# US 2019/0169293 A1 · Hanp-fc-containing molecular conjugate

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- Number: US 2019/0169293 A1
- Document ID: US20190169293A1
- Status: Published application
- Kind: A1
- Application number: 16/314,411
- Filed: 2017-06-30
- Published: 2019-06-06
- Earliest claimed: 2016-07-01
- Assignees: Daiichi Sankyo Company, Limited
- Applicants: Daiichi Sankyo Company, Limited
- Inventors: Mitsuhiro IWAMOTO; Shohei OISHI; Yukiko SEKIGUCHI; Hiroyuki CHAYA; Ryuki MIYAUCHI; Takeshi HONDA
- Web page: https://patentgrep.com/patent/US20190169293A1

## CPC

- C07K 16/283: {against Fc-receptors, e.g. CD16, CD32, CD64 (CD23 C07K16/2851)}
- A61K 38/22: Hormones (derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin A61K38/33, e.g. corticotropin A61K38/35)
- A61K 39/395: Antibodies (agglutinins A61K38/36 {; as drug carriers A61K47/50}); Immunoglobulins; Immune serum, e.g. antilymphocytic serum
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 47/68: the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61P 9/04: Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 9/12: Antihypertensives
- A61P 9/14: Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
- A61P 13/12: of the kidneys
- C07K 14/58: Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12N 15/09: Recombinant DNA-technology
- C12P 1/00: Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P 19/18: produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
- C12Y 302/01: Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y 302/01006: Endo-1,3(4)-beta-glucanase (3.2.1.6)
- 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 16/12: against material from bacteria
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES

## Abstract

The present invention provides a conjugate comprising a hANP peptide bonded via a polyethylene glycol linker to a glycan attached to Asn297 of a Fc-containing molecule (N297 glycan), or a pharmaceutically acceptable salt thereof, a medicament comprising the same as an active ingredient, a method for producing the same, etc.

## Claims

### Claim 1

A conjugate comprising a hANP peptide bonded via a polyethylene glycol linker (L(PEG)) to a glycan attached to Asn297 of a Fc-containing molecule (N297 glycan), or a pharmaceutically acceptable salt thereof, wherein:
the hANP peptide optionally lacks 1 to 5 amino acids consecutively from the N terminus and/or one C terminal amino acid in the amino acid sequence represented by SEQ ID NO: 1 and is optionally glycosylated at either one or both of its N terminus and C terminus;
the L(PEG) is a linker structure comprising 10 to 35 ethylene glycol structures and optionally comprising an additional binding structure and/or modifying structure;
the Fc-containing molecule is a molecule having an amino acid sequence corresponding to a human IgG Fc region and having no ability to bind specifically to a human biomolecule; and
the N297 glycan is a glycan N297-(Fuc)SG, N297-(Fuc)MSG1 or N297-(Fuc)MSG2 having a structure represented by the following formula:
wherein L(PEG) represents binding to carbonyl groups bonded to the 2-positions of sialic acid residues at the non-reducing ends of both the 1-3 and 1-6 branched chains of β-Man,
wherein L(PEG) represents binding to a carbonyl group bonded to the 2-position of a sialic acid residue at the non-reducing end of the 1-3 branched chain of β-Man, and
wherein L(PEG) represents binding to a carbonyl group bonded to the 2-position of a sialic acid residue at the non-reducing end of the 1-6 branched chain of β-Man.

### Claim 2 (depends on 1)

The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the hANP peptide is hANP(1-28), hANP(2-28), hANP(3-28), hANP(1-27), hANP(2-27) or hANP(3-27).

### Claim 3 (depends on 1)

The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the hANP peptide is a glycosylated peptide in which asparagine or glutamine with its side chain attached through a N-glycosidic bond to any of glycans AG5, AG7, AG9 and SG represented by the following formula is bonded to the N terminus or C terminus of the peptide:
[Formula 4]
Manα1-6
Manβ1-4GlcNAcβ1-4GlcNAcβ1-(N/Q)
Manα1-3 [AG5]
[Formula 5]
GlcNAcβ1-2Manα1-6
Manβ1-4GlcNAcβ1-4GlcNAcβ1-(N/Q)
GlcNAcβ1-2Manα1-3 [AG7]
[Formula 6]
Galβ1-4GlcNAcβ1-2Manα1-6
Manβ1-4GlcNAcβ1-4GlcNAcβ1-(N/Q)
Galβ1-4GlcNAcβ1-2Manα1-3 [AG9]
[Formula 7]
NeuAcα2-6Galβ1-4GlcNAcβ1-2Manα1-6
Manβ1-4GlcNAcβ1-4GlcNAcβ1-(N/Q)
NeuAcα2-6Galβ1-4GlcNAcβ1-2Manα1-3 [SG]
wherein “-(N/Q)” represents binding to the side chain of asparagine or glutamine through a N-glycosidic bond.

### Claim 4 (depends on 3)

The conjugate according to claim 3 or a pharmaceutically acceptable salt thereof, wherein the hANP peptide is hANP(1-28) in which asparagine with its side chain attached to a glycan SG through a N-glycosidic bond is bonded to the N terminus of the peptide.

### Claim 5 (depends on 1)

The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the L(PEG) is a linker structure comprising 25 to 30 ethylene glycol structures and comprising an amide bond and a 1,2,3-triazole ring as binding structures.

### Claim 6 (depends on 1)

The conjugate according to claim 1 or a pharmacologically acceptable salt thereof, wherein the L(PEG) is a linker structure represented by any of the following formulas:
wherein “hANP” represents the hANP peptide; “N297 GLY” represents binding to the non-reducing end of the N297-attached glycan; each linker structure has geometric isomeric structures formed during bond formation at the 1,2,3-triazole ring site, as shown in structures L(PEG)-X1 and L(PEG)-X2 (these are collectively referred to as L(PEG)-X (X being A, B, C, D, E, F, G or H)) in the formulas; and either only any one of the structures is present in the linkers in one molecule of the conjugate, or two structures coexist as linkers in one molecule of the conjugate.

### Claim 7 (depends on 1)

The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the Fc-containing molecule is a human IgG monoclonal antibody directed to a substance other than a human biogenic substance as an antigen, or a fragment or an engineered form of human IgG having a human IgG Fc region and lacking variable regions.

### Claim 8 (depends on 7)

The conjugate according to claim 7 or a pharmaceutically acceptable salt thereof, wherein the Fc-containing molecule is Fc derived from human IgG or CLCH consisting of human IgG constant regions.

### Claim 9 (depends on 7)

The conjugate according to claim 7 or a pharmaceutically acceptable salt thereof, wherein the Fc-containing molecule is an antibody consisting of a combination of a heavy chain consisting of an amino acid sequence from amino acid positions 20 to 474 of SEQ ID NO: 3 and a light chain consisting of an amino acid sequence from amino acid positions 21 to 234 of SEQ ID NO: 5 (mAb-A), CLCH consisting of a combination of a heavy chain consisting of an amino acid sequence from amino acid positions 20 to 349 of SEQ ID NO: 7 and a light chain consisting of an amino acid sequence from amino acid positions 21 to 125 of SEQ ID NO: 9 (CLCH-A), CLCH consisting of a combination of a heavy chain consisting of an amino acid sequence from amino acid positions 20 to 349 of SEQ ID NO: 11 and a light chain consisting of an amino acid sequence from amino acid positions 21 to 125 of SEQ ID NO: 9 (CLCH-B), a Fc fragment consisting of an amino acid sequence from amino acid positions 21 to 243 of SEQ ID NO: 15 (Fc-B), or a Fc fragment consisting of an amino acid sequence from amino acid positions 21 to 247 of SEQ ID NO: 17 (Fc-A), or an engineered form of any such antibody.

### Claim 10 (depends on 1)

The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein
the hANP peptide is hANP(1-28) or (SG-)Asn-hANP(1-28),
the PEG linker is L(PEG)-A, L(PEG)-B, L(PEG)-C, L(PEG)-D, L(PEG)-E, L(PEG)-F, L(PEG)-G or L(PEG)-H,
the Fc-containing molecule is mAb-A, CLCH-A, CLCH-B, Fc-A or Fc-B, and
the N297 glycan is N297-(Fuc)SG.

### Claim 11 (depends on 1)

The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the hANP peptide is (SG-)Asn-hANP(1-28), the PEG linker is L(PEG)-B, the Fc-containing molecule is Fc-A or Fc-B, and the N297 glycan is N297-(Fuc)SG.

### Claim 12 (depends on 1)

A medicament comprising the conjugate according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

### Claim 13 (depends on 12)

The medicament according to claim 12, wherein the medicament is a therapeutic agent or a prophylactic agent for a disease treatable by the activation of GC-A.

### Claim 14 (depends on 12)

The medicament according to claim 12, wherein the medicament is a therapeutic agent or a prophylactic agent for hypertension, acute heart failure, chronic heart failure, acute renal failure, or chronic renal failure.

### Claim 15 (depends on 1)

A method for producing the conjugate according to claim 1, comprising the following steps:
step A: the step of treating a Fc-containing molecule produced in an animal cell with hydrolase to produce a (Fucα1,6)GlcNAc-Fc-containing molecule;
step B1: the step of reacting a glycan donor molecule with the (Fucα1,6)GlcNAc-Fc-containing molecule in the presence of glycosyltransferase to synthesize a SG type glycan-remodeled Fc-containing molecule with an azide group introduced in the sialic acid, wherein the glycan donor contains SG(10) or MSG(9) with an azide group introduced in sialic acid and having oxazoline at the reducing end, or
step B2: the step of reacting a glycan donor molecule, the glycan donor molecule being (SG-)Asn or (MSG-)Asn with an azide group introduced in sialic acid thereof, with the (Fucα1,6)GlcNAc-Fc-containing molecule in the presence of two glycosyltransferases to synthesize a SG type glycan-remodeled Fc-containing molecule with the azide group introduced in the sialic acid; and
step C: the step of reacting a linker molecule having a hANP peptide on one side and DBCO on the other side with the SG type glycan-remodeled Fc-containing molecule with the azide group introduced in the sialic acid, prepared in step B to synthesize the conjugate according to claim 1.

### Claim 16 (depends on 14)

The production method according to claim 14, further comprising the step of purifying the (Fucα1,6)GlcNAc-Fc-containing molecule from the reaction solution of step A by a step involving purification using a hydroxyapatite column.

### Claim 17 (depends on 1)

A method for treating or preventing a disease treatable by the activation of GC-A, comprising administering the conjugate according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient to a subject in need of the administration.

### Claim 18 (depends on 1)

The conjugate according to claim 1 or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of a disease treatable by the activation of GC-A.