# US 2013/0156692 A1 · Vegf-specific capture agents, compositions, and methods of using and making

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- Number: US 2013/0156692 A1
- Document ID: US20130156692A1
- Status: Published application
- Kind: A1
- Application number: 13/601,023
- Filed: 2012-08-31
- Published: 2013-06-20
- Earliest claimed: 2011-08-31
- Assignees: Integrated Diagnostics, Inc.
- Inventors: Suresh Mark Pitram; Heather Dawn Agnew; Tsun Yin Lai; Rosemary Dyane Rohde; Paul Edward Kearney
- Web page: https://patentgrep.com/patent/US20130156692A1

## CPC

- C07K 14/47: from mammals
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 27/02: Ophthalmic agents
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 7/54: with at least one abnormal peptide link in the ring
- G01N 33/575: for cancer
- G01N 33/74: involving hormones {or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors}
- 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)
- G01N 2333/475: Assays involving growth factors
- G01N 2333/515: Angiogenesic factors; Angiogenin
- G01N 2800/102: Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
- G01N 2800/16: Ophthalmology
- 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
- 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)
- 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)

## Abstract

The present application provides biligand and triligand protein-catalyzed capture (PCC) agents that specifically bind VEGF, as well as the use of these capture agents as detection, diagnosis, and treatment agents.

## Claims

### Claim 1

(canceled)

### Claim 2

A stable, synthetic capture agent that specifically binds VEGF, wherein the capture agent comprises a designed anchor ligand, a designed secondary ligand, and, optionally, a designed tertiary ligand, and wherein the anchor ligand and secondary ligand selectively bind VEGF, wherein the capture agent is a biligand capture agent, wherein the anchor ligand comprises an amino acid sequence 95% identical to the amino acid sequence of SEQ ID NO:1 and wherein the secondary ligand comprises a formula of X2-X3-X4-X5-X6, wherein X2 is selected from the group consisting of D-arginine, D-tryptophan, D-leucine, D-valine, glycine, D-proline, D-isoleucine and D-alanine; wherein X3 is selected from the group consisting of D-proline, D-phenylalanine, glycine, D-arginine, D-lysine, D-histidine, D-tryptophan and D-tyrosine; wherein X4 is selected from the group consisting of D-isoleucine, D-leucine, D-proline, D-alanine, D-arginine, D-phenylalanine, D-threonine and D-histidine; wherein X5 is selected from the group consisting of D-isoleucine, glycine, D-valine, D-leucine, D-alanine, D-proline, D-lysine, D-glutamate, D-histidine and D-arginine; and wherein X6 is selected from the group consisting of D-arginine, D-lysine, D-tryptophan, D-tyrosine, D-proline, D-valine, glycine, D-isoleucine, D-alanine and D-glutamine.

### Claim 3 (depends on 2)

The biligand capture agent of claim 2, wherein the anchor ligand and secondary ligand are linked together via a 1,4-substituted-1,2,3-triazole residue (Tz4).

### Claim 4 (depends on 2)

The biligand capture agent of claim 2, wherein the capture agent has a structure selected from the group consisting of:

### Claim 5 (depends on 2)

The capture agent of claim 2, wherein the capture agent is a triligand capture agent wherein the anchor ligand comprises an amino acid sequence 95% identical to the amino acid sequence of SEQ ID NO:1 and wherein the secondary ligand comprises a formula of X2-X3-X4-X5-X6, wherein X2 is selected from the group consisting of D-arginine, D-tryptophan, D-leucine, D-valine, glycine, D-proline, D-isoleucine and D-alanine; wherein X3 is selected from the group consisting of D-proline, D-phenylalanine, glycine, D-arginine, D-lysine, D-histidine, D-tryptophan and D-tyrosine; wherein X4 is selected from the group consisting of D-isoleucine, D-leucine, D-proline, D-alanine, D-arginine, D-phenylalanine, D-threonine and D-histidine; wherein X5 is selected from the group consisting of D-isoleucine, glycine, D-valine, D-leucine, D-alanine, D-proline, D-lysine, D-glutamate, D-histidine and D-arginine; and wherein X6 is selected from the group consisting of D-arginine, D-lysine, D-tryptophan, D-tyrosine, D-proline, D-valine, glycine, D-isoleucine, D-alanine and D-glutamine, and
wherein the tertiary ligand comprises a formula of X2-X3-X4-X5-X6 wherein
X2 is selected from the group consisting of D-histidine, D-arginine and D-lysine; X3 is selected from the group consisting of D-threonine, D-asparagine, D-leucine, D-proline, D-isoleucine, D-alanine, and D-glutamate; X4 is selected from the group consisting of D-histidine, D-lysine, D-arginine, D-tryptophan, D-phenylalanine, D-proline, D-leucine and D-tyrosine; X5 is selected from the group consisting of D-valine, D-proline, D-histidine, D-phenylalanine, D-tryptophan, D-asparagine, D-glutamine, D-serine and D-tyrosine; and X6 is selected from the group consisting of D-arginine, D-tyrosine, D-asparagine, D-glutamine, D-leucine, D-proline, D-lysine and D-histidine;
X2 is selected from the group consisting of D-tyrosine, D-phenylalanine and D-tryptophan; X3 is selected from the group consisting of D-proline, D-alanine, glycine, D-leucine, D-lysine, D-arginine and D-histidine; X4 is selected from the group consisting of D-arginine, D-histidine, D-tryptophan, D-phenylalanine, D-glutamate, D-proline, D-serine and D-threonine; X5 is selected from the group consisting of D-proline, D-aspartate, D-lysine, D-arginine, D-tyrosine, D-histidine, D-alanine, D-valine, D-leucine and D-asparagine; and X6 is selected from the group consisting of D-histidine, D-lysine, D-asparagine, D-threonine, D-glutamine, D-leucine, D-aspartate, D-serine, D-tyrosine, D-arginine, D-tryptophan, D-glutamate and D-valine; or
X2 is selected from the group consisting of D-glutamate and D-aspartate; X3 is selected from the group consisting of D-glutamate, D-phenylalanine, D-tryptophan, D-histidine, D-lysine, D-asparagine and D-serine; X4 is selected from the group consisting of D-isoleucine, D-proline, D-alanine, D-arginine, D-serine, D-aspartate, D-asparagine, D-proline, D-phenylalanine, D-tyrosine and D-histidine; X5 is selected from the group consisting of D-arginine, D-tyrosine, glycine, D-tryptophan, D-lysine, D-histidine, D-alanine, D-asparagine and D-leucine; and X6 is selected from the group consisting of D-aspartate, D-proline, D-tryptophan, D-tyrosine, D-leucine, D-asparagine, D-serine and D-threonine.

### Claim 6 (depends on 5)

The triligand capture agent of claim 5, wherein the anchor ligand and secondary ligand are linked together via a 1,4-substituted-1,2,3-triazole residue (Tz4).

### Claim 7 (depends on 5)

The triligand capture agent of claim 5, wherein the secondary ligand and tertiary ligand are linked together via a 1,4-substituted-1,2,3-triazole residue (Tz4).

### Claim 8 (depends on 5)

The triligand capture agent of claim 5, wherein the capture agent has a structure selected from the group consisting of

### Claim 9 (depends on 2)

The capture agent of claim 2, wherein binding of said capture agent to VEGF inhibits VEGF activity.

### Claim 10 (depends on 2)

The capture agent of claim 2, wherein said capture agent inhibits binding of VEGF to VEGF receptor (VEGFR).

### Claim 11 (depends on 2)

The capture agent of claim 2, wherein the capture agent is stable at a temperature of about −80° C. to about 40° C.

### Claim 12 (depends on 2)

The capture agent of claim 2, wherein the capture agent is stable at room temperature.

### Claim 13 (depends on 2)

The capture agent of claim 2, wherein the capture agent is stable in blood serum or blood plasma for at least 24 hours.

### Claim 14 (depends on 2)

The capture agent of claim 2, wherein the capture agent is stable at a pH in the range of from about 3 to about 12.

### Claim 15 (depends on 2)

The capture agent of claim 2, wherein the capture agent is labeled with a label selected from the group consisting of biotin and copper-DOTA.

### Claim 16 (depends on 2)

The use of a capture agent of claim 2 as a detection agent for detecting VEGF in a biological sample.

### Claim 17 (depends on 2)

A method of detecting VEGF in a biological sample using an immunoassay, wherein the immunoassay utilizes a capture agent of claim 2, and wherein said capture agent replaces an antibody or its equivalent in the immunoassay.

### Claim 18 (depends on 17)

The method of claim 17, wherein the immunoassay is selected from the group of Western blot, pull-down assay, dot blot, and ELISA.

### Claim 19 (depends on 2)

A method of treating a condition associated with increased VEGF expression and/or activity in a subject in need thereof, comprising administering a therapeutically effective amount of a capture agent of claim 2.

### Claim 20 (depends on 19)

The method of claim 19, wherein said condition is selected from the group consisting of cancer, proliferative retinopathy, disease pathology of wet form age-related macular degeneration (AMD), or rheumatoid arthritis.

### Claim 21 (depends on 2)

A method of inhibiting VEGF activity in a subject comprising administering to the subject a capture agent of claim 2.

### Claim 22 (depends on 2)

The capture agent of claim 2, wherein the capture agent is labeled with the detectable moiety consisting of 64Cu DOTA, 68Ga DOTA, 18F, 64Cu, 68Ga, 89Zr, 124I, 86Y, 94mTc, 110mIn, 11C or 76Br.

### Claim 23 (depends on 2)

A method of diagnosing a VEGF expressing cancer in a subject, the method comprising the steps of: a) administering to the subject the VEGF capture agent of claim 2, linked to a detectable moiety; and b) detecting the moiety linked to the VEGF capture agent in the subject; wherein detection of the moiety diagnoses a VEGF-expressing cancer in the subject.

### Claim 24 (depends on 23)

The method of claim 23, wherein the capture agent is labeled with the detectable moiety consisting of 64Cu DOTA, 68Ga DOTA, 18F, 64Cu, 68Ga, 89Zr, 124I, 86Y, 94mTc, 110In, 11C or 76Br.

### Claim 25 (depends on 2, 5)

A method of monitoring treatment of a subject receiving VEGF-directed therapy comprising administering to the patient a small-molecule positron-emission-tomography ligand (PET ligand) that is bound to the VEGF capture agent of claim 2 or claim 5 on or near a VEGF-expressing cancer in the subject.

### Claim 26 (depends on 2)

A method of detecting VEGF in a sample comprising a) exposing the sample to the VEGF capture agent of claim 2, linked to a detectable moiety; and b) detecting the moiety linked to the VEGF capture agent in the subject; wherein detection of the moiety diagnoses a VEGF-expressing cancer in the subject.

### Claim 27 (depends on 26)

The method of claim 26, wherein the capture agent is labeled with the detectable moiety consisting of 64Cu DOTA, 68Ga DOTA, 18F, 64Cu, 68Ga, 89Zr, 124I, 86Y, 94mTc, 110In, 11C or 76Br.

### Claim 28 (depends on 26)

The method of claim 26, wherein the moiety linked to the VEGF capture agent is detected using PET or SPECT.

### Claim 29 (depends on 24)

The method of claim 24, wherein the moiety linked to the VEGF capture agent is detected using PET or SPECT.

### Claim 30 (depends on 4)

The capture agent of claim 4, wherein R is a pegylated reporter tag or an N-terminal capping group.

### Claim 31 (depends on 30)

The capture agent of claim 30, wherein the pegylated reporter tag is selected from the group consisting of biotin-PEG and DOTA-PEG.

### Claim 32 (depends on 30)

The capture agent of claim 30, wherein the N-terminal capping group is an acetyl group.

### Claim 33 (depends on 8)

The capture agent of claim 8, wherein R is a pegylated reporter tag or an N-terminal capping group.

### Claim 34 (depends on 33)

The capture agent of claim 33, wherein the pegylated reporter tag is selected from the group consisting of biotin-PEG and DOTA-PEG.

### Claim 35 (depends on 33)

The capture agent of claim 33, wherein the N-terminal capping group is an acetyl group.

### Claim 36 (depends on 15)

The capture agent of claim 15, wherein the label is bound to the capture agent by a linker.

### Claim 37 (depends on 36)

The capture agent of claim 36, wherein the linker is a PEG linker.

### Claim 38 (depends on 31)

The capture agent of claim 31, wherein the biotin-PEG is biotin-PEG3.

### Claim 39 (depends on 34)

The capture agent of claim 34, wherein the biotin-PEG is biotin-PEG3.

### Claim 40 (depends on 2)

The biligand capture agent of claim 2, wherein the capture agent has a structure selected from:

### Claim 41 (depends on 40)

The capture agent of claim 40, wherein R is a pegylated reporter tag or an N-terminal capping group.

### Claim 42 (depends on 41)

The capture agent of claim 41, wherein the pegylated reporter tag is selected from the group consisting of biotin-PEG and DOTA-PEG.

### Claim 43 (depends on 42)

The capture agent of claim 42, wherein the biotin-PEG is biotin-PEG3.

### Claim 44 (depends on 41)

The capture agent of claim 41, wherein the N-terminal capping group is an acetyl group.