# US 2013/0158095 A1 · Eml4-alk fusion gene

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- Number: US 2013/0158095 A1
- Document ID: US20130158095A1
- Status: Published application
- Kind: A1
- Application number: 13/468,730
- Filed: 2012-05-10
- Published: 2013-06-20
- Earliest claimed: 2006-10-11
- Assignees: CureGene K.K.; Astellas Pharma Inc.
- Inventors: Hiroyuki Mano; Sadao Kuromitsu; Nobuaki Shindo; Takatoshi Soga; Takashi Furutani
- Web page: https://patentgrep.com/patent/US20130158095A1

## CPC

- C12N 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- A61K 31/506: not condensed and containing further heterocyclic rings
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/47: from mammals
- C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases}
- C12Q 1/485: {involving kinase}
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C07K 2319/00: Fusion polypeptide
- C12N 2310/14: interfering nucleic acids [NA]
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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)
- 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 present inventors found that a fusion gene present in some cancer patients is an oncogene. The present invention relates to a polypeptide as a novel fusion protein, a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, a transformed cell comprising the vector, a method for detecting the fusion protein or polynucleotide, a method for screening a therapeutic agent for cancer, and a method for treating cancer that is shown to be positive for the fusion gene. Further, the present invention relates kit, primer set, and probe useful in the detection of cancer that is shown to be positive for the fusion gene.

## Claims

### Claim 1

An isolated polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or 7 and having a kinase activity.

### Claim 2 (depends on 1)

The isolated polypeptide according to claim 1 comprising the amino acid sequence represented by SEQ ID NO: 2 or 7.

### Claim 3 (depends on 1)

An isolated polynucleotide encoding the polypeptide according to claim 1.

### Claim 4 (depends on 3)

An expression vector comprising the polynucleotide according to claim 3.

### Claim 5 (depends on 4)

A cell transformed with the expression vector according to claim 4.

### Claim 6 (depends on 1, 5)

A method for producing the polypeptide according to claim 1, comprising culturing a transformed cell according to claim 5 under conditions suitable for polypeptide expression and collecting the polypeptide from the cell.

### Claim 7

(canceled)

### Claim 8

(canceled)

### Claim 9 (depends on 1)

A kit for detecting a fusion gene of EML4 gene and ALK gene, comprising sense and antisense primers designed to specifically amplify a polynucleotide encoding the polypeptide according to any of claim 1.

### Claim 10 (depends on 3)

A primer set for detecting a fusion gene of EML4 gene and ALK gene, comprising an antisense primer comprising nucleic acid molecule hybridizing under stringent conditions to i) the polynucleotide according to claim 3, ii) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 4, and/or iii) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 5, and a sense primer comprising a nucleic acid molecule hybridizing under stringent conditions to complementary strands to the above i) to iii).

### Claim 11

A primer set of a sense primer comprising an oligonucleotide with at least any 16 consecutive bases located at base Nos. 1 to 1759 in SEQ ID NO: 1 and an antisense primer comprising an oligonucleotide complementary to an oligonucleotide with at least any 16 consecutive bases located at base Nos. 1760 to 3926 in SEQ ID NO: 1, or a primer set comprising complementary strands thereof, wherein the sense and antisense primers give amplification products of 1 kb or less in size.

### Claim 12 (depends on 11)

The primer set of the sense primer and the antisense primer in according to claim 11 selected from a group consisting of (1)-(11) below or complementary strands thereof;
(1) SEQ ID NOs: 8 and 9, (2) SEQ ID NOs: 61 and 62, (3) SEQ ID NOs: 63 and 64, (4) SEQ ID NOs: 65 and 66, (5) SEQ ID NOs: 67 and 68, (6) SEQ ID NOs: 69 and 70, (7) SEQ ID NOs: 71 and 72, (8) SEQ ID NOs: 73 and 74, (9) SEQ ID NOs: 75 and 76, (10) SEQ ID NOs: 77 and 78, and (11) SEQ ID NOs: 79 and 80.

### Claim 13

A primer set of a sense primer comprising an oligonucleotide with at least any 16 consecutive bases located at base Nos. 1 to 2242 in SEQ ID NO: 6 and an antisense primer comprising an oligonucleotide complementary to an oligonucleotide with at least any 16 consecutive bases located at base Nos. 2243 to 3933 in SEQ ID NO: 6, or a primer set comprising complementary strands thereof, wherein the sense and antisense primers give amplification products of 1 kb or less in size.

### Claim 14 (depends on 13)

The primer set of the sense primer and the antisense primer in according to claim 13 selected from a group consisting of (1)-(10) below or complementary strands thereof;
(1) SEQ ID NOs: 81 and 82, (2) SEQ ID NOs: 83 and 84, (3) SEQ ID NOs: 85 and 86, (4) SEQ ID NOs: 87 and 88, (5) SEQ ID NOs: 89 and 90, (6) SEQ ID NOs: 91 and 92, (7) SEQ ID NOs: 93 and 94, (8) SEQ ID NOs: 95 and 96, (9) SEQ ID NOs: 97 and 98, and (10) SEQ ID NOs: 99 and 100.

### Claim 15

A primer set of a sense primer comprising an oligonucleotide with at least any 16 consecutive bases located at base Nos. 1 to 3629 in SEQ ID NO: 4 and an antisense primer comprising an oligonucleotide complementary to an oligonucleotide with at least any 16 consecutive bases located at base Nos. 3630 to 3979 in SEQ ID NO: 4, or a primer set comprising complementary strands thereof, wherein the sense and antisense primers give amplification products of 1 kb or less in size.

### Claim 16 (depends on 15)

The primer set of the sense primer and the antisense primer in according to claim 15 selected from a group consisting of (1)-(10) below or complementary strands thereof;
(1) SEQ ID NOs: 15 and 16, (2) SEQ ID NOs: 17 and 18, (3) SEQ ID NOs: 19 and 20, (4) SEQ ID NOs: 21 and 22, (5) SEQ ID NOs: 23 and 24, (6) SEQ ID NOs: 25 and 26, (7) SEQ ID NOs: 27 and 28, (8) SEQ ID NOs: 29 and 30, (9) SEQ ID NOs: 31 and 32, and (10) SEQ ID NOs: 33 and 34.

### Claim 17

A primer set of a sense primer comprising an oligonucleotide with at least any 16 consecutive bases located at base Nos. 1 to 579 in SEQ ID NO: 5 and an antisense primer comprising an oligonucleotide complementary to an oligonucleotide with at least any 16 consecutive bases located at base Nos. 580 to 853 in SEQ ID NO: 5, or a primer set comprising complementary strands thereof.

### Claim 18 (depends on 17)

The primer set of the sense primer and the antisense primer in according to claim 17 selected from a group consisting of (1)-(11) below or complementary strands thereof;
(1) SEQ ID NOs: 35 and 36, (2) SEQ ID NOs: 37 and 38, (3) SEQ ID NOs: 39 and 18, (4) SEQ ID NOs: 41 and 20, (5) SEQ ID NOs: 43 and 22, (6) SEQ ID NOs: 45 and 46, (7) SEQ ID NOs: 47 and 26, (8) SEQ ID NOs: 49 and 28, (9) SEQ ID NOs: 51 and 52, (10) SEQ ID NOs: 53 and 54, and (11) SEQ ID NOs: 55 and 34.

### Claim 19 (depends on 3)

A probe for detecting the polynucleotide of the present invention, comprising a nucleic acid molecule with at least 32 consecutive bases hybridizing under stringent conditions to i) the polynucleotide according to claim 3, ii) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 4, iii) a polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 5, or iv) complementary strands to the above i) to iii), and comprising positions 1744 to 1775 of the nucleotide sequence represented by SEQ ID NO: 1, positions 2227 to 2258 of the nucleotide sequence represented by SEQ ID NO: 6, positions 3614 to 3645 of the nucleotide sequence represented by SEQ ID NO: 4, positions 564 to 595 of the nucleotide sequence represented by SEQ ID NO: 5; or complementary strands thereof.

### Claim 20 (depends on 1)

A method for screening a substance inhibiting the polypeptide according to claim 1, comprising the steps of (1) bringing test substances into contact with the polypeptide or a cell expressing the polypeptide, (2) analyzing whether the polypeptide is inhibited or not, and (3) selecting a substance inhibiting the polypeptide.

### Claim 21 (depends on 20)

The screening method according to claim 20, further comprising the step of confirming that the selected test substance has a therapeutic activity against cancer that is shown to be positive for a fusion gene of EML4 gene and ALK gene.

### Claim 22 (depends on 1)

A method for treating cancer that is shown to be positive for a fusion gene of EML4 gene and ALK gene, comprising administering an effective amount of a substance inhibiting the polypeptide according to claim 1 to a target in need of treatment of cancer that is shown to be positive for a fusion gene of EML4 gene and ALK gene.

### Claim 23 (depends on 1, 22)

The method for treating cancer that is shown to be positive for a fusion gene of EML4 gene and ALK gene according to claim 22, wherein the substance inhibiting the polypeptide according to any of claim 1 is 5-chloro-N4-[2-(isopropylsulfonyl)phenyl]-N2-{2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl}pyrimidine-2,4-diamine or 2-[(5-bromo-2-{[2-methoxy-4-(4-methylpiperazin-1-yl)phenyl]amino}pyrimidin-4-yl)amino]-N-methylbenzene sulfonamide.

### Claim 24 (depends on 1)

A double-stranded nucleic acid having an inhibitory activity against the expression of the polypeptide according to claim 1, wherein a double-stranded portion is designed on the basis of bases at positions selected from a group consisting of (1) 1743 to 1761, (2) 1744 to 1762, (3) 1750 to 1768, (4) 1753 to 1771, (5) 1756 to 1774, and (6) 1757 to 1775 in SEQ ID NO: 1.

### Claim 25 (depends on 24)

A method for treating cancer that is shown to be positive for a fusion gene of EML4 gene and ALK gene, comprising administering an effective amount of the double-stranded nucleic acid according to claim 24 to a target in need of treatment of cancer that is shown to be positive for a fusion gene of EML4 gene and ALK gene.