# US 10,308,926 B2 · Stablized EZH2 peptides

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

- Number: US 10,308,926 B2
- Document ID: US10308926B2
- Status: Granted
- Kind: B2
- Application number: 14/772,143
- Filed: 2014-03-13
- Published: 2016-03-10
- Granted: 2019-06-04
- Earliest claimed: 2013-03-15
- Assignees: Dana-Farber Cancer Institute, Inc.
- Applicants: Dana-Farber Cancer Institute, Inc.
- Inventors: Loren D. Walensky; Gregory H. Bird; Woojin Kim; Stuart Orkin
- Web page: https://patentgrep.com/patent/US10308926B2

## CPC

- C12N 9/96: Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
- C07K 14/47: from mammals
- C12N 9/1007: {Methyltransferases (general) (2.1.1.)}
- 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)
- C12Y 201/01043: Histone-lysine N-methyltransferase (2.1.1.43)
- 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)
- 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)
- C12Y: ENZYMES

## Abstract

Provided herein are polypeptides containing stabilized therapeutic peptides related to enhancer of zeste homolog 2 (EZH2), histone lysine N-methyltransferase. Also provided are compositions containing these polypeptides and methods of using such peptides in the treatment of cancer that include administering to a subject one of the polypeptides.

## Claims

### Claim 1

An internally cross-linked polypeptide that inhibits the interaction between Enhancer of Zeste Homolog 2 (EZH2) and Embryonic Ectoderm Development (EED), the internally cross-linked polypeptide comprising the amino acid sequence FSSNRX1KILX2RTEILNQEWKQRRIQPV (SEQ ID NO:26), wherein X1 and X2 are each a non-natural amino acid with an olefinic side chain, and wherein the internally cross-linked polypeptide is cell penetrant.

### Claim 2 (depends on 1)

The internally cross-linked polypeptide of claim 1, wherein X1 and X2 are each (S)-2-(4′-pentenyl) alanine (S5).

### Claim 3 (depends on 2)

The internally cross-linked polypeptide of claim 2, wherein the internally cross-linked polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 26.

### Claim 4 (depends on 3)

A pharmaceutical composition comprising the internally cross-linked polypeptide of claim 3 and a pharmaceutically acceptable carrier.

### Claim 5 (depends on 4)

The pharmaceutical composition of claim 4, further comprising an inhibitor of EZH2 enzymatic activity.

### Claim 6 (depends on 5)

The pharmaceutical composition of claim 5, wherein the inhibitor of EZH2 enzymatic activity is GSK126.

### Claim 7 (depends on 1)

A method of treating an EZH2-overexpressing or EZH2-dependent cancer in a human subject in need thereof, the method comprising administering to the human subject an effective amount of the internally cross-linked polypeptide of claim 1.

### Claim 8 (depends on 7)

The method of claim 7, further comprising administering to the human subject an inhibitor of EZH2 enzymatic activity.

### Claim 9

An internally cross-linked polypeptide that inhibits the interaction between Enhancer of Zeste Homolog 2 (EZH2) and Embryonic Ectoderm Development (EED), the internally cross-linked polypeptide comprising the amino acid sequence FSSNRX1KILX2RTQILNQEWKQRRIQPV (SEQ ID NO:27), wherein X1 and X2 are each a non-natural amino acid with an olefinic side chain, and wherein the internally cross-linked polypeptide is cell penetrant.

### Claim 10 (depends on 9)

The internally cross-linked polypeptide of claim 9, wherein X1 and X2 are each (S)-2-(4′-pentenyl) alanine (S5).

### Claim 11 (depends on 10)

The internally cross-linked polypeptide of claim 10, wherein the internally cross-linked polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 27.

### Claim 12 (depends on 11)

A pharmaceutical composition comprising the internally cross-linked polypeptide of claim 11, and a pharmaceutically acceptable carrier.

### Claim 13 (depends on 12)

The pharmaceutical composition of claim 12, further comprising an inhibitor of EZH2 enzymatic activity.

### Claim 14 (depends on 13)

The pharmaceutical composition of claim 13, wherein the inhibitor of EZH2 enzymatic activity is GSK126.

### Claim 15 (depends on 10)

A method of treating an EZH2-overexpressing or EZH2-dependent cancer in a human subject in need thereof, the method comprising administering to the human subject an effective amount of the internally cross-linked polypeptide of claim 10.

### Claim 16 (depends on 15)

The method of claim 15, further comprising administering to the human subject an inhibitor of EZH2 enzymatic activity.

### Claim 17 (depends on 16)

The method of claim 16, wherein the inhibitor of EZH2 enzymatic activity is GSK126.

### Claim 18 (depends on 15)

The method of claim 15, wherein the cancer is a leukemia, a breast cancer, a prostate cancer, a sarcoma, or a neuroblastoma.

### Claim 19 (depends on 12)

A method of treating an EZH2-overexpressing or EZH2-dependent cancer in a human subject in need thereof, the method comprising administering to the human subject an effective amount of the pharmaceutical composition of claim 12.

### Claim 20 (depends on 19)

The method of claim 19, further comprising administering to the human subject an inhibitor of EZH2 enzymatic activity.

### Claim 21 (depends on 20)

The method of claim 20, wherein the inhibitor of EZH2 enzymatic activity is GSK126.

### Claim 22 (depends on 19)

The method of claim 19, wherein the cancer is a leukemia, a breast cancer, a prostate cancer, a sarcoma, or a neuroblastoma.

### Claim 23

An internally cross-linked polypeptide that inhibits the interaction between Enhancer of Zeste Homolog 2 (EZH2) and Embryonic Ectoderm Development (EED), the internally cross-linked polypeptide comprising the amino acid sequence FSSNRX1KILX2RTX3ILNX4EWKQRRIQPV (SEQ ID NO: 56), wherein X1, X2, X3, and X4 are each a non-natural amino acid with an olefinic side chain, and wherein the internally cross-linked polypeptide is cell penetrant.

### Claim 24 (depends on 23)

The internally cross-linked polypeptide of claim 23, wherein X1, X2, X3, and X4 are each (S)-2-(4′-pentenyl) alanine (S5).

### Claim 25 (depends on 23)

The internally cross-linked polypeptide of claim 23, wherein the internally cross-linked polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 56.

### Claim 26 (depends on 25)

A pharmaceutical composition comprising the internally cross-linked polypeptide of claim 25 and a pharmaceutically acceptable carrier.

### Claim 27 (depends on 26)

The pharmaceutical composition of claim 26, further comprising an inhibitor of EZH2 enzymatic activity.

### Claim 28 (depends on 27)

The pharmaceutical composition of claim 27, wherein the inhibitor of EZH2 enzymatic activity is GSK126.

### Claim 29 (depends on 23)

A method of treating an EZH2-overexpressing or EZH2-dependent cancer in a human subject in need thereof, the method comprising administering to the human subject an effective amount of the internally cross-linked polypeptide of claim 23.

### Claim 30 (depends on 29)

The method of claim 29, further comprising administering to the human subject an inhibitor of EZH2 enzymatic activity.

### Claim 31 (depends on 30)

The method of claim 30, wherein the inhibitor of EZH2enzymatic activity is GSK126.

### Claim 32

An internally cross-linked polypeptide that inhibits the interaction between Enhancer of Zeste Homolog 2 (EZH2) and Embryonic Ectoderm Development (EED), the internally cross-linked polypeptide comprising an amino acid sequence set forth in FSSNRX1KILX2RTEILNQEWKQRRIQPV (SEQ ID NO:26), except that one or more amino acids at positions 2, 3, 9, 13, 14, 17, 21 or 23 of SEQ ID NO: 26 are substituted, and wherein X1, and X2, are each a non-natural amino acid with an olefinic side chain, and wherein the internally cross-linked polypeptide is cell penetrant.

### Claim 33 (depends on 32)

The internally cross-linked polypeptide of claim 32, wherein the amino acids at positions 13 and 17, are each substituted with a non-natural amino acid with an olefinic side chain.

### Claim 34 (depends on 32)

The internally cross-linked polypeptide of claim 32, wherein the internally cross-linked polypeptide consists of an amino acid sequence set forth in SEQ ID NO:26, but with 1 to 6 amino acid substitutions at positions selected from the group consisting of positions: 2, 3, 9, 13, 14, 17, 21, and 23, of SEQ ID NO: 26.

### Claim 35 (depends on 32)

The internally cross-linked polypeptide of claim 32, wherein the internally cross-linked polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 26 but with 1 to 4 amino acid substitutions at positions 2, 3, 9, 13, 14, 17, 21 or 23.

### Claim 36 (depends on 32)

The internally cross-linked polypeptide of claim 32, wherein the internally cross-linked polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 26 but with 1 amino acid substitution at position 2, 3, 9, 13, 14, 17, 21 or 23.

### Claim 37 (depends on 32)

The internally cross-linked polypeptide of claim 32, wherein the non-natural amino acid is S5 [(S)-2-4′-pentenyl) alanine].

### Claim 38 (depends on 32)

A pharmaceutical composition comprising the internally cross-linked polypeptide of claim 32 and a pharmaceutically acceptable carrier.

### Claim 39 (depends on 38)

The pharmaceutical composition of claim 38, further comprising an inhibitor of EZH2 enzymatic activity.

### Claim 40 (depends on 39)

The pharmaceutical composition of claim 39, wherein the inhibitor of EZH2 enzymatic activity is GSK126.

### Claim 41 (depends on 32)

A method of treating an EZH2-overexpressing or EZH2-dependent cancer in a human subject in need thereof, the method comprising administering to the human subject an effective amount of the internally cross-linked polypeptide of claim 32.

### Claim 42 (depends on 41)

The method of claim 41, further comprising administering to the human subject an inhibitor of EZH2 enzymatic activity.

### Claim 43 (depends on 42)

The method of claim 42, wherein the inhibitor of EZH2 enzymatic activity is GSK126.