# US 10,370,442 B2 · vNAR recombinant monoclonal antibodies that neutralize vascular endophelial growth factor VEGF

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,370,442 B2
- Document ID: US10370442B2
- Status: Granted
- Kind: B2
- Application number: 15/304,516
- Filed: 2014-04-17
- Published: 2017-05-18
- Granted: 2019-08-06
- Earliest claimed: 2014-04-17
- Assignees: Teraclon Idf, S.L.
- Applicants: Teraclon Idf, S.L.
- Inventors: Carolina Elosua Portugal; Maria Teresa Mata Gonzalez; Tanya Amanda Camacho Villegas; Araceli Olguin Jimenez; Alexei Fedorovish Licea Navarro; Jorge Fernando Paniagua Solis
- Web page: https://patentgrep.com/patent/US10370442B2

## CPC

- C07K 16/22: against growth factors {; against growth regulators}
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 7/04: Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
- A61P 7/06: Antianaemics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 27/02: Ophthalmic agents
- A61P 27/06: Antiglaucoma agents or miotics
- A61P 27/12: for cataracts
- A61P 31/10: Antimycotics
- A61P 35/00: Antineoplastic agents
- A61P 39/06: Free radical scavengers or antioxidants
- C07K 2317/20: characterized by taxonomic origin
- C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention refers to the selection, isolation and purification of proteins belonging to variable regions named VHNAR or vNAR, originated from IgNAR-type immunoglobulins of elasmobranches with antigen receptor abilities. The clones from which they originate are named VEGFvNAR V32R and V19; and the antibodies are named V32R and V19. Their amino acid sequences and tertiary structures were elucidated, and their ability to neutralize the vascular endothelial growth factor (VEGF) activity was determined. During the development of the invention these proteins were optimized for expression in a production model of E. coli at industrial level. The invention involves the use of these antibodies in general to treat conditions related to angiogenesis or neovascularization and in particular to treat neovascularization-related ophthalmic conditions by topical administration.

## Claims

### Claim 1

An isolated anti-VEGF vNAR protein selected from the group consisting of V19 comprising the amino acid sequence of SEQ ID NO: 4 and V32R comprising the amino acid sequence of SEQ ID NO: 6, wherein the anti-VEGF vNAR protein recognizes human VEGF and has enhanced affinity for a target molecule VEGF.

### Claim 2 (depends on 1)

The anti-VEGF vNAR protein of claim 1, wherein V19 comprises various β sheets connected by loops, with a highly flexible variable region consisting of two β sheets interconnected by a loop and a monomer consisting of two groups of beta sheets with a single disulfide bridge bonding the two groups of sheets, comprising SEQ ID NO: 4.

### Claim 3 (depends on 1)

The anti-VEGF vNAR protein of claim 1, wherein V32R comprises β sheets and a monomer consisting of two groups of beta sheets with a single disulfide bridge bonding the two groups of sheets, comprising SEQ ID NO: 6.

### Claim 4 (depends on 1)

The isolated anti-VEGF vNAR protein of claim 1, wherein the V19 or the V32R:
a) originates from Orectolobus maculatus or Heterodontus jrancisci; and
b) binds to and neutralizes the activity of the vascular endothelial growth factor (VEGF) in the eye.

### Claim 5 (depends on 1)

A pharmaceutical composition comprising the isolated anti-VEGF vNAR protein selected from the group consisting of V19 and V32R of claim 1, and a pharmaceutically acceptable excipient.

### Claim 6 (depends on 5)

The pharmaceutical composition of claim 5, wherein it is formulated for topical administration.

### Claim 7 (depends on 5)

The pharmaceutical composition of claim 5, wherein the composition is formulated for topical ophthalmic administration.

### Claim 8 (depends on 5)

The pharmaceutical composition of claim 5, wherein the composition is formulated for topical administration in a VEGF-related eye disease.

### Claim 9 (depends on 8)

The pharmaceutical composition of claim 8, wherein the VEGF-related eye disease is selected from wet age-related macular degeneration, diabetic retinopathy, or neovascular glaucoma.

### Claim 10

A medicament comprising an effective amount of an isolated anti-VEGF vNAR protein comprising the amino acid sequence of SEQ ID NO: 4 (V19) or SEQ ID NO: 6 (V32R), administrable by a topical ophthalmic route.

### Claim 11 (depends on 10)

A method treating a VEGF-related eye disease, wherein the disease is also a pathology related to an angiogenic process, where excessive angiogenesis occurs when diseased cells produce abnormal amounts of VEGF or VEGF receptors, the method comprising applying the medicament of claim 10.

### Claim 12 (depends on 11)

The method of claim 11, wherein the angiogenic process is selected from the group consisting of age related macular degeneration, diabetic retinopathy, and neovascular glaucoma.

### Claim 13 (depends on 12)

The method of claim 12, wherein the angiogenic process is diabetic retinopathy.

### Claim 14 (depends on 12)

The method of claim 12, wherein the angiogenic process is wet age-related macular degeneration.

### Claim 15 (depends on 12)

The method of claim 12, wherein the angiogenic process is neovascular glaucoma.

### Claim 16 (depends on 1)

A method for preparing a medicament comprising an anti-VEGF vNAR protein of claim 1, the method comprising the step of culturing a host cell to produce the anti-VEGF vNAR protein V19 comprising the amino acid sequence of SEQ ID NO: 4 or the anti-VEGF vNAR protein V32R comprising the amino acid sequence of SEQ ID NO: 6.

### Claim 17 (depends on 16)

The method of claim 16, wherein the ophthalmic condition is wet age-related macular degeneration, diabetic retinopathy, or neovascular glaucoma.

### Claim 18 (depends on 1)

An isolated anti-VEGF vNAR protein selected from the group consisting of V19 and V32R of claim 1, wherein,
(a) it originates from Orectolobus maculatus or Heterodontus francisci;
(b) it binds to and neutralizes the activity of the vascular endothelial growth factor (VEGF) in the eye;
(c) wherein the following vNAR V19 residues in CDR zone are able to interact with the following VEGF residues:
i. ARG 101 of V19 interacts with GLU 17 of VEGF,
ii. GLU103 of V 19 interacts with ARG 10 of VEGF; and,
(d) wherein the following vNAR V32R residues in CDR zone are able to interact with the following VEGF residues:
i. GLU 98 of V32R interacts with ARG 43 and GLN 24 of VEGF;
ii. ARG 91 of V32R interacts with GLN 24 and ASP 21 of VEGF;
iii. HIS 90 of V32R interacts with PHE 23 of VEGF;
iv. LYS 95 of V32R interacts with GLU 25 of VEGF;
v. ARG 100 of V32R interacts with GLU 54 of VEGF; and,
vi. TYR104 of V32R interacts with CYS55 of VEGF.

### Claim 19 (depends on 1)

A process for producing an anti-VEGF vNAR protein of claim 1, said process comprising the steps of:
a) obtaining a vector comprising the DNA sequence of SEQ ID NO: 3 encoding V19 or the DNA sequence of SEQ ID NO: 5 encoding V32R;
b) obtaining a host cell comprising said vector; and,
c) culturing the host cell to produce the anti-VEGF vNAR protein V19 comprising the amino acid sequence of SEQ ID NO: 4 or the anti-VEGF vNAR protein V32 comprising the amino acid sequence of SEQ ID NO: 6.