# US 5,703,039 A · Chimeric toxins

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- Number: US 5,703,039 A
- Document ID: US5703039A
- Status: Granted
- Kind: A
- Application number: 08/483,726
- Filed: 1995-06-07
- Granted: 1997-12-30
- Earliest claimed: 1990-03-02
- Assignees: The University Hospital
- Inventors: Diane P. Williams; John R. Murphy
- Web page: https://patentgrep.com/patent/US5703039A

## CPC

- C12N 15/62: DNA sequences coding for fusion proteins
- A61P 35/00: Antineoplastic agents
- C07K 14/34: from Corynebacterium (G)
- C07K 14/485: Epidermal growth factor [EGF], i.e. urogastrone
- C07K 14/5406: {IL-4}
- C07K 14/5412: {IL-6}
- C07K 14/55: IL-2
- 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)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/55: containing a fusion with a toxin, e.g. diphteria toxin
- C07K 2319/75: containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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 chimeric toxin comprising protein fragments joined together by peptide bonds, the chimeric toxin comprising, in sequential order, beginning at the amino terminal end of the chimeric toxin, (a) the enzymatically active Fragment A of diphtheria toxin, (b) a first fragment including the cleavage domain 1.sub.1 adjacent the Fragment A of diphtheria toxin, (c) a second fragment comprising at least a portion of the hydrophobic transmembrane region of Fragment B of diphtheria toxin, the second fragment having a deletion of at least 50 diphtheria toxin amino acid residues, the deletion being C-terminal to the portion of the transmembrane region, and the second fragment not including domain 1.sub.2, and (d) a third fragment comprising a portion of a cell-specific polypeptide ligand, the portion including at least a portion of the binding domain of the polypeptide ligand, the portion of the binding domain being effective to cause the chimeric toxin to bind selectively to a predetermined class of cells to be attacked by the enzymatically active Fragment A, the chimeric toxin possessing any of, greater toxicity than that of a toxin comprised of DAB.sub.486 fused to the third fragment, a lower K.sub.d for the sites on cells of the predetermined class to which the chimeric toxin binds than that of a toxin comprised of DAB.sub.486 fused to the third fragment, greater resistance to proteolytic degradation than that exhibited by a toxin comprised of DAB.sub.486 fused to the third fragment, greater resistance to the inhibition of its cytotoxicity by the cell-specific polypeptide ligand than that exhibited by DAB.sub.486 fused to the third fragment, the ability to inhibit protein synthesis to a given degree by a period of exposure that is shorter than the period of exposure required by DAB.sub.489 fused to the third fragment to inhibit protein synthesis to the same degree, or the ability to effect a more rapid onset of the inhibition of protein synthesis than that exhibited by DAB.sub.486 fused to the third fragment.

## Claims

### Claim 1

A pharmaceutical composition, comprising a therapeutically effective amount of a chimeric toxin which binds selectively to a predetermined class of cells, said chimeric toxin comprising protein fragments joined together by peptide bonds, said chimeric toxin comprising, sequentially from N-terminus to C-terminus,
(a) a first fragment which is the enzymatically active fragment A of native diphtheria toxin and the 11 cleavage domain of native diphtheria toxin;
(b) a second fragment comprising at least a portion of the hydrophobic transmembrane region of native diphtheria toxin effective to deliver said fragment A into the cytosol of the predetermined class of cells;
(c) a third fragment comprising the sequence of native diphtheria toxin fragment B amino acids C-terminal to the hydrophobic transmembrane region of native diphtheria toxin which is amino acids 372-535, minus the generalized eucaryotic binding domain of native diptheria toxin which is amino acids 486-535, and minus the 1.sub.2 cleavage domain of native diphtheria toxin which is amino acids 461-471, and further minus at least 50 native diphtheria toxin amino acids between amino acid residue 386 of native diphtheria toxin and the generalized eucaryotic binding site of native diphtheria toxin, provided that the 1.sub.2 domain and the at least 50 amino acids deleted total no more than 99 amino acids; and
(d) a fourth fragment comprising at least a portion of the binding domain of a cell-specific polypeptide ligand effective to cause said chimeric toxin to bind selectively to the predetermined class of cells which bear a receptor to the ligand;
wherein said chimeric toxin possesses greater toxicity to the predetermined class of cells than that of a toxin comprising DAB.sub.486 fused to said fourth fragment, and
a pharmaceutically acceptable carrier.

### Claim 2 (depends on 1)

The pharmaceutical composition of claim 1, wherein said third fragment comprises the sequence of native diphtheria toxin fragment B amino acids C-terminal to the hydrophobic transmembrane region of native diphtheria toxin which is amino acids 372-535, minus the generalized eucaryotic binding domain of native diphtheria toxin which is amino acids 486-535, and minus the 1.sub.2 cleavage domain of native diphtheria toxin which is amino acids 461-471, and further minus at least 80 native diphtheria toxin amino acids between amino acid residue 386 of native diphtheria toxin and the generalized eucaryotic binding site of native diphtheria toxin, provided that the 1.sub.2 domain and the at least 80 amino acids deleted total no more than 99 amino acids.

### Claim 3 (depends on 1)

The pharmaceutical composition of claim 1, wherein said third fragment further comprises amino acid residues His.sub.484 and Ala.sub.485 of native diphtheria toxin.

### Claim 4 (depends on 1)

The pharmaceutical composition of claim 1, wherein said chimeric toxin lacks any diphtheria toxin amino acids C-terminal to amino acid residue 386 of native diphtheria toxin.

### Claim 5 (depends on 1)

The pharmaceutical composition of claim 1, wherein the length and composition of said third fragment render said chimeric toxin at least about four times as toxic to the predetermined class of cells as that of a toxin comprising DAB.sub.486 fused to said fourth fragment.

### Claim 6 (depends on 1)

A pharmaceutical composition of claim 1, wherein the length and composition of said third fragment render said chimeric toxin at least about 10 times as toxic to the predetermined class of cells as that of a toxin comprising DAB.sub.486 fused to said fourth fragment.

### Claim 7 (depends on 1)

A pharmaceutical composition of claim 1, wherein said fourth fragment comprises a portion of the binding domain of EGF effective to cause said chimeric toxin to bind selectively to the predetermined class of cells which bear a receptor to EGF.

### Claim 8 (depends on 7)

A pharmaceutical composition of claim 7, wherein said fourth fragment comprises EGF.

### Claim 9 (depends on 1)

A pharmaceutical composition of claim 1, wherein said fourth fragment comprises at least a portion of the binding domain of IL-2 effective to cause said chimeric toxin to bind selectively to the predetermined class of cells which bear a receptor to IL-2.

### Claim 10 (depends on 9)

A pharmaceutical composition of claim 9, wherein said fourth fragment comprises amino acids 2 to 133 of human IL-2.

### Claim 11 (depends on 1)

A pharmaceutical composition of claim 1, wherein said fourth fragment comprises at least a portion of the binding domain of IL-4 effective to cause said chimeric toxin to bind selectively to the predetermined class of cells which bear a receptor to IL-4.

### Claim 12 (depends on 11)

A pharmaceutical composition of claim 11, wherein said fourth fragment comprises IL-4.

### Claim 13 (depends on 1)

A pharmaceutical composition of claim 1, wherein said fourth fragment comprises at least a portion of the binding domain of IL-6 effective to cause said chimeric toxin to bind selectively to the predetermined class of cells which bear a receptor to IL-6.

### Claim 14 (depends on 13)

A pharmaceutical composition of claim 13, wherein said fourth fragment comprises IL-6.

### Claim 15

A pharmaceutical composition, comprising a therapeutically effective amount of a chimeric toxin which binds selectively to a predetermined class of cells, said chimeric toxin comprising protein fragments joined together by peptide bonds, said chimeric toxin comprising, sequentially from N-terminus to C-terminus,
(a) a first fragment which is the enzymatically active fragment A of native diphtheria toxin and the 1.sub.1 cleavage domain of native diphtheria toxin;
(b) a second fragment comprising at least a portion of the hydrophobic transmembrane region of native diphtheria toxin effective to deliver said fragment A into the cytosol of the predetermined class of cells;
(c) a third fragment comprising the sequence of native diphtheria toxin fragment B amino acids C-terminal to the hydrophobic transmembrane region of native diphtheria toxin which is amino acids 372-535, minus the generalized eucaryotic binding domain of native diphtheria toxin which is amino acids 486-535, and minus a sequence of from 61 to 99 native diphtheria toxin amino acids, which includes the 1.sub.2 region of native diphtheria toxin which is amino acids 461-471, the deleted sequence being N-terminal with respect to the generalized eucaryotic binding site of native diphtheria toxin and C-terminal with respect to amino acid residue 386 of native diphtheria toxin; and
(d) a fourth fragment comprising at least a portion of the binding domain of a cell-specific polypeptide ligand effective to cause said chimeric toxin to bind selectively to the predetermined class of cells which bear a receptor to said ligand;
wherein said chimeric toxin possesses greater toxicity to the predetermined class of cells than that of a toxin comprising DAB.sub.486 fused to said fourth fragment, and
a pharmaceutically acceptable carrier.

### Claim 16 (depends on 15)

A pharmaceutical composition of claim 15, wherein said third fragment further comprises amino acid residues His.sub.484 and Ala.sub.485 of native diphtheria toxin.

### Claim 17 (depends on 16)

A chimeric toxin according to claim 16, wherein the deleted sequence of from 61 to 97 native diphtheria toxin amino acids is immediately N-terminal to amino acid residue His.sub.484 of native diphtheria toxin.

### Claim 18 (depends on 17)

A pharmaceutical composition of claim 17, wherein the sequence deleted contains from 80 to 97 native diphtheria toxin amino acids.

### Claim 19 (depends on 18)

A pharmaceutical composition of claim 18, wherein the deleted sequence contains 97 amino acids.

### Claim 20 (depends on 19)

A pharmaceutical composition of claim 19, wherein said first, second, and third fragments together consist of DAB.sub.389.

### Claim 21 (depends on 15)

A pharmaceutical composition of claim 15, wherein said chimeric toxin lacks any diphtheria toxin amino acids C-terminal to amino acid residue 386 of native diphtheria toxin.

### Claim 22 (depends on 15)

A pharmaceutical composition of claim 15, wherein the length and composition of said third fragment render said chimeric toxin at least about four times as toxic to the predetermined class of cells as that of a toxin comprising DAB.sub.486 fused to said fourth fragment.

### Claim 23 (depends on 15)

A pharmaceutical composition of claim 15, wherein the length and composition of said third fragment render said chimeric toxin at least about 10 times as toxic to the predetermined class of cells as that of a toxin comprising DAB.sub.486 fused to said fourth fragment.

### Claim 24 (depends on 15)

A pharmaceutical composition of claim 15, wherein said fourth fragment comprises a portion of the binding domain of EGF effective to cause said chimeric toxin to bind selectively to the predetermined class of cells which bear a receptor to EGF.

### Claim 25 (depends on 24)

A pharmaceutical composition of claim 24, wherein said fourth fragment comprises EGF.

### Claim 26 (depends on 15)

A pharmaceutical composition of claim 15, wherein said fourth fragment comprises at least a portion of the binding domain of IL-2 effective to cause said chimeric toxin to bind selectively to the predetermined class of cells which bear a receptor to IL-2.

### Claim 27 (depends on 26)

A pharmaceutical composition of claim 26, wherein said fourth fragment comprises amino acids 2 to 133 of human IL-2.

### Claim 28 (depends on 15)

A pharmaceutical composition of claim 15, wherein said fourth fragment comprises at least a portion of the binding domain of IL-4 effective to cause said chimeric toxin to bind selectively to the predetermined class of cells which bear a receptor to IL-4.

### Claim 29 (depends on 28)

A pharmaceutical composition of claim 28, wherein said fourth fragment comprises IL-4.

### Claim 30 (depends on 15)

A pharmaceutical composition of claim 15, wherein fourth fragment comprises at least a portion of the binding domain of IL-6 effective to cause said chimeric toxin to bind selectively to the predetermined class of cells which bear a receptor to IL-6.

### Claim 31 (depends on 30)

A chimeric toxin according to claim 30, wherein said fourth fragment comprises IL-6.

### Claim 32

A pharmaceutical composition, comprising a therapeutically effective amount of the chimeric toxin DAB.sub.389 -IL-2(2-133), and a pharmaceutically acceptable carrier.

### Claim 33

A pharmaceutical composition, comprising a therapeutically effective amount of the chimeric toxin DAB.sub.389 -EGF, and a pharmaceutically acceptable carrier.

### Claim 34 (depends on 1, 15, 32, 33)

A pharmaceutical composition as in any one of claims 1, 15, 32 or 33, which is in the form of an injectable composition.

### Claim 35 (depends on 1, 15, 32, 33)

A pharmaceutical composition as in any one of claims 1, 15, 32 or 33, which is in the form of a timed release implant.

### Claim 36 (depends on 1, 15, 32, 33)

A pharmaceutical composition as in any one of claims 1, 15, 32 or 33 which is in the form of a topical cream.

### Claim 37 (depends on 1, 15, 32)

A method of treating a mammal suffering from a medical disorder characterized by the presence of an unwanted class of cells, comprising the step of administering a pharmaceutical composition as in any one of claims 1, 15, 32, or 33, to the mammal, such that the chimeric toxin will selectively bind to said cells.

### Claim 38 (depends on 37)

The method of claim 37, wherein the mammal is a human.

### Claim 39 (depends on 37)

The method of claim 37, wherein the medical disorder is cancer.