

### Claim 17 (depends on 16)

The cell culture of claim 16, wherein the site specific DNA recombinase is provided by transfection of an expression vector encoding the DNA recombinase into the cells of the cell culture.

### Claim 18 (depends on 16)

The cell culture of claim 16, wherein the sequence encoding said reporter is deleted in the cells of the cell culture.

### Claim 19 (depends on 18)

The cell culture of claim 18, wherein the gene of interest is expressed and the corresponding protein of interest (POI) is secreted into the cell culture media.

### Claim 20

A DNA construct comprising (a) promoter; (b) a library containing a plurality of genes of interest; and (c) a reporter gene flanked by a DNA recombinase recognition sequence (DRRS).

### Claim 21 (depends on 20)

The DNA construct of claim 20, wherein the genes of interest in the library comprise an intron with a splice acceptor sequence followed by a membrane association sequence.

### Claim 22 (depends on 21)

The DNA construct of claim 21, wherein the splice acceptor sequence provides a means for alternative splicing wherein the expressed genes of interest can encode membrane associated or secreted proteins of interest.

### Claim 23 (depends on 20)

The DNA construct of claim 20, wherein the library is an antibody library.

### Claim 24 (depends on 23)

The DNA construct of claim 23, wherein the antibody library is an affinity maturation library.

### Claim 25 (depends on 24)

A cell library comprising the DNA construct of claim 24.

### Claim 26 (depends on 25)

A cell culture comprising the cell library of claim 25.

### Claim 27 (depends on 1)

A cell line screening method comprising,
(a) providing host cells transfected with a DNA construct according to claim 1;
(b) culturing the transfected cells wherein the reporter protein is expressed and a protein of interest may also be secreted into the cell culture media;
(c) screening the transfected host cells and selecting host cells expressing a reporter molecule;
(d) exposing the selected cells to a DNA recombinase, wherein following such exposure the cells no longer express the reporter protein; and
(e) screening for cells that secrete a protein of interest into the cell culture media.

### Claim 28 (depends on 27)

The cell line screening method of claim 27, wherein the reporter molecule is a membrane anchored reporter (MAR).

### Claim 29 (depends on 27)

The cell line screening method of claim 27, wherein the reporter molecule is an intracellular reporter.

### Claim 30 (depends on 27)

The cell line screening method of claim 27, wherein the DNA construct comprises a DRSS selected from the group consisting of an FRT sequence (DRRS for flippase or FLP), a LoxP (DRRS for Cre), and an attB or attP (DRRS for φC31 integrase).

### Claim 31 (depends on 28)

The cell line screening method of claim 28, wherein the MAR is any protein that can be detected on the cell surface.

### Claim 32 (depends on 31)

The cell line screening method of claim 31, wherein the MAR is detected on the cell surface by FACS or MACS.

### Claim 33 (depends on 27)

The cell line screening method of claim 27, wherein the gene of interest encoded by the DNA construct has two or more exons and one or more introns.

### Claim 34 (depends on 33)

The cell line screening method of claim 33, wherein an intron of the gene of the interest comprises a splice acceptor sequence followed by a membrane association sequence.

### Claim 35 (depends on 27)

The cell line screening method of claim 27, wherein the transfected host cell is exposed to a site specific DNA recombinase.

### Claim 36 (depends on 27)

The cell line screening method of claim 27, wherein the protein of interest is an antibody.

### Claim 37 (depends on 20)

A cell line screening method comprising the steps of:
(a) providing host cells in culture media;
(b) transfecting the host cells with a DNA construct according to claim 20;
(c) screening the transfected host cells and selecting host cells expressing a reporter molecule;
(d) exposing the selected host cells to a DNA recombinase;
(e) culturing the host cells in cell culture media; and
(e) screening the cell culture media for a protein of interest with a desired property.

### Claim 38 (depends on 37)

The cell line screening method of claim 37, wherein the reporter molecule is a membrane anchored reporter (MAR).

### Claim 39 (depends on 37)

The cell line screening method of claim 37, wherein the reporter molecule is an intracellular reporter.

### Claim 40 (depends on 37)

The cell line screening method of claim 37, wherein the DNA construct comprises a DRSS selected from the group consisting of an FRT sequence (DRRS for flippase or FLP), an LoxP (DRRS for Cre), an attB or attP (DRRS for φC31 integrase).

### Claim 41 (depends on 38)

The cell line screening method of claim 38, wherein the MAR is any protein that can be detected on the cell surface.

### Claim 42 (depends on 41)

The cell line screening method of claim 41, wherein the MAR is detected on the cell surface by FACS or MACS.

### Claim 43 (depends on 37)

The cell line screening method of claim 37, wherein the gene of interest encoded by the DNA construct has two or more exons and one or more introns.

### Claim 44 (depends on 43)

The cell line screening method of claim 43, wherein an intron of the gene of the interest comprises a splice acceptor sequence followed by a membrane association sequence.

### Claim 45 (depends on 37)

The cell line screening method of claim 37, wherein the transfected host cell is exposed to a site specific DNA recombinase.

### Claim 46 (depends on 37)

The cell line screening method of claim 37, wherein the protein of interest is an antibody.

### Claim 47 (depends on 20)

A method of screening a cell library screening for a protein of interest comprising switching a cell having a membrane anchored reporter protein into a cell that secretes a protein of interest into culture media, comprising:
(a) providing host cells in culture media;
(b) transfecting the host cells with a DNA construct according to claim 20 to generate a cell library expressing a library of membrane-anchored protein of interest;
(c) screening the cell library and selecting host cells expressing a membrane-anchored protein of interest with a desired property;
(c) exposing the selected host cells to a DNA recombinase;
(d) screening the cell culture media for the protein of interest for the protein of interest with desired property; and

### Claim 48 (depends on 47)

The screening method of claim 47, wherein the library is an antibody library.

### Claim 49 (depends on 47)

The screening method of claim 47, wherein the desired property is binding of an antigen.

### Claim 50 (depends on 47)

The screening method of claim 47, wherein the desired property is bioactivity.