# US 10,077,290 B2 · Stabilized antiviral fusion helices

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- Number: US 10,077,290 B2
- Document ID: US10077290B2
- Status: Granted
- Kind: B2
- Application number: 14/367,973
- Filed: 2012-12-31
- Published: 2014-12-18
- Granted: 2018-09-18
- Earliest claimed: 2011-12-29
- Assignees: Dana-Farber Cancer Institute, Inc.
- Applicants: Dana-Farber Cancer Institute, Inc.
- Inventors: Loren D. Walensky; Gregory H. Bird
- Web page: https://patentgrep.com/patent/US10077290B2

## CPC

- C07K 14/005: from viruses
- A61K 39/12: Viral antigens
- A61K 39/155: Paramyxoviridae, e.g. parainfluenza virus
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61P 31/14: for RNA viruses
- A61P 37/04: Immunostimulants
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- G01N 33/68: involving proteins, peptides or amino acids {(involving lipoproteins G01N33/92)}
- 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)
- C12N 2760/18522: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2760/18533: Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
- C12N 2760/18534: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2760/18571: Demonstrated in vivo effect
- G01N 2333/135: Respiratory syncytial virus
- G01N 2500/02: Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
- 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)
- 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

Internally cross-linked peptides useful for interfering with Respiratory Syncytial Virus (RSV) infection is based on RSV-F protein. These peptides are capable of reducing infection in cellular and animal models.

## Claims

### Claim 1

An internally cross-linked polypeptide comprising the amino acid sequence A0B0C0D0E0F0G0 A1B1C1D1E1F1G1A2B2C2D2E2F2G2A3B3C3D3E3F3G3A4B4C4D4E4F4G4 (SEQ ID NO:1) wherein:
A0 is Phe;
B0 is Asp or Glu;
C0 is Ala, Val, Ile, Leu, or Ser;
D0 is Ser;
E0 is Ile;
F0 is Ser or Thr;
G0 is Gln or Asn;
A1 is Val;
B1 is Asn or Gln;
C1 is Glu or Asp;
D1 is Lys;
E1 is Ile;
F1 is Asn or Gln;
G1 is Gln or Asn;
A2 is Ser;
B2 is Leu;
C2 is Ala, Val, Leu, Ser, or Ile;
D2 is Phe;
E2 is Ile;
F2 is Arg or Lys;
G2 is Lys;
A3 is Ser;
B3 is Asp;
C3 is Glu or Asp;
D3 is Leu;
E3 is Leu;
F3 is His, Arg, or Lys;
G3 is Asn or Gln;
A4 is Val;
B4 is Asn;
C4 is Ala, Leu, Ile, Ser, or Val;
D4 is Gly;
E4 is Lys;
F4 is Ser or Thr; and
G4 is Thr or Ser; wherein
zero to five amino acids at positions corresponding to A0, D0, E0, A1, D1, E1, A2, B2, D2, E2, A3, B3, D3, E3, A4, B4, D4, and E4 are substituted by a conservative amino acid;
two to four amino acids of SEQ ID NO:1 separated by two, three, or six amino acids are replaced with non-natural amino acids with olefinic side chains, and:
if two amino acids of SEQ ID NO:1 are replaced with non-natural amino acids, then the side chains of the two amino acids are joined by an internal staple;
if three amino acids of SEQ ID NO:1 are replaced with non-natural amino acids, then the side chains of the three amino acids are joined by internal staples and/or an internal stitch; or
if four amino acids of SEQ ID NO:1 are replaced with non-natural amino acids, then the side chains of the four amino acids are joined by internal staples, internal stiches, or a combination of internal staples and stiches.

### Claim 2 (depends on 1)

The internally cross-linked peptide of claim 1, wherein:
A0 is Phe,
B0 is Asp,
C0 is Ala or Ser,
D0 is Ser,
E0 is Ile,
F0 is Ser,
G0 is Gln,
A1 is Val,
B1 is Asn,
C1 is Glu,
D1 is Lys,
E1 is Ile,
F1 is Asn,
G1 is Gln,
A2 is Ser,
B2 is Leu,
C2 is Ala,
D2 is Phe,
E2 is Ile,
F2 is Arg,
G2 is Lys,
A3 is Ser,
B3 is Asp,
C3 is Glu,
D3 is Leu,
E3 is Leu,
F3 is His,
G3 is Asn,
A4 is Val,
B4 is Asn,
C4 is Ala or Val,
D4 is Gly,
E4 is Lys,
F4 is Ser, and
G4 is Thr,
wherein two to four amino acids of SEQ ID NO:1 separated by three or six amino acids are replaced with non-natural amino acids with olefinic side chains.

### Claim 3 (depends on 1)

The cross-linked peptide of claim 1, wherein the amino acid sequence is FDASISQVNEKINQSLAFIRKSDELLHNVNAGKST (SEQ ID NO:2), wherein two to four amino acids of SEQ ID NO:2 separated by three or six amino acids are replaced with non-natural amino acids with olefinic side chains.

### Claim 4 (depends on 1)

The cross-linked peptide of claim 1, wherein the internal staple replacing the side chains of the two amino acids separated by two or six amino acids comprises an internal staple selected from Table 1.

### Claim 5 (depends on 4)

The cross-linked peptide of claim 4, wherein the internally cross-linked peptide is selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 14, 15, 16, 17, and 18.

### Claim 6 (depends on 1)

The cross-linked peptide of claim 1, wherein the internal staples and/or the internal stitch replacing the side chains of the three amino acids comprise an internal stitch selected from Table 1.

### Claim 7 (depends on 1)

The cross-linked peptide of claim 1, wherein the internal staples and/or the internal stitch replacing the side chains of the three amino acids comprises at least two internal staples.

### Claim 8 (depends on 1)

The cross-linked peptide of claim 1, wherein the internal staples and/or the internal stitch replacing the side chains of the three amino acids comprises a combination of at least one internal staple and an internal stitch.

### Claim 9 (depends on 6)

The cross-linked peptide of claim 6, wherein the internal stitch replaces the side chain of a first amino acid and a second and a third amino acid thereby cross-linking the first amino acid to the second and third amino acid via an internal cross-link, wherein the first and second amino acid are separated by two, three, or six amino acids, the first and the third amino acids are separated by two, three, or six amino acids, and the second and third amino acids are distinct amino acids.

### Claim 10 (depends on 6)

The cross-linked peptide of claim 6, wherein the internal stitch replacing the side chains of the three amino acids cross-links a pair of amino acids separated by two, three, or six amino acids.

### Claim 11 (depends on 1)

The cross-linked peptide of claim 1, wherein four amino acids of SEQ ID NO:1 are replaced with non-natural amino acids with olefinic side chains, each pair of the four amino acids are separated by three or six amino acids, and wherein the side chains of the four amino acids are joined by two distinct internal staples.

### Claim 12 (depends on 11)

The cross-linked peptide of claim 11, wherein a first of the two distinct internal staples cross-links a first pair of amino acids separated by three or six amino acids, and a second of the at least two distinct internal staples cross-links a second pair of amino acids separated by three or six amino acids.

### Claim 13 (depends on 1)

The cross-linked peptide of claim 1, wherein the internally cross-linked peptide is selected from the group consisting of SEQ ID NOs: 6, 19, 20, 21, 22, 23, 24, 25, and 26.

### Claim 14 (depends on 13)

The cross-linked peptide of claim 13, wherein the peptide comprises FD8′SISQVNX′KINQSLAFI8″KSDELLX″NVNAGKST (SEQ ID NO:20), wherein there is a first internal crosslink between 8′ and X′ and a second internal crosslink between 8″ and X″.

### Claim 15 (depends on 14)

The cross-linked peptide of claim 14, wherein the first cross-link is a C11 alkylene between the alpha carbons of 8′ and X′ and the second cross-link is a C11 alkylene between the alpha carbons of 8″ and X″.

### Claim 16 (depends on 14)

The cross-linked peptide of claim 14, wherein the alpha carbons of 8′, X′, 8″ and X″ are substituted with a methyl group.

### Claim 17 (depends on 1)

The cross-linked peptide of claim 1, wherein the internal staples, internal stiches, or the combination of internal staples and internal stitches replacing the side chains of the at least four amino acids comprises at least one staple and at least one stitch.

### Claim 18 (depends on 17)

The cross-linked peptide of claim 17, wherein the at least one staple cross-links a pair of amino acids separated by two, three, or six amino acids and the at least one stitch cross-links a first amino acid to a second amino acid and a third amino acid, wherein the first and second amino acid are separated by two, three, or six amino acids, the first and the third amino acids are separated by two, three, or six amino acids, and the second and third amino acids are distinct amino acids.

### Claim 19 (depends on 17)

The cross-linked peptide of claim 17, wherein the at least one staple is selected from Table 1.

### Claim 20 (depends on 1)

A method of treating Respiratory Syncytial Virus (RSV) in a human subject, the method comprising:
selecting a human subject at risk of or with RSV infection; and
administering to the human subject an effective amount of the peptide of claim 1.

### Claim 21 (depends on 20)

The method of claim 20, comprising:
assessing a level of RSV or a symptom associated with RSV in the human subject before and after treatment; and
continuing treatment until a decrease in the level of RSV or the symptom associated with RSV in the human subject.

### Claim 22 (depends on 1)

A compound comprising the internally cross-linked polypeptide of claim 1.

### Claim 23 (depends on 22)

The compound of claim 22, wherein the compound comprises polyethylene glycol or spermine.

### Claim 24 (depends on 22)

The compound of claim 22, wherein the polyethylene glycol is linked to the cross-linked polypeptide through a biodegradable linker.

### Claim 25

A peptide of 35 amino acids in length that is at least 80% identical to FDASISQVNEKINQSLAFIRKSDELLHNVNAGKST (SEQ ID NO:2), wherein the peptide comprises a stabilized alpha-helix with non-natural amino acids comprising a hydrocarbon staple between relative positions: (1) i and i+3, (2) i′ and i′+4, (3) i″ and i″+7, or (4) i′ and i′+4 and i″ and i″+7 of SEQ ID NO:2, and wherein the peptide binds recombinant RSV 5-helix bundle protein.

### Claim 26 (depends on 25)

The peptide of claim 25, wherein the amino acids at positions 1, 4, 5, 8, 11, 12, 15, 16, 18, 19, 21, 22, 23, 25, 26, and 29-33 of SEQ ID NO:2 are unaltered.

### Claim 27 (depends on 25)

The peptide of claim 25, wherein the peptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2.

### Claim 28 (depends on 25)

The peptide of claim 25, comprising the amino acid sequence set forth in any one of SEQ ID NOs.: 10-14, 17, or 19-24.

### Claim 29 (depends on 25)

The peptide of claim 25, wherein the peptide binds recombinant RSV 5-helix bundle protein with greater affinity than a peptide of SEQ ID NO:2.

### Claim 30 (depends on 25)

The peptide of claim 25, wherein the non-natural amino acid amino acids are: S5, R8, or S5 and R8.

### Claim 31 (depends on 25)

A method of immunizing a human subject against RSV, the method comprising:
selecting a human subject at risk for RSV infection; and
administering to the human subject an effective amount of the peptide of claim 25.

### Claim 32

A peptide comprising an amino acid sequence that is at least 80% identical to FDASISQVNEKINQSLAFIRKSDELLHNVNAGKST (SEQ ID NO:2), wherein either one or both of the amino acids at positions 21 and 31 of SEQ ID NO: 2 is unaltered, the peptide comprises a stabilized alpha-helix with non-natural amino acids comprising a hydrocarbon staple between relative positions: (1) i and i+3, (2) i′ and i′+4, (3) i″ and i″+7, or (4) i′ and i′+4 and i″ and i″+7 of SEQ ID NO:2, and the peptide binds recombinant RSV 5-helix bundle protein.

### Claim 33 (depends on 32)

The peptide of claim 32, wherein the amino acids at positions 1, 4, 5, 8, 11, 12, 15, 16, 18, 19, 21, 22, 23, 25, 26, and 29-33 of SEQ ID NO:2 are unaltered.

### Claim 34 (depends on 32)

The peptide of claim 32, wherein the peptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2.

### Claim 35 (depends on 32)

The peptide of claim 32, comprising the amino acid sequence set forth in any one of SEQ ID NOs.: 10-14, 17, or 19-24.

### Claim 36 (depends on 32)

The peptide of claim 32, wherein the peptide binds recombinant RSV 5-helix bundle protein with greater affinity than a peptide of SEQ ID NO:2.

### Claim 37 (depends on 32)

The peptide of claim 32, wherein the non-natural amino acid amino acids are: S5, R8, or S5 and R8.