# US 6,589,730 B1 · Methods for identifying protein-protein interactions by selective transduction

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- Number: US 6,589,730 B1
- Document ID: US6589730B1
- Status: Granted
- Kind: B1
- Application number: 09/193,445
- Filed: 1998-11-17
- Granted: 2003-07-08
- Earliest claimed: 1997-08-29
- Assignees: Selective Genetics, Inc.
- Inventors: David Larocca; Andrew Baird; Paul Kassner
- Web page: https://patentgrep.com/patent/US6589730B1

## CPC

- C40B 40/02: Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
- A61K 47/6901: {Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors}
- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 1/6897: involving reporter genes operably linked to promoters
- C07K 2319/00: Fusion polypeptide
- C12N 2799/021: for the expression of a heterologous nucleic acid
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)
- 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)
- 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
- 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

A method for identifying specific protein-protein (i.e., ligand/anti-ligand) interactions that internalize and are detected by transgene expression. A ligand displaying genetic package that carries a reporter or selectable marker and presents a ligand or putative ligand on its surface is utilized to screen cells displaying known or putative anti-ligands for the ability to successfully internalize the ligand displaying genetic package.

## Claims

### Claim 1

A method of identifying an internalizing ligand or anti-ligand of an internalizing ligand/anti-ligand pair, comprising:
(a) contacting one or more ligand displaying genetic packages with a cell(s), wherein the ligand displaying genetic packages comprise a candidate internalizing ligand of an internalizing ligand/anti-ligand pair, wherein the ligand displaying genetic packages carry a gene encoding a detectable product which is expressed upon internalization of the ligand displaying genetic package, and wherein the cell(s) expresses an anti-ligand-receptor fusion protein on its surface;
(b) detecting the detectable product expressed by the cell(s); and
(c) recovering a nucleic acid molecule encoding the internalizing ligand and/or a nucleic acid molecule encoding the internalizing anti-ligand from the cell(s) expressing the detectable product, and
thereby identifying the internalizing ligand and/or anti-ligand of an internalizing ligand/anti-ligand pair.

### Claim 2 (depends on 1)

The method of claim 1, further comprising isolating cells that express the detectable product.

### Claim 3 (depends on 2)

The method of claim 2, wherein the cells are isolated by flow cytometry.

### Claim 4 (depends on 1)

The method of claim 1, wherein the ligand displaying package comprise a library of ligand displaying packages.

### Claim 5 (depends on 1)

The method of claim 1, wherein the cells express a library of anti-ligand-receptor fusions.

### Claim 6 (depends on 4, 5)

The method of claim 4 or 5, wherein the library is a cDNA library.

### Claim 7 (depends on 4, 5)

The method of claim 4 or 5, wherein the library is an antibody gene library.

### Claim 8 (depends on 4, 5)

The method of claim 4 or 5, wherein the library is a random peptide gene library.

### Claim 9 (depends on 4, 5)

The method of claim 4 or 5, wherein the library is a mutein library.

### Claim 10 (depends on 1)

The method of claim 1, wherein the detectable product is selected from the group consisting of green fluorescent protein, β-galactosidase, secreted alkaline phosphatase, chloramphenicol acetyltransferase, luciferase, human growth hormone and neomycin phosphotransferase.

### Claim 11 (depends on 1)

The method of claim 1, wherein the ligand displaying genetic packages comprise bacteriophage.

### Claim 12 (depends on 11)

The method of claim 11, wherein the bacteriophage are filamentous phage.

### Claim 13 (depends on 11, 12)

The method of claim 11 or 12, wherein the bacteriophage carries a genome vector.

### Claim 14

A method of identifying an internalizing ligand and/or anti-ligand of an internalizing ligand/anti-ligand pair, comprising:
(a) contacting one or more ligand displaying genetic packages with a cell(s), wherein the ligand displaying genetic packages comprise a candidate internalizing ligand of an internalizing ligand/anti-ligand pair, wherein the ligand displaying genetic packages carry a gene encoding a detectable product which is expressed upon internalization of the ligand displaying genetic package, and wherein the cell(s) expresses an anti-ligand-receptor fusion protein on its surface;
(b) incubating the cell(s) under selective conditions; and
(c) recovering a nucleic acid molecule encoding the internalizing ligand and/or a nucleic acid molecule encoding the internalizing anti-ligand from cell(s) which grow under the selective conditions, and
thereby identifying the internalizing ligand and/or anti-ligand of an internalizing ligand/anti-ligand pair.

### Claim 15 (depends on 14)

The method of claim 14, further comprising isolating cells that express the detectable product.

### Claim 16 (depends on 15)

The method of claim 15, wherein the cells are isolated by flow cytometry.

### Claim 17 (depends on 14)

The method of claim 14, wherein the ligand displaying packages comprises a library of ligand displaying packages.

### Claim 18 (depends on 14)

The method of claim 14, wherein the cells express a library of anti-ligand-receptor fusions.

### Claim 19 (depends on 17, 18)

The method of claim 17 or 18, wherein the library is a cDNA library.

### Claim 20 (depends on 17, 18)

The method of claim 17 or 18, wherein the library is an antibody gene library.

### Claim 21 (depends on 17, 18)

The method of claim 17 or 18, wherein the library is a random peptide gene library.

### Claim 22 (depends on 17, 18)

The method of claim 17 or 18, wherein the library is a mutein library.

### Claim 23 (depends on 14)

The method of claim 14, wherein the ligand displaying package comprises a bacteriophage.

### Claim 24 (depends on 23)

The method of claim 23, wherein the bacteriophage are filamentous phage.

### Claim 25 (depends on 23, 24)

The method of claim 23 or 24, wherein the bacteriophage carries a genome vector.