# US 8,541,565 B2 · Polynucleotide encoding anti-CD14 antibody fusion protein

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- Number: US 8,541,565 B2
- Document ID: US8541565B2
- Status: Granted
- Kind: B2
- Application number: 13/547,548
- Filed: 2012-07-12
- Published: 2012-11-01
- Granted: 2013-09-24
- Earliest claimed: 2005-06-03
- Assignees: Mochida Pharmaceutical Co., Ltd.
- Applicants: Shoji Furusako; Kazuyuki Nakayama; Yoshitaka Hosaka; Tetsushi Kawahara; Masaki Nakamura; Takashi Takeuchi
- Inventors: Shoji Furusako; Kazuyuki Nakayama; Yoshitaka Hosaka; Tetsushi Kawahara; Masaki Nakamura; Takashi Takeuchi
- Web page: https://patentgrep.com/patent/US8541565B2

## CPC

- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- A61K 39/39541: {against normal tissues, cells}
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 7/02: Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/04: Antibacterial agents
- C07K 14/81: Protease inhibitors
- 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)
- A61K 2039/505: {comprising antibodies}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2319/00: Fusion polypeptide
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

A protein comprising (I) an anti-CD14 antibody or its active fragment, or a derivative thereof and (II) an inhibitor for a protease, or its active fragment, or a derivative thereof is provided.

## Claims

### Claim 1

At least a first polynucleotide and a second polynucleotide which together encode a fusion protein, wherein said first and second polynucleotides are a part of the same nucleic acid molecule or a different nucleic acid molecule, and wherein said first polynucleotide encodes a polypeptide comprising (Ia) and (II), below, and said second polynucleotide encodes a polypeptide comprising (Ib), below, or said first polynucleotide encodes a polypeptide comprising (Ia), below, and said second polynucleotide encodes a polypeptide comprising (Ib) and (II), below,
wherein (Ia) is
a heavy chain of an anti-CD14 antibody or its CD14 binding fragment, wherein said antibody or the fragment has an antigen binding activity and an inhibitory activity for LPS-induced IL-6 production in a TLR expressing cell, and
wherein (Ib) is
a light chain of an anti-CD14 antibody or its CD14 binding fragment, wherein said antibody or the fragment has an antigen binding activity and an inhibitory activity for LPS-induced IL-6 production in a TLR expressing cell, and
wherein (II) is
an inhibitor for a protease recited in 1), 2) or 3) below;
1) a polypeptide consisting of any one of amino acid sequences selected from the group consisting of a human UTI domain 2 (hereinafter referred to “UTI-D2”) represented by from 474th to 543rd amino acid residues of SEQ ID NO 10 and a mutant of human UTI domain 2 with 3 amino acid substitutions (R11S/Q19K/Y46D) (hereinafter referred to “UTI-D2(3)”) represented by from 474th to 543rd amino acid residues of SEQ ID NO 12,
2) a mutant of UTI-D2, wherein the mutant has one or more mutations, and wherein the one or more mutations are selected from the group consisting of:
a deletion of the 474th amino acid of SEQ ID NO: 10,
a deletion of the 475th amino acid of SEQ ID NO: 10,
a substitution of the 484th amino acid of SEQ ID NO: 10,
a substitution of the 492nd amino acid of SEQ ID NO: 10, and
a substitution of the 519th amino acid of SEQ ID NO: 10
to the amino acid sequence recited in 1) above, wherein said mutant has an anticoagulant action, or an inhibitory action for a blood coagulation factor,
3) a polypeptide consisting of any one of amino acid sequences selected from the group consisting of the amino acid sequences of the mutant UTI domain 2 of SEQ ID NOS: 34, 36, 42, 43, 46, 47, 48, 49, 62 and 63.

### Claim 2 (depends on 1)

The at least first and second polynucleotides according to claim 1, wherein the first polynucleotide encodes a polypeptide comprising (Ia) and (II), and the second polynucleotide encodes a polypeptide comprising (Ib).

### Claim 3 (depends on 2)

The at least first and second polynucleotides according to claim 2, wherein the heavy chain of the anti-CD 14 antibody or its CD 14 binding fragment in (Ia) comprises a CDR1 consisting of SEQ ID NO:173, a CDR2 consisting of SEQ ID NO:174, and a CDR3 consisting of SEQ ID NO:175 as the CDR1, CDR2, and CDR3 in a heavy chain variable region, and the light chain of the anti-CD14 antibody or its CD14 binding fragment in (Ib) comprises a CDR1 consisting of SEQ ID NO:176, a CDR2 consisting of SEQ ID NO:177, a CDR3 consisting of SEQ:ID:NO:178 as the CDR1, CDR2, and CDR3 in a light chain variable region.

### Claim 4 (depends on 2)

The first and second polynucleotides according to claim 2, wherein the fusion protein is obtained by binding (I) the anti-CD14 antibody or its CD14 binding fragment consisting of (Ia) the heavy chain comprising a CDR1 consisting of SEQ ID NO:173, a CDR2 consisting of SEQ ID NO:174, and a CDR3 consisting of SEQ ID NO:175 as the CDR1, CDR2, and CDR3 in a heavy chain variable region, and (Ib) the light chain comprising a CDR1 consisting of SEQ ID NO:176, a CDR2 consisting of SEQ ID NO:177, a CDR3 consisting of SEQ ID NO:178 as the CDR1, CDR2, and CDR3 in a light chain variable region,
with (II) the polypeptide consisting of any one of amino acid sequences selected from the group consisting of UTI-D2 and UTI-D2(3), by using a linker.

### Claim 5

A polynucleotide comprising a polynucleotide, which encodes for a polypeptide comprising:
(Ia) a heavy chain of an anti-CD 14 antibody or its CD14 binding fragment, wherein said antibody or the fragment has an antigen binding activity and an inhibitory activity for LPS-induced IL-6 production in a TLR expressing cell, and
(II) an inhibitor for a protease recited in 1), 2) or 3) below;
1) a polypeptide consisting of any one of amino acid sequences selected from the group consisting of UTI-D2 and UTI-D2(3),
2) a mutant of UTI-D2, wherein the mutant has one or more mutations, and wherein the one or more mutations are selected from the group consisting of:
a deletion of the 474th amino acid of SEQ ID NO:10,
a deletion of the 475th amino acid of SEQ ID NO:10,
a substitution of the 484th amino acid of SEQ ID NO:10,
a substitution of the 492nd amino acid of SEQ ID NO:10, and
a substitution of the 519th amino acid of SEQ ID NO:10
to the amino acid sequence recited in 1) above, wherein said mutant has an anticoagulant action, or an inhibitory action for a blood coagulation factor,
3) a polypeptide consisting of any one of amino acid sequences selected from the group consisting of the amino acid sequences of the mutant UTI domain 2 of SEQ ID NOS:27-106.

### Claim 6 (depends on 5)

The polynucleotide according to claim 5, wherein the heavy chain of the anti-CD14 antibody or its CD14 binding fragment in (Ia) comprises a CDR1 consisting of SEQ ID NO:173, a CDR2 consisting of SEQ ID NO:174, and a CDR3 consisting of SEQ ID NO:175 as the CDR1, CDR2, and CDR3 in a heavy chain variable region.

### Claim 7 (depends on 5)

The polynucleotide according to claim 5, wherein the polypeptide is obtained by binding (Ia) the heavy chain of the anti-CD14 antibody or its CD14 binding fragment comprising a CDR1 consisting of SEQ ID NO:173, a CDR2 consisting of SEQ ID NO:174, and a CDR3 consisting of SEQ ID NO:175 as the CDR1, CDR2, and CDR3 in a heavy chain variable region, with (II) the polypeptide consisting of any one of amino acid sequences selected from the group consisting of UTI-D2 and UTI-D2(3), by using a linker.

### Claim 8 (depends on 2)

The at least first and second polynucleotides according to claim 2, wherein the protease inhibitor in (II) is fused to the C terminal side of the heavy chain of the anti-CD 14 antibody or its CD14 binding fragment in (Ia).

### Claim 9 (depends on 5)

The polynucleotide according to claim 5, wherein the protease inhibitor in (II) is fused to the C terminal side of the heavy chain of the anti-CD14 antibody or its CD14 binding fragment in (Ia).

### Claim 10 (depends on 2)

The at least first and second polynucleotide of claim 2, wherein said first and second polynucleotide are part of the same nucleic acid molecule.

### Claim 11 (depends on 4)

The at least first and second polynucleotide of claim 4, wherein said first and second polynucleotide are part of the same nucleic acid molecule.

### Claim 12 (depends on 2)

At least a first vector and a second vector, wherein the first vector comprises the first polynucleotide of claim 2 and wherein the second vector comprises the second polynucleotide of claim 2.

### Claim 13 (depends on 4)

At least a first vector and a second vector, wherein the first vector comprises the first polynucleotide of claim 4 and wherein the second vector comprises the second polynucleotide of claim 4.

### Claim 14 (depends on 5)

A vector comprising the polynucleotide of claim 5.

### Claim 15 (depends on 7)

A vector comprising the polynucleotide of claim 7.

### Claim 16 (depends on 10)

A vector comprising the nucleic acid molecule of claim 10.

### Claim 17 (depends on 11)

A vector comprising the nucleic acid molecule of claim 11.

### Claim 18 (depends on 2)

A cell comprising the first and second polynucleotides of claim 2.

### Claim 19 (depends on 4)

A cell comprising the first and second polynucleotides of claim 4.

### Claim 20 (depends on 5)

A cell comprising the polynucleotides of claim 5.

### Claim 21 (depends on 7)

A cell comprising the polynucleotides of claim 7.

### Claim 22 (depends on 10)

A cell comprising the nucleic acid molecule of claim 10.

### Claim 23 (depends on 11)

A cell comprising the nucleic acid molecule of claim 11.

### Claim 24 (depends on 12)

A method of producing a fusion protein comprising:
co-transfecting the first and second vectors of claim 12 into a host cell;
culturing said host cell under conditions suitable for expressing said fusion protein; and
recovering said fusion protein.

### Claim 25 (depends on 13)

A method of producing a fusion protein comprising:
co-transfecting the first and second vectors of claim 13 into a host cell;
culturing said host cell under conditions suitable for expressing said fusion protein; and
recovering said fusion protein.

### Claim 26 (depends on 14)

A method of producing a fusion protein comprising:
transfecting the vector of claim 14 into a host cell;
culturing said host cell under conditions suitable for expressing said fusion protein; and
recovering said fusion protein.

### Claim 27 (depends on 15)

A method of producing a fusion protein comprising:
transfecting the vector of claim 15 into a host cell;
culturing said host cell under conditions suitable for expressing said fusion protein; and
recovering said fusion protein.

### Claim 28 (depends on 16)

A method of producing a fusion protein comprising:
transfecting the vector of claim 16 into a host cell;
culturing said host cell under conditions suitable for expressing said fusion protein; and
recovering said fusion protein.

### Claim 29 (depends on 17)

A method of producing a fusion protein comprising:
transfecting the vector of claim 17 into a host cell; culturing said host cell under conditions suitable for expressing said fusion protein; and recovering said protein.

### Claim 30 (depends on 2)

A therapeutic agent comprising the at least first and second polynucleotide of claim 2.