# US 10,370,440 B2 · Reconstituted polypeptides

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- Number: US 10,370,440 B2
- Document ID: US10370440B2
- Status: Granted
- Kind: B2
- Application number: 14/701,331
- Filed: 2015-04-30
- Published: 2016-01-28
- Granted: 2019-08-06
- Earliest claimed: 2002-06-06
- Assignees: Research Corporation Technologies, Inc.
- Applicants: Research CorporationTechnologies, Inc.
- Inventors: Shohei Koide
- Web page: https://patentgrep.com/patent/US10370440B2

## CPC

- C07K 16/18: against material from animals or humans
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 2317/565: Complementarity determining region [CDR]
- 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)

## Abstract

The present invention provides modified fibronectin type III (Fn3) molecules, and nucleic acid molecules encoding the modified Fn3 molecules. Also provided are methods of preparing these molecules, and kits to perform the methods.

## Claims

### Claim 1

A fibronectin type III (Fn3) monobody binding pair comprising:
(a) a first fibronectin type III (Fn3) monobody polypeptide comprising two to six β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin
(i) with a loop region linked between the β-strand domains, and
(ii) wherein a polypeptide tail region may be attached to one or both terminal β-strand domains, and
(b) a second Fn3 monobody polypeptide comprising two to six β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin
(i) with a loop region linked between the β-strand domains, and
(ii) wherein a polypeptide tail region may be attached to one or both terminal β-strand domains,
wherein the first Fn3 monobody polypeptide has a binding affinity for the second Fn3 monobody polypeptide with a dissociation constant of about 10−9 moles/liter.

### Claim 2 (depends on 1)

The binding pair of claim 1, wherein the loop region of the first Fn3 monobody polypeptide, the second Fn3 monobody polypeptide or both is capable of catalyzing a chemical reaction with a catalyzed rate constant (Kcat), as measured in the binding reaction of the corresponding uncut, full-length monobody, and an uncatalyzed rate constant (Kuncat) such that the ratio of Kcat/Kuncat is greater than 10.

### Claim 3 (depends on 1)

The binding pair of claim 1, wherein the loop region linked between the β-strand domains of the first Fn3 monobody polypeptide comprise amino acid residues:
i) from 15 to 16 inclusive in an AB loop;
ii) from 22 to 30 inclusive in a BC loop;
iii) from 39 to 45 inclusive in a CD loop;
vi) from 51 to 55 inclusive in a DE loop;
v) from 60 to 66 inclusive in an EF loop; or
vi) from 76 to 87 inclusive in an FG loop.

### Claim 4 (depends on 1)

The binding pair of claim 1, wherein a loop region varies from a corresponding wild-type loop region by deletion of one to all except one amino acid in the loop region, insertion of one to 25 amino acids, and/or replacement of at least one amino acid in the loop region.

### Claim 5 (depends on 4)

The binding pair of claim 4, wherein the loop region varies from a corresponding wild-type Fn3 loop region by deletion of one to all except one amino acid and/or replacement of at least one amino acid.

### Claim 6 (depends on 4)

The binding pair of claim 4, wherein the loop region varies from a corresponding wild-type Fn3 loop region by insertion of one to 25 amino acids.

### Claim 7 (depends on 1)

The binding pair of claim 1, wherein the first polypeptide comprises a first cysteine and the second polypeptide comprises a second cysteine, and wherein the first cysteine and the second cysteine form a disulfide bond.

### Claim 8 (depends on 7)

The binding pair of claim 7, wherein the first cysteine is located in a loop region.

### Claim 9 (depends on 7)

The binding pair of claim 7, herein the second cysteine is located in a loop region.

### Claim 10 (depends on 7)

The binding pair of claim 7, wherein the first cysteine is located in one of the β-strand domains.

### Claim 11 (depends on 7)

The binding pair of claim 7, wherein the second cysteine is located in one of the β-strand domains.

### Claim 12 (depends on 1)

The binding pair of claim 1, wherein the loop region linked between the β-strand domains of the second Fn3 monobody polypeptide comprise amino acid residues:
i) from 15 to 16 inclusive in an AB loop;
ii) from 22 to 30 inclusive in a BC loop;
iii) from 39 to 45 inclusive in a CD loop;
vi) from 51 to 55 inclusive in a DE loop;
v) from 60 to 66 inclusive in an EF loop; or
vi) from 76 to 87 inclusive in an FG loop.

### Claim 13 (depends on 1)

The binding pair of claim 1, wherein the loop region linked between the β-strand domains of each of the first and second Fn3 monobody polypeptides comprise amino acid residues:
i) from 15 to 16 inclusive in an AB loop;
ii) from 22 to 30 inclusive in a BC loop;
iii) from 39 to 45 inclusive in a CD loop;
vi) from 51 to 55 inclusive in a DE loop;
v) from 60 to 66 inclusive in an EF loop; or
vi) from 76 to 87 inclusive in an FG loop.

### Claim 14 (depends on 1)

The binding pair of claim 1, wherein the first Fn3 monobody polypeptide comprises two 3-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the β-strand domains.

### Claim 15 (depends on 14)

The binding pair of claim 14, wherein the second Fn3 monobody polypeptide comprises: a) two β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the β-strand domains; b) three β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains and the second and third β-strand domains; c) four β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second 3-strand domains, the second and third β-strand domains and the third and fourth β-strand domains; d) five β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains, the third and fourth β-strand domains and the fourth and fifth β-strand domains; or e) six β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains, the third and fourth β-strand domains, the fourth and fifth β-strand domains and the fifth and sixth β-strand domains.

### Claim 16 (depends on 1)

The binding pair of claim 1, wherein the first Fn3 monobody polypeptide comprises three β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains and the second and third β-strand domains.

### Claim 17 (depends on 16)

The binding pair of claim 16, wherein the second Fn3 monobody polypeptide comprises: a) two β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the β-strand domains; b) three β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains and the second and third β-strand domains; c) four β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second 3-strand domains, the second and third β-strand domains and the third and fourth β-strand domains; d) five β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains, the third and fourth β-strand domains and the fourth and fifth β-strand domains; or e) six β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains, the third and fourth β-strand domains, the fourth and fifth β-strand domains and the fifth and sixth β-strand domains.

### Claim 18 (depends on 1)

The binding pair of claim 1, wherein the first Fn3 monobody polypeptide comprises four β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains and the third and fourth β-strand domains.

### Claim 19 (depends on 18)

The binding pair of claim 18, wherein the second Fn3 monobody polypeptide comprises: a) two β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the β-strand domains; b) three β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains and the second and third β-strand domains; c) four β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains and the third and fourth β-strand domains; d) five β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains, the third and fourth β-strand domains and the fourth and fifth β-strand domains; or e) six β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains, the third and fourth β-strand domains, the fourth and fifth β-strand domains and the fifth and sixth β-strand domains.

### Claim 20 (depends on 1)

The binding pair of claim 1, wherein the first Fn3 monobody polypeptide comprises five β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains, the third and fourth β-strand domains and the fourth and fifth β-strand domains.

### Claim 21 (depends on 20)

The binding pair of claim 20, wherein the second Fn3 monobody polypeptide comprises: a) two β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the β-strand domains; b) three β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains and the second and third β-strand domains; c) four β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second 3-strand domains, the second and third β-strand domains and the third and fourth β-strand domains; d) five β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains, the third and fourth β-strand domains and the fourth and fifth β-strand domains; or e) six β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains, the third and fourth β-strand domains, the fourth and fifth β-strand domains and the fifth and sixth β-strand domains.

### Claim 22 (depends on 1)

The binding pair of claim 1, wherein the first Fn3 monobody polypeptide comprises six β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains, the third and fourth β-strand domains, the fourth and fifth β-strand domains and the fifth and sixth β-strand domains.

### Claim 23 (depends on 22)

The binding pair of claim 22, wherein the second Fn3 monobody polypeptide comprises: a) two β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the β-strand domains; b) three β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains and the second and third β-strand domains; c) four β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second 3-strand domains, the second and third β-strand domains and the third and fourth β-strand domains; d) five β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains, the third and fourth β-strand domains and the fourth and fifth β-strand domains; or e) six β-strand domains selected from an A, B, C, D, E, F and G β-strand domain of the 10th Fn3 domain of fibronectin with a loop region linked between the first and second β-strand domains, the second and third β-strand domains, the third and fourth β-strand domains, the fourth and fifth β-strand domains and the fifth and sixth β-strand domains.

### Claim 24 (depends on 1)

The binding pair of claim 1, wherein the first Fn3 monobody polypeptide further comprises a first auxiliary domain, and the second Fn3 monobody polypeptide comprises a second auxiliary domain.

### Claim 25 (depends on 24)

The binding pair of claim 24, wherein the first auxiliary region comprises a first cysteine and the second auxiliary region comprises a second cysteine, and wherein the first cysteine and the second cysteine form a disulfide bond.

### Claim 26 (depends on 24)

The binding pair of claim 24, wherein the auxiliary domains are a natural protein/peptide pair, a peptide-binding protein and its target peptide; or two fragments of a protein that have been artificially generated.

### Claim 27 (depends on 24)

The binding pair of claim 24, wherein the auxiliary domains are a pair of coiled coils or a C-intein and N-intein pair.