# US 10,308,917 B2 · Nucleotide sequences encoding VEGF antagonist compositions of FLT-1 and uses thereof

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- Number: US 10,308,917 B2
- Document ID: US10308917B2
- Status: Granted
- Kind: B2
- Application number: 15/218,667
- Filed: 2016-07-25
- Published: 2016-11-10
- Granted: 2019-06-04
- Earliest claimed: 2010-08-06
- Assignees: Genzyme Corporation
- Applicants: Genzyme Corporation
- Inventors: James Stefano; Clark Pan; Huawei Qiu; Michael O'Callaghan; Gloria Matthews
- Web page: https://patentgrep.com/patent/US10308917B2

## CPC

- C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7)
- C07K 14/71: for growth factors; for growth regulators
- C12Y 207/10001: Receptor protein-tyrosine kinase (2.7.10.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C12Y: ENZYMES

## Abstract

The invention provides compositions and methods for treating a disease or disorder associate with vascular endothelial growth factor (VEGF). Specifically, the invention relates to an oligomerized VEGF binding domain to provide VEGF antagonism, and thereby treat diseases associated thereof.

## Claims

### Claim 1

A nucleotide sequence comprising a coding sequence encoding a monomer that comprises the second VEGF binding domain (D2) of FMS-like tyrosine kinase-1 (FLT-1), wherein said monomer comprises the amino acid sequence of SEQ ID NO:1, or an amino acid sequence with at least 95% sequence identity thereto; wherein the monomer further comprises one or more cysteine residues; wherein the one or more cysteine residues are C-terminal to the amino acid sequence of SEQ ID NO:1; wherein at least two of said monomer can form an oligomer; wherein the oligomer exhibits enhanced stability compared to soluble FLT-1 (sFLT01).

### Claim 2 (depends on 1)

The nucleotide sequence of claim 1, wherein said nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:2.

### Claim 3 (depends on 1)

An expression vector comprising the nucleotide sequence of claim 1, operably linked to expression control elements.

### Claim 4 (depends on 3)

A host cell comprising the expression vector of claim 3.

### Claim 5 (depends on 4)

A method for producing one or more monomers that comprise the second VEGF binding domain (D2) of FMS-like tyrosine kinase-1 (FLT-1), said method comprising:
(a) providing host cells according to claim 4; and
(b) culturing the host cells under conditions whereby the monomer encoded by the coding sequence present in the expression vector is expressed.

### Claim 6 (depends on 5)

The method of claim 5, wherein the method further comprises (c) recovering one or more monomers from the host cells.

### Claim 7

A method of producing a VEGF antagonist oligomer wherein said oligomer comprises a plurality of monomers linked to each other, wherein each monomer comprises the second VEGF binding domain (D2) of FMS-like tyrosine kinase-1 (FLT-1), wherein each monomer further comprises one or more cysteine residues, wherein the one or more cysteine residues are C-terminal to the second VEGF binding domain of FLT-1; said method comprising:
(a) providing host cells comprising an expression vector that comprises a coding sequence encoding said monomer, wherein said monomer comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence with at least 95% sequence identity thereto;
(b) culturing the host cells under conditions whereby the monomer encoded by the coding sequence present in the expression vector is expressed;
(c) recovering one or more monomers from the host cells; and
(d) linking said monomers to produce the oligomer; wherein the oligomer exhibits enhanced stability compared to soluble FLT-1 (sFLT01).

### Claim 8 (depends on 7)

The method of claim 7, wherein said monomers are linked using copper oxidation to form an oligomer.

### Claim 9 (depends on 7)

The method of claim 7, wherein said monomers are linked to each other through a non-protein polymer.

### Claim 10 (depends on 9)

The method of claim 9, wherein said monomers are linked to each other through a multifunctional PEG.

### Claim 11 (depends on 7)

The method of claim 7, wherein the oligomer is a dimer.

### Claim 12 (depends on 7)

The method of claim 7, wherein the oligomer is a trimer.

### Claim 13 (depends on 7)

The method of claim 7, wherein the oligomer is a tetramer.