# US 10,766,928 B2 · Targeted conformationally-constrained kinked endosomal disrupting peptides

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- Number: US 10,766,928 B2
- Document ID: US10766928B2
- Status: Granted
- Kind: B2
- Application number: 15/480,120
- Filed: 2017-04-05
- Published: 2017-08-03
- Granted: 2020-09-08
- Earliest claimed: 2012-10-05
- Assignees: University of Kansas
- Applicants: The University of Kansas
- Inventors: Blake R. Peterson
- Web page: https://patentgrep.com/patent/US10766928B2

## CPC

- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- A61K 47/554: {the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid}
- A61K 47/65: Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
- A61K 47/6811: {the drug being a protein or peptide, e.g. transferrin or bleomycin}
- A61K 47/6849: {the antibody targeting a receptor, a cell surface antigen or a cell surface determinant}
- A61K 47/6851: {the antibody targeting a determinant of a tumour cell}
- A61K 47/6889: {Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment}
- C07K 16/2887: {against CD20}
- C07K 16/32: against translation products of oncogenes
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2319/06: containing a lysosomal/endosomal localisation signal
- C07K 2319/74: containing a fusion for binding to a cell surface 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)

## Abstract

A peptide can have a sequence of one of SEQ ID NOs: 78-91. A conformationally-constrained kinked peptide includes: a conformationally-constraining portion and a kinked portion linked to the conformationally-constraining portion that conformationally constrains the kinked portion having a peptide sequence of one of SEQ NOs: 78-97. A cell-targeting compound can include a conformationally-constrained kinked peptide having a peptide sequence of one of SEQ ID NOs: 78-97. The peptide sequence can be one of SEQ ID NOs: 78-97, or 78-91, or 92-97. A cell-targeting compound can include a conformationally-constrained kinked peptide linked to a branched linker with one branch arm linked to a specific targeting moiety and one branch arm linked to a general targeting moiety. The specific targeting moiety can be an antibody. The general targeting moiety can be a lipid or cholesterol derivative.

## Claims

### Claim 1

A peptide sequence comprising:
one of SEQ ID NOs: 92-97.

### Claim 2

A peptide sequence, comprising:
one of SEQ ID NOs: 5-38, 40-54, 61-69, or 78-91.

### Claim 3 (depends on 2)

The peptide sequence of claim 2, comprising:
one of SEQ ID NOs: 78-91.

### Claim 4 (depends on 1)

A cell-targeting compound comprising:
one or more peptides having a conformationally-constraining portion, and a kinked portion linked through a peptide linker to the conformationally-constraining portion that conformationally constrains the kinked portion, the kinked portion, peptide linker and conformationally-constraining portion includes the peptide sequence of claim 1; and
at least one targeting moiety linked to an end of the one or more peptides.

### Claim 5 (depends on 4)

The cell-targeting compound of claim 4, wherein at least one targeting moiety is on the C-terminus of the peptide.

### Claim 6 (depends on 4)

The cell-targeting compound of claim 4, wherein at least one targeting moiety is on the N-terminus of the peptide.

### Claim 7 (depends on 4)

The cell-targeting compound of claim 4, comprising one of Formulae 2-2C, 3-3C, or 4-4C, wherein:
ED-KP is an endosomal-disrupting kinked peptide having one or more amino acids independently selected from proline and glycine;
CC-Peptide includes a peptide having one or more 2-aminoisobutyric acid residues that conformationally-constrains the ED-KP;
Peptide independently includes natural, unnatural, essential or non-essential aromatic, aliphatic, or other amino acids, or having L or D configuration;
Xaa, Xaa1, and Xaa2 are independently one or more natural or non-natural amino acids, essential amino acids, or non-essential amino acids, or amino acids having L or D configuration;
L1 and L2 are independently linkers;
n1 and n3 are independently an integer greater than 0 and less than 50;
n2 and n4 are independently 0-50;
Z1 and Z2 are independently a targeting moiety, cargo moiety, or nothing, wherein at least one is a targeting moiety;
Y1 and Y2 are independently nothing or a linker, or a linker having a cargo moiety; and
X1 and X2 are independently nothing, a coupling group, one or more beta-alanine residues, or a polypeptide,
Formula 2=Z1—Y1—X1—(CC-Peptide)n1-(Peptide)n2-(ED-KP)n3-(Peptide)n4-X2—Y2—Z2;
Formula 2A =Z1—Y1—X1—(CC-Peptide)n1-(L1)n2-(ED-KP)n3-(L2)n4-X2—Y2—Z2;
Formula 2B =Z1—Y1—X1—(CC-Peptide)n1-(Xaa)n2-(ED-KP)n3-(Xaa)n4-X2—Y2—Z2;
Formula 2C =Z1—Y1—X1—(CC-Peptide)n1-(Xaa1)n2-(ED-KP)n3-(Xaa2)n4-X2—Y2—Z2;
Formula 3=Z1—Y1—X1—(CC-Peptide)n1-(Peptide)n2-(ED-KP)n3-(Peptide)n4;
Formula 3A =Z1—Y1—X1—(CC-Peptide)n1-(L1)n2-(ED-KP)n3-(L2)n4;
Formula 3B =Z1—Y1—X1—(CC-Peptide)n1-(Xaa)n2-(ED-KP)n3-(Xaa)n4;
Formula 3C =Z1—Y1—X1—(CC-Peptide)n1-(Xaa1)n2-(ED- KP)n3-(Xaa2)n4;
Formula 4=(CC-Peptide)n1-(Peptide)n2-(ED-KP)n3-(Peptide)n4-X2—Y2—Z2;
Formula 4A =(CC-Peptide)n1-(L1)n2-(ED-KP)n3-(L2)4-X2—Y2—Z2;
Formula 4B =(CC-Peptide)n1-(Xaa)n2-(ED-KP)n3-(Xaa)n4-X2—Y2—Z2; and
Formula 4C =(CC-Peptide)n1-(Xaa1)n2-(ED-KP)n3-(Xaa2)n4-X2—Y2—Z2.

### Claim 8 (depends on 4)

The cell-targeting compound of claim 4, comprising one of Formulae 6-6C, 7-7C, or 8-8C, wherein:
ED-KP is an endosomal-disrupting kinked peptide having one or more amino acids independently selected from proline and glycine;
CCM includes a moiety having one or more 2-aminoisobutyric acid residues that conformationally constrains the ED-KP;
Peptide independently includes natural, unnatural, essential or non-essential aromatic, aliphatic, or other amino acids, or having L or D configuration;
Xaa, Xaa1, and Xaa2 are independently one or more natural or non-natural amino acids, essential amino acids, or non-essential amino acids, or amino acids having L or D configuration;
L1 and L2 are independently linkers;
n1 and n3 are independently an integer greater than 0 and less than 50;
n2 and n4 are independently 0-50;
Z1 and Z2 are independently a targeting moiety, cargo moiety, or nothing, wherein at least one is a targeting moiety;
Y1 and Y2 are independently nothing or a linker, or a linker having a cargo moiety; and
X1 and X2 are independently nothing, a coupling group, one or more beta-alanine residues, or a polypeptide,
Formula 6=Z1—Y1—X1—(CCM)n1-(Peptide)n2-(ED-KP)n3-(Peptide)n4-X2—Y2—Z2;
Formula 6A =Z1—Y1—X1—(CCM)n1-(L1)n2-(ED-KP)n3-(L2)n4-X2—Y2—Z2;
Formula 6B =Z1—Y1—X1—(CCM)n1-(Xaa)n2-(ED-KP)n3-(Xaa)n4-X2—Y2—Z2;
Formula 6C =Z1—Y1—X1—(CCM)n1-(Xaa1)n2-(ED-KP)n3-(Xaa2)n4-X2—Y2—Z2;
Formula 7=Z1—Y1—X1—(CCM)n1-(Peptide)n2-(ED-KP)n3-(Peptide)n4;
Formula 7A =Z1—Y1—X1—(CCM)n1-(L1)n2-(ED-KP)n3-(L2)n4;
Formula 7B =Z1—Y1—X1—(CCM)n1-(Xaa)n2-(ED-KP)n3-(Xaa)n4;
Formula 7C =Z1—Y1—X1—CCM)n1-(Xaa1)n2-(ED-KP)n3-(Xaa2)n4;
Formula 8=(CCM)n1-(Peptide)n2-(ED-KP)n3-(Peptide)n4-X2—Y2—Z2;
Formula 8A =(CCM)n1-(L1)n2-(ED-KP)n3-(L2)n4-X2—Y2—Z2;
Formula 8B =(CCM)n1-(Xaa)n2-(ED-KP)n3-(Xaa)n4-X2—Y2—Z2; and
Formula 8C =(CCM)n1-(Xaa1)n2-(ED-KP)n3-(Xaa2)n4-X2—Y2—Z2.

### Claim 9 (depends on 4)

The cell-targeting compound of claim 4, comprising one of Formulae 10-10C, 11-11C, or 12-12C wherein:
KP and KP1 are independently one or more amino acids independently selected from proline and glycine that can cause the peptide to kink;
Aib is a 2-aminoisobutyric acid residue;
Xaa, Xaa1, and Xaa2 are independently one or more natural or non-natural amino acids, essential amino acids, or non-essential amino acids, or amino acids having L or D configuration;
Z1 and Z2 are independently a targeting moiety, cargo moiety, or nothing, wherein at least one is a targeting moiety;
Y1 and Y2 are independently nothing or a linker, or a linker having a cargo moiety;
X1 and X2 are independently nothing, a coupling group, one or more beta-alanine residues, or a polypeptide; and
n1 and n3 are independently an integer greater than 0 and less than or equal to 50;
n2 n4, n5, n6, and n7 are independently 0-50,
Formula 10=Z1—Y1—X1—(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-X2—Y2—Z2;
Formula 11=Z1—Y1—X1—(Aib)n1-(Xaal)n2-(KP)n3-(Xaa2)n4;
Formula 12=(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-X2—Y2—Z2;
Formula 10A =Z1—Y1—X1—(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-X2—Y2—Z2;
Formula 11A =Z1—Y1—X1—(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5;
Formula 12A =(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-X2—Y2—Z2;
Formula 10B =Z1—Y1—X1—(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-(Xaa3)n6-X2—Y2—Z2;
Formula 11B =Z1—Y1—X1—(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-(Xaa3)n6;
Formula 12B =(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-(Xaa3)n6-X2—Y2—Z2;
Formula 10C =Z1—Y1—X1—(Xaa4)n7-(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-X2—Y2—Z2;
Formula 11C =Z1—Y1—X1—(Xaa4)n7-(Aib)n1-(Xaal)n2-(KP)n3-(Xaa2)n4-(KP1)n5; and
Formula 12C =(Xaa4)7-(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-X2—Y2—Z2.

### Claim 10 (depends on 4)

The cell-targeting compound of claim 4, further comprising:
at least one cargo moiety linked to the one or more peptides.

### Claim 11 (depends on 4)

The cell-targeting compound of claim 4, wherein the at least one targeting moiety is linked to the one or more peptides through a branched linker.

### Claim 12 (depends on 11)

The cell-targeting moiety of claim 11, wherein a first arm of the branched linker is linked to a specific targeting moiety and a second arm of the branched linker is linked to a general targeting moiety.

### Claim 13 (depends on 12)

The cell targeting moiety of claim 12, wherein the specific targeting moiety is specific to a protein or portion thereof that is associated with a cell membrane and the general targeting moiety generally associates with the cell membrane.

### Claim 14 (depends on 13)

The cell targeting moiety of claim 13, wherein the specific targeting moiety is an antibody, or fragment thereof, and the general targeting moiety is a lipid or a cholesterol or cholesterol derivative.

### Claim 15 (depends on 3)

A cell-targeting compound comprising:
one or more peptides having a conformationally-constraining portion, and a kinked portion linked through a peptide linker to the conformationally-constraining portion that conformationally constrains the kinked portion, the kinked portion, peptide linker and conformationally-constraining portion includes the peptide sequence of claim 3; and
at least one targeting moiety linked to an end of the one or more peptides.

### Claim 16 (depends on 15)

The cell-targeting compound of claim 15, wherein at least one targeting moiety is on the C-terminus of the peptide.

### Claim 17 (depends on 15)

The cell-targeting compound of claim 15, wherein at least one targeting moiety is on the N-terminus of the peptide.

### Claim 18 (depends on 15)

The cell-targeting compound of claim 15, comprising one of Formulae 2-2C, 3-3C, or 4-4C, wherein:
ED-KP is an endosomal-disrupting kinked peptide having one or more amino acids independently selected from proline and glycine;
CC-Peptide includes a peptide having one or more 2-aminoisobutyric acid residues that conformationally-constrains the ED-KP;
Peptide independently includes natural, unnatural, essential or non-essential aromatic, aliphatic, or other amino acids, or having L or D configuration;
Xaa, Xaa1, and Xaa2 are independently one or more natural or non-natural amino acids, essential amino acids, or non-essential amino acids, or amino acids having L or D configuration;
L1 and L2 are independently linkers;
n1 and n3 are independently an integer greater than 0 and less than 50;
n2 and n4 are independently 0-50;
Z1 and Z2 are independently a targeting moiety, cargo moiety, or nothing, wherein at least one is a targeting moiety;
Y1 and Y2 are independently nothing or a linker, or a linker having a cargo moiety; and
Y1 and X2 are independently nothing, a coupling group, one or more beta-alanine residues, or a polypeptide,
Formula 2 =Z1—Y1—X1—(CC-Peptide)n1-(Peptide)n2-(ED-KP)n3-(Peptide)n4-X2—Y2—Z2;
Formula 2A =Z1—Y1—X1—(CC-Peptide)1-(L1)n2-(ED-KP)n3-(L2)-X1—Y2—Z2;
Formula 2B =Z1—Y1—X1—(CC-Peptide)n1-(Xaa)n2-(ED-KP)n3-(Xaa)n4-X2—Y2—Z2;
Formula 2C =Z1—Y1—X1—(CC-Peptide)n1-(Xaa1)n2-(ED-KP)n3-(Xaa2)n4-X2—Y2—Z2;
Formula 3=Z1—Y1—X1—(CC-Peptide)n1-(Peptide)n2-(ED-KP)n3-(Peptide)n4;
Formula 3A =Z1—Y1—X1—(CC-Peptide)n1-(L1)n2-(ED-KP)n3-(L2)n4;
Formula 3B =Z1—Y1—X1—(CC-Peptide)n1-(Xaa)n2-(ED-KP)n3-(Xaa)n4;
Formula 3C =Z1—Y1—X1—(CC-Peptide)n1-(Xaa1)n2-(ED-KP)n3-(Xaa2)n4;
Formula 4=(CC-Peptide)n1-(Peptide)n2-(ED-KP)n3-(Peptide)n4-X2—Y2—Z2;
Formula 4A =(CC-Peptide)n1-(L1)n2-(ED-KP)n3-(L1)n4-X2—Y2—Z2;
Formula 4B =(CC-Peptide)n1-(Xaa)n2-(ED-KP)n3-(Xaa)n4-X2—Y2—Z2; and
Formula 4C =(CC-Peptide)n1-(Xaa1)n2-(ED-KP)n3-(Xaa2)n4-X2—Y2—Z2.

### Claim 19 (depends on 15)

The cell-targeting compound of claim 15, comprising one of Formulae 6-6C, 7-7C, or 8-8C, wherein:
ED-KP is an endosomal-disrupting kinked peptide having one or more amino acids independently selected from proline and glycine;
CCM includes a moiety having one or more 2-aminoisobutyric acid residues that conformationally constrains the ED-KP;
Peptide independently includes natural, unnatural, essential or non-essential aromatic, aliphatic, or other amino acids, or having L or D configuration;
Xaa, Xaa1, and Xaa2 are independently one or more natural or non-natural amino acids, essential amino acids, or non-essential amino acids, or amino acids having L or D configuration;
L1 and L2 are independently linkers;
n1 and n3 are independently an integer greater than 0 and less than 50;
n2 and n4 are independently 0-50;
Z1 and Z2 are independently a targeting moiety, cargo moiety, or nothing, wherein at least one is a targeting moiety;
Y1 and Y2 are independently nothing or a linker, or a linker having a cargo moiety; and
X1 and X2 are independently nothing, a coupling group, one or more beta-alanine residues, or a polypeptide,
Formula 6 =Z1—Y1—X1—(CCM)n1-(Peptide)n2-(ED-KP)n3-(Peptide)n4-X2—Y2—Z2;
Formula 6A =Z1—Y1—X1—(CCM)n1-(L1)n2-(ED-KP)n3-(L2)n4-X2—Y2—Z2;
Formula 6B =Z1—Y1—X1—(CCM)n1-(Xaa)n2-(ED-KP)n3-(Xaa)n4-X2—Y2—Z2;
Formula 6C =Z1—Y1—X1—(CCM)n1-(Xaa1)n2-(ED-KP)n3-(Xaa2)n4-X2—Y2—Z2;
Formula 7=Z1—Y1—X1—(CCM)n1-(Peptide)n2-(ED-KP)n3-(Peptide)n4;
Formula 7A =Z1—Y1—X1—(CCM)n1-(L1)n2-(ED-KP)n3-(L2)n4;
Formula 7B =Z1—Y1—X1—(CCM)n1-(Xaa)n2-(ED-KP)n3-(Xaa)n4;
Formula 7C =Z1—Y1—X1-CCM)n1-(Xaa1)n2-(ED-KP)n3-(Xaa2)n4;
Formula 8 =(CCM)n1-(Peptide)n2-(ED-KP)n3-(Peptide)n4-X2—Y2—Z2;
Formula 8A =(CCM)n1-(L1)n2-(ED-KP)n3-(L2)n4-X2—Y2—Z2;
Formula 8B =(CCM)n1-(Xaa)n2-(ED-KP)n3-(Xaa)n4-X2—Y2—Z2; and
Formula 8C =(CCM)n1-(Xaa1)n2-(ED-KP)n3-(Xaa2)n4-X2—Y2—Z2.

### Claim 20 (depends on 15)

The cell-targeting compound of claim 15, comprising one of Formulae 10-10C, 11-11C, or 12-12C wherein:
KP and KP1 are independently one or more amino acids independently selected from proline and glycine that can cause the peptide to kink;
Aib is a 2-aminoisobutyric acid residue;
Xaa, Xaa1, and Xaa2 are independently one or more natural or non-natural amino acids, essential amino acids, or non-essential amino acids, or amino acids having L or D configuration;
Z1 and Z2 are independently a targeting moiety, cargo moiety, or nothing, wherein at least one is a targeting moiety;
Y1 and Y2 are independently nothing or a linker, or a linker having a cargo moiety;
X1 and X2 are independently nothing, a coupling group, one or more beta-alanine residues, or a polypeptide; and
n1 and n3 are independently an integer greater than 0 and less than or equal to 50;
n2 n4, n5, n6, and n7 are independently 0-50,
Formula 10 =Z1—Y1—X1—(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-X2—Y2—Z2;
Formula 11 =Z1—Y1—X1—(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4;
Formula 12 =(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-X2—Y2—Z2;
Formula 10A =Z1—Y1—X1—(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-X2—Y2—Z2;
Formula 11A =Z1—Y1—X1—(Aib)n1-(Xaal)n2-(KP)n3-(Xaa2)n4-(KP1)n5;
Formula 12A =(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-X2—Y2—Z2;
Formula 10B =Z1—Y1—X1—(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-(Xaa3)n6-X2—Y2—Z2;
Formula 11B =Z1—Y1—X1—(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-(Xaa3)n6;
Formula 12B =(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-(Xaa3)n6-X2—Y2—Z2;
Formula 10C =Z1—Y1—X1—(Xaa4)n7-(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-X2—Y2—Z2;
Formula 11C =Z1—Y1—X1—(Xaa4)n7-(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5; and
Formula 12C =(Xaa4)n7-(Aib)n1-(Xaa1)n2-(KP)n3-(Xaa2)n4-(KP1)n5-X2—Y2—Z2.

### Claim 21 (depends on 15)

The cell-targeting compound of claim 15, further comprising:
at least one cargo moiety linked to the one or more peptides.

### Claim 22 (depends on 15)

The cell-targeting compound of claim 15, wherein the at least one targeting moiety is linked to the one or more peptides through a branched linker.

### Claim 23 (depends on 22)

The cell-targeting moiety of claim 22, wherein a first arm of the branched linker is linked to a specific targeting moiety and a second arm of the branched linker is linked to a general targeting moiety.

### Claim 24 (depends on 23)

The cell targeting moiety of claim 23, wherein the specific targeting moiety is specific to a protein or portion thereof that is associated with a cell membrane and the general targeting moiety generally associates with the cell membrane.

### Claim 25 (depends on 24)

The cell targeting moiety of claim 24, wherein the specific targeting moiety is an antibody, or fragment thereof, and the general targeting moiety is a lipid or cholesterol derivative.