# US 6,720,410 B2 · Nucleic acids encoding enamel matrix related polypeptides

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- Number: US 6,720,410 B2
- Document ID: US6720410B2
- Status: Granted
- Kind: B2
- Application number: 09/950,739
- Filed: 2001-09-13
- Published: 2002-08-08
- Granted: 2004-04-13
- Earliest claimed: 1995-10-19
- Assignees: Biora AB
- Inventors: Radim Cerny; Ivan Slaby; Lars Hammarström; Tilmann Wurtz; Cheng Dan Fong
- Web page: https://patentgrep.com/patent/US6720410B2

## CPC

- C07K 14/47: from mammals
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- 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)
- 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)

## Abstract

The invention relates to novel nucleic acid fragments encoding polypeptides which are capable of mediating contact between enamel and cell surface. The invention also relates to expression vectors containing the nucleic acid fragments according to the invention for production of the protein, organisms containing said expression vector, methods for producing the polypeptide, compositions comprising the polypeptides, antibodies or antibody fragments recognizing the polypeptides, and methods for treating various hard tissue diseases or disorders.

## Claims

### Claim 1

An at least partially purified nucleic acid encoding a polypeptide which comprises an amino acid subsequence S having a length of 20 amino acids, said subsequence S comprising at least one sequence element selected from the group consisting of the tetrapeptides DGEA (Asp-Gly-Glu-Ala), VTKG (Val-Thr-Lys-Gly), EKGE (Glu-Lys-Gly-Glu) and DKGE (Asp-Lys-Gly-Glu),
which subsequence S has a percentage sequence identity of at least 80% with at least one 20 amino acid reference subsequence of a reference sequence selected from the group consisting of (a) the amino acid sequence shown in SEQ ID NO:2, and (b) the amino acid sequence shown in SEQ ID:4, the reference subsequence likewise comprising said sequence element,
where said polypeptide exhibits at least one of the following activities when administered in an effective amount to a subject:
(i) binds to enamel;
(ii) binds to ameloblasts;
(iii) mediates contact between the enamel and the surface of a cell;
(iv) competitively inhibits contact between an extracellular matrix protein and the surface of a cell;
(v) promotes mineralization of bone, enamel, dentin or cementum; or
(vi) promotes formation of the enamel matrix or of hard tissues of mesenchymal origin.

### Claim 2 (depends on 1)

The nucleic acid of claim 1 where said polypeptide comprises the amino acid sequence SEQ ID NO:2.

### Claim 3 (depends on 1)

The nucleic acid claim 1 where said polypeptide comprises the amino acid sequence SEQ ID NO:4.

### Claim 4 (depends on 1)

The nucleic acid of claim 1 where said polypeptide has activity (i).

### Claim 5 (depends on 1)

The nucleic acid of claim 1 where said polypeptide has activity (ii).

### Claim 6 (depends on 1)

The nucleic acid of claim 1 where said polypeptide has activity (iii).

### Claim 7 (depends on 1)

The nucleic acid of claim 1 where said polypeptide has activity (iv).

### Claim 8 (depends on 1)

The nucleic acid of claim 1 where said polypeptide has activity (v).

### Claim 9 (depends on 1)

The nucleic acid of claim 1 where said polypeptide has activity (vi).

### Claim 10 (depends on 1)

The nucleic acid of claim 1, said subsequence S comprising at least one sequence element selected from the group consisting of the tetrapeptides DGEA (Asp-Gly-Glu-Ala), VTKG (Val-Thr-Lys-Gly), EKGE (Glu-Lys-Gly-Glu) and DKGE (Asp-Lys-Gly-Glu).

### Claim 11 (depends on 8)

The nucleic acid of claim 8 where said polypeptide promotes mineralization of teeth.

### Claim 12 (depends on 1)

The nucleic acid of claim 1 where said polypeptide comprises the amino acid sequence 1-407 in SEQ ID NO:2 except for the changes of Gln33 to Arg, Ala36 to Gly, Ala175 to Gly, Ala 239 to Gly and Ala295 to Pro.

### Claim 13 (depends on 1)

The nucleic acid of claim 1 where said polypeptide comprises the amino acid sequence 1-324 in SEQ ID NO:4 except for the changes of Ala92 to Gly, Ala156 to Gly and Ala212 to Pro.

### Claim 14

An at least partially purified nucleic acid, at least 18 nucleotides long, encoding a polypeptide, said nucleic acid being (I) a nucleic acid whose coding strand hybridizes with the noncoding strand of (a) a nucleic acid having the coding strand nucleotide sequence shown in SEQ ID NO: 1, or (b) a nucleic acid having the coding strand nucleotide sequence shown in SEQ ID NO:3 under stringent conditions, said conditions being 5 mM monovalent ions (0.1×SSC), neutral pH and 65° C., or (II) a nucleic acid which encodes the same polypeptide as a nucleic acid of (I) above,
where said polypeptide exhibits at least one of the following activities when administered in an effective amount to a subject:
(i) binds to enamel;
(ii) binds to ameloblasts;
(iii) mediates contact between the enamel and the surface of a cell;
(iv) competitively inhibits contact between an extracellular matrix protein and the surface of a cell;
(v) promotes mineralization of bone, enamel, dentin or cementum; or
(vi) promotes formation of the enamel matrix or of hard tissues of mesenchymal origin.

### Claim 15 (depends on 14)

The nucleic acid of claim 14 where said polypeptide has activity (i).

### Claim 16 (depends on 14)

The nucleic acid of claim 14 where said polypeptide has activity (ii).

### Claim 17 (depends on 14)

The nucleic acid of claim 14 where said polypeptide has activity (iii).

### Claim 18 (depends on 14)

The nucleic acid of claim 14 where said polypeptide has activity (iv).

### Claim 19 (depends on 14)

The nucleic acid of claim 14 where said polypeptide has activity (v).

### Claim 20 (depends on 14)

The nucleic acid of claim 14 where said polypeptide has activity (vi).

### Claim 21 (depends on 14)

The nucleic acid of claim 14 where said polypeptide comprises at least one sequence element selected from the group consisting of the tetrapeptides DGEA (Asp-Gly-Glu-Ala), VTKG (Val-Thr-Lys-Gly), EKGE (Glu-Lys-Gly-Glu) and DKGE (Asp-Lys-Gly-Glu).

### Claim 22 (depends on 14)

The nucleic acid of claim 14 where said nucleic acid is at least 21 nucleotides long.

### Claim 23 (depends on 14)

The nucleic acid of claim 14 where said nucleic acid is at least 48 nucleotides long.

### Claim 24 (depends on 14)

The nucleic acid of claim 14 where said nucleic acid is at least 75 nucleotides long.

### Claim 25 (depends on 14)

The nucleic acid of claim 14 where said nucleic acid is at least 99 nucleotides long.

### Claim 26 (depends on 14)

The nucleic acid of claim 14 where the polypeptide is encoded by (I).

### Claim 27 (depends on 26)

The nucleic acid of claim 26 where (I) hybridizes with (a).

### Claim 28 (depends on 26)

The nucleic acid of claim 26 where (I) hybridizes with (b).

### Claim 29

An at least partially purified nucleic acid encoding a polypeptide which is at least six amino acids long, and which is bound by an antibody which also binds amelin-1, having the amino acid sequence of SEQ ID NO:2, or amelin-2, having the amino acid sequence of SEQ ID NO:4, said polypeptide mediating contact between enamel and cell surfaces.

### Claim 30

An at least partially purified nucleic acid encoding a polypeptide comprises at least one sequence element selected from the group consisting of the tetrapeptides DGEA (Asp-Gly-Glu-Ala), VTKG (Val-Thr-Lys-Gly), EKGE (Glu-Lys-Gly-Glu) and DKGE (Asp-Lys-Gly-Glu),
where said polypeptide has a percentage amino acid sequence identity of at least 80% with SEQ ID NO:2 or SEQ ID NO:4, and
where said polypeptide exhibits at least one of the following activities when administered in an effective amount to a subject:
(i) binds to enamel;
(ii) binds to ameloblasts;
(iii) mediates contact between the enamel and the surface of a cell;
(iv) competitively inhibits contact between an extracellular matrix protein and the surface of a cell;
(v) promotes mineralization of bone, enamel, dentin or cementum; or
(vi) promotes formation of the enamel matrix or of hard tissues of mesenchymal origin.

### Claim 31 (depends on 30)

The nucleic acid of claim 30 where said polypeptide has activity (i).

### Claim 32 (depends on 30)

The nucleic acid of claim 30 where said polypeptide has activity (ii).

### Claim 33 (depends on 30)

The nucleic acid of claim 30 where said polypeptide has activity (iii).

### Claim 34 (depends on 30)

The nucleic acid of claim 30 where said polypeptide has activity (iv).

### Claim 35 (depends on 30)

The nucleic acid of claim 30 where said polypeptide has activity (v).

### Claim 36 (depends on 30)

The nucleic acid of claim 30 where said polypeptide has activity (vi).

### Claim 37 (depends on 30)

The nucleic acid of claim 30 where said percentage identity is at least 85%.

### Claim 38 (depends on 30)

The nucleic acid of claim 30 where said percentage identity is at least 90%.

### Claim 39 (depends on 30)

The nucleic acid of claim 30 where said polypeptide comprises at least one sequence element selected from the group consisting of the tetrapeptides DGEA (Asp-Gly-Glu-Ala), VTKG (Val-Thr-Lys-Gly), EKGE (Glu-Lys-Gly-Glu) and DKGE (Asp-Lys-Gly-Glu).

### Claim 40 (depends on 30)

The nucleic acid of claim 30 where said sequences are aligned, and percentage identity calculated, using the Wisconsin sequence analysis program set (Genetics Computer Group, Inc.), and the default parameter settings therefor, as they existed in the version of said program set which was current on Oct. 14, 1995.

### Claim 41 (depends on 14)

The nucleic acid of claim 14 where said polypeptide has a percentage amino acid sequence identity of at least 80% with SEQ ID NO:2 or SEQ ID NO:4.

### Claim 42 (depends on 1)

The nucleic acid of claim 1 where said polypeptide has a percentage amino acid sequence identity of at least 80% with SEQ ID NO:2 or SEQ ID NO:4.

### Claim 43 (depends on 1)

The nucleic acid of claim 1 where said nucleic acid is (I) a nucleic acid whose coding strand hybridizes with the noncoding strand of (a) a nucleic acid having the coding strand nucleotide sequence shown in SEQ ID NO:1, or (b) a nucleic acid having the coding strand nucleotide sequence shown in SEQ ID NO:3 under stringent conditions, said conditions being 5 mM monovalent ions (0.1×SSC), neutral pH and 65° C., or (II) a nucleic acid which encodes the same polypeptide as a nucleic acid of (I) above.

### Claim 44 (depends on 43)

The nucleic acid of claim 43 where said polypeptide has a percentage amino acid sequence identity of at least 80% with SEQ ID NO:2 or SEQ ID NO:4.

### Claim 45 (depends on 14)

The nucleic acid of claim 14, in labeled form.

### Claim 46 (depends on 1)

A replicable expression vector which carries and is capable of mediating the expression of a nucleic acid as defined in claim 1.

### Claim 47 (depends on 46)

A cell comprising an expression vector according to claim 46.

### Claim 48 (depends on 1)

A method of producing a polypeptide comprising cultivating a cell according to claim 1 under conditions suitable for expressing the polypeptide, and recovering the polypeptide.

### Claim 49 (depends on 14)

A method of producing a polypeptide comprising cultivating a cell comprising an expression vector which carries and is capable of mediating the expression of a nucleic acid as defined in claim 14, under conditions suitable for expressing the polypeptide, and recovering the polypeptide.

### Claim 50 (depends on 30)

A method of producing a polypeptide comprising cultivating a cell comprising an expression vector which carries and is capable of mediating the expression of a nucleic acid as defined in claim 30, under conditions suitable for expressing the polypeptide, and recovering the polypeptide.

### Claim 51 (depends on 8)

A method of repairing a lesion in a tooth, the method comprising administering to a patient in need thereof an effective amount of a polypeptide encoded by a nucleic acid of claim 8.

### Claim 52 (depends on 8)

A method of joining two bond elements, the method comprising administering to a patient in need thereof an effective amount of a polypeptide encoded by a nucleic acid of claim 8.

### Claim 53 (depends on 8)

A method of promoting or provoking the mineralization of hard tissue selected from the group consisting of bond, enamel, dentin and cementum, the method comprising administering to a patient in need thereof an effective amount of a polypeptide according to claim 8.