# US 9,493,523 B2 · RNF8-FHA domain-modified protein and method of producing the same

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- Number: US 9,493,523 B2
- Document ID: US9493523B2
- Status: Granted
- Kind: B2
- Application number: 13/810,025
- Filed: 2011-05-06
- Published: 2013-06-06
- Granted: 2016-11-15
- Earliest claimed: 2010-07-14
- Assignees: GeneFrontier Corporation
- Applicants: Kanehisa Kojoh; Shizue Katoh; Akira Miyakoshi; Mikiko Nakamura
- Inventors: Kanehisa Kojoh; Shizue Katoh; Akira Miyakoshi; Mikiko Nakamura
- Web page: https://patentgrep.com/patent/US9493523B2

## CPC

- C07K 14/47: from mammals
- C12N 9/93: {Ligases (6)}
- C12Y 603/02019: Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2318/20: Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics
- 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)
- C12Y: ENZYMES

## Abstract

Provided is an antigen-binding protein prepared merely by a method of in vitro selection using the RNF8-FHA domain, which has no intramolecular disulfide bond and functions in cells as it is. One to four loops extending from the FHA domain are randomized, and a recognition site for a target molecule is artificially created on the FHA domain surface to construct an RNF8-FHA domain library. Using the library, an antigen-binding protein is efficiently selected in vitro.

## Claims

### Claim 1

A method of producing an artificial antibody against a predetermined antigen, comprising:
(i) preparing a RNF8-FHA domain library comprising randomized-loop polypeptides obtained by randomizing two or more of the following loops (a′) to (d′) in the E3 ubiquitin ligase RNF8-FHA domain comprising the amino acid sequence of SEQ ID NO:1:
(a′) a loop consisting of the 41st-46th amino acid residues of the RNF8 protein shown by SEQ ID NO:1,
(b′) a loop consisting of the 49th-62nd amino acid residues of the RNF8 protein shown by SEQ ID NO:1,
(c′) a loop consisting of the 78th-83rd amino acid residues of the RNF8 protein shown by SEQ ID NO:1,
(d′) a loop consisting of the 108th-118th amino acid residues of the RNF8 protein shown by SEQ ID NO:1,
wherein each of the randomized-loop polypeptides comprises the amino acid sequence shown by SEQ ID NO:1 except that a random sequence is introduced into two or more loops selected from the group consisting of (a′) to (d′), and no amino acid residues are deleted from, substituted for, inserted into, or added to any regions of the amino acid sequence other than loops (a′), (b′), (c′), and (d′), and
(ii) selecting a randomized-loop polypeptide that binds to the antigen as the artificial antibody from among the randomized-loop polypeptides of the RNF8-FHA domain polypeptide library,
wherein unmodified RNF8-FHA domain has substantially no binding affinity for the antigen.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the number of amino acid residues in the random sequence is 3-12 residues.

### Claim 3 (depends on 2)

The method according to claim 2, wherein selecting a randomized-loop polypeptide that binds to the antigen from the randomized-loop polypeptides is performed by in vitro selection.

### Claim 4 (depends on 1)

The method according to claim 1, wherein selecting a randomized-loop polypeptide that binds to the antigen from the randomized-loop polypeptides is performed by in vitro selection.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the random sequence is introduced into the two or more loops in each of the randomized-loop polypeptides by replacing a region in each of the loops with the random sequence, wherein the region and the random sequence have the same number of amino acid residues.

### Claim 6 (depends on 1)

The method according to claim 1, wherein the random sequence is introduced into the two or more loops in each of the randomized-loop polypeptides by replacing each of the loops with the random sequence.

### Claim 7 (depends on 2)

The method according to claim 2, wherein the random sequence is introduced into the two or more loops in each of the randomized-loop polypeptides by replacing a region in each of the loops with the random sequence, wherein the region and the random sequence have the same number of amino acid residues.

### Claim 8 (depends on 2)

The method according to claim 2, wherein the random sequence is introduced into the two or more loops in each of the randomized-loop polypeptides by replacing each of the loops with the random sequence.