# US 10,308,684 B2 · Cell penetrating stapled peptide, manufacturing method therefor, and use thereof

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- Number: US 10,308,684 B2
- Document ID: US10308684B2
- Status: Granted
- Kind: B2
- Application number: 15/529,967
- Filed: 2015-11-27
- Published: 2017-11-30
- Granted: 2019-06-04
- Earliest claimed: 2014-11-28
- Assignees: Seoul National University R&Db Foundation
- Applicants: Seoul National University R&Db Foundation
- Inventors: Jaehoon Yu; Soonsil Hyun; Yan Lee; Seonju Lee
- Web page: https://patentgrep.com/patent/US10308684B2

## CPC

- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 38/10: Peptides having 12 to 20 amino acids {(A61K38/043 - A61K38/046 take precedence)}
- A61K 38/12: Cyclic peptides {, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C (A61K38/043 - A61K38/046 take precedence)}
- A61K 47/42: Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein (oligopeptides having up to five amino acids {A61K47/183}; polyamino acids A61K47/34)
- A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65)
- A61K 47/6455: {Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids}
- C07K 1/1075: {by covalent attachment of amino acids or peptide residues}
- C07K 1/1077: {by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids}
- C07K 1/113: without change of the primary structure
- C07K 14/001: {by chemical synthesis}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- 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 2310/14: interfering nucleic acids [NA]
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to a stapled peptide, a preparation method thereof and the use thereof, and more specifically to an amphipathic alpha-helical stapled peptide comprising hydrophobic amino acids and hydrophilic amino acids, a preparation method thereof, and the use thereof for intracellular delivery of an active substance.

## Claims

### Claim 1

A method for intracellular delivery of a biologically active substance, which comprises delivering a composition comprising an amphipathic alpha-helical stapled peptide comprising hydrophobic amino acids and hydrophilic amino acids, wherein the stapled peptide comprises any one of the following sequences:
(SEQ ID NO: 1)
X1KKLLKLX2KKLLKLAG,
(SEQ ID NO: 2)
LKKX1LKLLKKX2LKLAG,
(SEQ ID NO: 3)
LKKLX1KLLKKLX2KLAG,
(SEQ ID NO: 4)
LKKLLKX1LKKLLKX2AG,
(SEQ ID NO: 5)
LKKLLKLX1KKLLKLX2G,
(SEQ ID NO: 6)
LKKLLKLLX1KLLKLAX2,
(SEQ ID NO: 7)
LKHLLHLX1KHLLKLX2G,
and
(SEQ ID NO: 8)
CLKKLLKLX1KKLLKLX2G,
wherein X1 is (R)-2-(7′-octenyl) alanine, and X2 is (S)-2-(4′-pentenyl) alanine.

### Claim 2 (depends on 1)

The method of claim 1, wherein the stapled peptide further comprises cysteine at an N-terminus or C-terminus thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein the biologically active substance is DNA, RNA, siRNA, an aptamer, a protein, an antibody, or a low-molecular weight compound.

### Claim 4 (depends on 1)

The method of claim 1, wherein the biologically active substance and the stapled peptide are contained in the composition at a molar ratio of 1:2 to 1:100.

### Claim 5

An amphipathic alpha-helical stapled peptide comprising any one of the following sequences:
(SEQ ID NO: 1)
X1KKLLKLX2KKLLKLAG,
(SEQ ID NO: 2)
LKKX1LKLLKKX2LKLAG,
(SEQ ID NO: 3)
LKKLX1KLLKKLX2KLAG
(SEQ ID NO: 4)
LKKLLKX1LKKLLKX2AG,
(SEQ ID NO: 5)
LKKLLKLX1KKLLKLX2G,
(SEQ ID NO: 6)
LKKLLKLLX1KLLKLAX2,
(SEQ ID NO: 7)
LKHLLHLX1KHLLKLX2G,
and
(SEQ ID NO: 8)
CLKKLLKLX1KKLLKLX2G,
wherein X1 is (R)-2-(7′-octenyl) alanine, and X2 is (S)-2-(4′-pentenyl) alanine.

### Claim 6 (depends on 5)

The amphipathic alpha-helical stapled peptide of claim 5, further comprising cysteine at an N-terminus or C-terminus of the peptide.