# US 2019/0316133 A1 · Anti-Fibrinogen Aptamers and Uses Thereof

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- Number: US 2019/0316133 A1
- Document ID: US20190316133A1
- Status: Published application
- Kind: A1
- Application number: 16/312,457
- Filed: 2017-07-06
- Published: 2019-10-17
- Earliest claimed: 2016-07-06
- Assignees: Laboratoire Francais Du Fractionnement Et Des Biotechnologies
- Applicants: Laboratoire Francais du Fractionnement et des Biotechnologies
- Inventors: Alexander SEIFERT
- Web page: https://patentgrep.com/patent/US20190316133A1

## CPC

- A61K 38/363: {Fibrinogen}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 47/12: Carboxylic acids; Salts or anhydrides thereof
- A61K 47/183: {Amino acids, e.g. glycine, EDTA or aspartame}
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers}
- C12Q 1/68: involving nucleic acids
- G01N 33/5002: {Partitioning blood components}
- C12N 2310/16: Aptamers
- C12N 2320/50: Methods for regulating/modulating their activity
- 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
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The invention relates to aptamers which specifically bind to fibrinogen and their use in the purification of said protein.

## Claims

### Claim at position 1

-16. (canceled)

### Claim 17

An aptamer which specifically binds to fibrinogen in a pH-dependent manner.

### Claim 18 (depends on 17)

The aptamer of claim 17 which does not bind to fibrinogen at a pH higher than 7.0 but specifically binds to fibrinogen at an acid pH value.

### Claim 19 (depends on 18)

The aptamer of claim 18, wherein the acid pH value is 6.0 to 6.6.

### Claim 20

An aptamer capable of specifically binding to fibrinogen, wherein:
the aptamer comprises a polynucleotide having at least 70% of sequence identity with the nucleotide sequence of SEQ ID No 66, or
the aptamer comprises the nucleotide moiety of formula (II):
5′-[SEQ ID NO: 79]-[X1]SEQ ID NO: 77]-[X2]- [SEQ
ID NO: 78]-3′ (III)
wherein:
[X2] and [X1] are independently a nucleotide or an oligonucleotide of 2 to 5 nucleotides in length,
[SEQ ID NO:77] is an oligonucleotide of SEQ ID NO:77 (namely GTTGGTAGGG),
[SEQ ID NO:78] is an oligonucleotide of SEQ ID NO:78 (namely GGTGTAT), and
[SEQ ID NO:79] is an oligonucleotide of SEQ ID NO:79 (namely TGT).

### Claim 21 (depends on 20)

The aptamer of claim 20, wherein the aptamer has at least 70% sequence identity with SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, or SEQ ID NO:74.

### Claim 22 (depends on 20)

The aptamer of claim 20, wherein the aptamer is of formula (I)
5′-[NUC1]m-[CENTRAL]-[NUC2]n-3′
Wherein
n and m are integers independently selected from 0 and 1,
[NUC1] is a polynucleotide comprising from 2 to 40 nucleotides,
[NUC2] is a polynucleotide comprising from 2 to 40 nucleotides, and
[CENTRAL] is a polynucleotide having at least 70% of sequence identity with SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73 SEQ ID NO:74, SEQ ID NO:94, or SEQ ID NO:95.

### Claim 23 (depends on 20)

The aptamer of claim 20, wherein the aptamer comprises SEQ ID NO:66, or differs from SEQ ID NO:66 by 1 to 14 nucleotide modifications at nucleotide positions selected from 1, 2, 11-25, 32-35, 42, 45-47, 50 and 54-58, wherein the numbering refers to nucleotide numbering in SEQ ID NO:66.

### Claim 24 (depends on 23)

The aptamer of claim 23, wherein the aptamer comprises SEQ ID NO:66, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, or SEQ ID NO:93.

### Claim 25 (depends on 20)

The aptamer of claim 20, wherein the aptamer comprises at least 70% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:67.

### Claim 26 (depends on 20)

The aptamer of claim 20, wherein the aptamer specifically binds to human plasma fibrinogen or recombinant human fibrinogen.

### Claim 27 (depends on 20)

An affinity ligand capable of specifically binding to fibrinogen, comprising an aptamer as defined in claim 20 and at least one moiety selected from a means of detection and a means of immobilization onto a support.

### Claim 28 (depends on 27)

A solid affinity support comprising thereon a plurality of affinity ligands as defined in claim 27.

### Claim 29 (depends on 28)

A method for preparing a purified fibrinogen composition from a starting fibrinogen-containing composition, comprising:
a) contacting said starting composition with an affinity support as defined in claim 28, in conditions suitable to form a complex between (i) the affinity ligands immobilized on said support and (ii) fibrinogen;
b) releasing fibrinogen from said complex; and
c) recovering a purified fibrinogen composition.

### Claim 30 (depends on 29)

The method of claim 29 wherein step a) is performed at a pH lower than 7.0, and step b) is performed at a pH above 7.0.

### Claim 31 (depends on 30)

The method of claim 30, wherein step a) is performed at a pH of 6.0 to 6.6, and step b) is performed at a pH of 7.2 to 7.6.

### Claim 32 (depends on 29)

The method of claim 29, wherein steps a), b), and c) are performed by chromatography technology.

### Claim 33 (depends on 27)

A method of purifying fibrinogen, detecting fibrinogen, blood plasma fractionation, or preparing a composition comprising fibrinogen that is stable in liquid form, comprising using the affinity ligand of claim 27.

### Claim 34 (depends on 20)

A blood plasma fractionation process comprising:
(a) an affinity chromatography step to recover fibrinogen wherein the affinity ligand is an aptamer which specifically binds to fibrinogen as defined in claim 20,
(b) an affinity chromatography step to recover immunoglobulins (Ig) wherein the affinity ligand specifically binds to immunoglobulins, and
(c) optionally a purification step of albumin,
wherein steps (a), (b) and (c) can be performed in any order.

### Claim 35 (depends on 20)

A composition comprising fibrinogen which is stable in liquid form, which is obtainable by a method comprising the steps of:
providing a blood plasma or a cryosupernatant fraction of blood plasma,
purifying said blood plasma or said cryosupernatant fraction of blood plasma by separation on affinity chromatography gel using an affinity ligand selected from aptamers as defined in claim 20,
collecting the purified adsorbed fraction comprising fibrinogen, and
optionally, adding pharmaceutically acceptable excipients, preferably arginine and/or citrate such as citrate salt.

### Claim 36 (depends on 17)

A method for preparing a purified fibrinogen composition from a starting fibrinogen-containing composition comprising:
a) contacting said starting composition with an affinity support on which aptamers as defined in claim 17 are immobilized, in conditions suitable to form a complex between (i) said aptamers immobilized on the support and (ii) fibrinogen;
b) releasing fibrinogen from said complex; and
c) recovering a purified fibrinogen composition.