# US 6,037,329 A · Compositions containing nucleic acids and ligands for therapeutic treatment

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- Number: US 6,037,329 A
- Document ID: US6037329A
- Status: Granted
- Kind: A
- Application number: 08/718,904
- Filed: 1996-09-24
- Granted: 2000-03-14
- Earliest claimed: 1994-03-15
- Assignees: Selective Genetics, Inc.
- Inventors: J. Andrew Baird; Lois Ann Chandler; Barbara A. Sosnowski
- Web page: https://patentgrep.com/patent/US6037329A

## CPC

- C07K 14/503: {basic FGF [bFGF]}
- A61K 38/168: {from plants}
- A61K 41/0042: {Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion}
- A61K 47/642: {the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate}
- A61K 47/6425: {the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C07K 14/415: from plants
- C07K 14/475: Growth factors; Growth regulators
- C07K 14/52: Cytokines; Lymphokines; Interferons
- C12N 15/62: DNA sequences coding for fusion proteins
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C07K 2319/00: Fusion polypeptide
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/04: containing an ER retention signal such as a C-terminal HDEL motif
- C07K 2319/09: containing a nuclear localisation signal
- C07K 2319/21: containing a His-tag
- C07K 2319/50: containing protease site
- 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
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C12N 2799/026: where the vector is derived from a baculovirus
- 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

Preparations of conjugates of a receptor-binding internalized ligand and a cytocide-encoding agent and compositions containing such preparations are provided. The conjugates contain a polypeptide that is reactive with an FGF receptor, such as bFGF, or another heparin-binding growth factor, cytokine, or growth factor coupled to a nucleic acid binding domain. One or more linkers may be used in the conjugation. The linker is selected to increase the specificity, toxicity, solubility, serum stability, or intracellular availability, and promote nucleic acid condensation of the targeted moiety. The conjugates are complexed with a cytocide-encoding agent, such as DNA encoding saporin. Conjugates of a receptor-binding internalized ligand to a nucleic acid molecule are also provided.

## Claims

### Claim 1

A gene delivery composition having the formula:
polypeptide that binds to a fibroblast growth factor (FGF) receptor-nucleic acid binding domain-nucleic acid molecule, wherein:
the nucleic acid binding domain being chemically conjugated or fused to the polypeptide that binds to an FGF receptor,
the nuclcic acid molecule being bound to the nucleic acid binding domain; and wherein the gene delivery composition binds to an FGF receptor and is internalized specifically in cells bearing the FGF receptor.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the nucleic acid molecule is a cytocide-encoding agent.

### Claim 3 (depends on 2)

The composition of claim 2 wherein the cytocide encoding agent encodes a ribosome inactivating protein.

### Claim 4 (depends on 3)

The composition of claim 3 wherein the ribosome inactivating protein is saporin.

### Claim 5 (depends on 3)

The composition of claim 3 wherein the ribosome inactivating protein is gelonin.

### Claim 6 (depends on 2)

The composition of claim 2 wherein the cytocide encoding agent encodes a protein that inhibits elongation factor 2.

### Claim 7 (depends on 6)

The composition of claim 6 wherein the protein is diphtheria toxin.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the nucleic acid molecule is a prodrug-encoding agent.

### Claim 9 (depends on 1, 2)

The composition of either of claims 1, 2, or 8 wherein the polypeptide that binds to the FGF receptor is FGF-2 and the nucleic acid binding domain is poly-L-lysine.

### Claim 10 (depends on 1, 2)

The composition of either of claims 1, 2, or 8 wherein the nucleic acid binding domain is a transcription factor.

### Claim 11 (depends on 1, 2)

The composition of either of claims 1, 2, or 8 wherein the nucleic acid binding domain is a protein selected from the group consisting of AP-1, Sp-1, rev, GCN4, .lambda.cro, .lambda.cI, TFIIA, myoD, retinoic acid receptor, glucocosteroid receptor, SV40 large T antigen, and GAL4 polypeptides.

### Claim 12 (depends on 1, 2)

The composition of either of claims 1, 2, or 8, wherein the nucleic binding domain is a polycation.

### Claim 13 (depends on 12)

The composition of claim 12 wherein the polycation is selected from the group consisting of poly-L-lysine, protamine, histone and spermine.

### Claim 14 (depends on 2)

The composition of claim 2 wherein the cytocide-encoding agent further comprises a tissue-specific promoter operably linked thereto.

### Claim 15 (depends on 8)

The composition of claim 8 wherein the prodrug-encoding agent further comprises a tissue-specific promoter operably linked thereto.

### Claim 16 (depends on 14, 15)

The composition of either of claims 14 or 15 wherein the tissue-specific promoter is selected from the group consisting of alpha-crystalline promoter, tyrosinase promoter, .alpha.-fetoprotein promoter, prostate specific antigen promoter, CEA promoter, .alpha.-actin promoter, VEGF receptor promoter, erbB-2 promoter, C-myc promoter, cyclin D promoter, FGF receptor promoter and gamma-crystalline promoter.

### Claim 17 (depends on 1)

The composition of claim 1, further comprising a tissue-specific promoter operably linked thereto.

### Claim 18 (depends on 17)

The composition of claim 17 wherein the tissue specific promoter is a promoter selected from the group consisting of VEGF receptor promoter, tek promoter, tic promoter, urokinase receptor promoter, E-selectin promoter, P-selectin promoter, VCAM-1 promoter, endoglin promoter, endosialin promoter, alpha.sub.v integrin promoter, .beta..sub.3 integrin promoter, endothelin-1 promoter, ICAM-3 promoter, E9 promoter, von Willebrand Factor promoter, CD-44 promoter, CD40 promoter, vascular endothelial cadherin promoter, notch 4 promoter and high molecular weight melanoma-associated antigen promoter.

### Claim 19 (depends on 1, 2)

The composition of either of claims 1, 2, or 8, further comprising between one to six linkers that are selected from the group consisting of a cleavable linker, a linker that provides a sorting signal, a linker that reduces steric hindrance and a linker that contributes to a condensing ability of the nucleic acid binding domain, the addition of said one to six linkers resulting in one of the following formulas:
polypeptide that binds to an FGF receptor-L-nucleic acid binding domain-nucleic acid molecule,
polypeptide that binds to an FGF receptor-L-nucleic acid binding domain-cytocide encoding agent, or the formula:
polypeptide that binds to an FGF receptor-L-nucleic acid binding domain-prodrug encoding agent wherein:
L is one to six linkers; and
wherein the conjugate retains the ability to bind to an FGF receptor and internalize the nucleic acid molecule, cytocide-encoding agent or prodrug encoding agent, and wherein the cytocide-encoding agent is bound to the nucleic acid binding domain.

### Claim 20 (depends on 19)

The composition of claim 19 wherein the linker is selected from the group consisting of a GlySer linker, a SerGly linker, or an AlaAlaProAla (SEQ ID NO: 51) linker.

### Claim 21 (depends on 20)

The composition of claim 20, wherein the linker is encoded by a sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50.

### Claim 22 (depends on 19)

The composition of claim 19 wherein the linker is a disulfide bond.

### Claim 23 (depends on 1, 5, 6, 7, 8, 9, 10, 11)

A method of delivering a nucleic acid molecule to a cell, comprising contacting a cell with the composition according to any one of claims 1 and 5-11, wherein the nucleic acid molecule is internalized in the cell.

### Claim 24

A composition having the formula:
polypeptide that binds to an fibroblast growth factor (FGF) receptor-nucleic acid molecule-nucleic acid binding domain, wherein:
the nucleic acid molecule being conjugated to the polypeptide that binds to an FGF receptor; and wherein the nucleic acid molecule is bound to the nucleic acid binding domain; and wherein the composition binds to an FGF receptor and is internalized specifically in cells bearing the FGF receptor.

### Claim 25 (depends on 1, 2, 8)

The composition of either of claims 1, 2, 8, or 24, wherein the polypeptide that binds to an FGF receptor is selected from the group consisting of an FGF-1 polypeptide, an FGF-2 polypeptide, an FGF-3 polypeptide, an FGF-4 polypeptide, an FGF-5 polypeptide, an FGF-6 polypeptide, an FGF-7 polypeptide, an FGF-8 polypeptide and an FGF-9 polypeptide.

### Claim 26 (depends on 1, 2, 8)

The composition of either of claims 1, 2, 8, or 24, wherein the polypeptide that binds to an FGF receptor is an FGF-2 polypeptide.

### Claim 27 (depends on 26)

The composition of claim 26, wherein FGF-2 of SEQ ID NO: 11 has a serine residue at position 96.

### Claim 28 (depends on 1, 2, 8)

The composition of either of claims 1, 2, 8, or 24, wherein the polypeptide that binds to an FGF receptor is an FGF-7 polypeptide.

### Claim 29 (depends on 19)

The composition of claim 19, wherein the polypeptide that binds to an FGF receptor is selected from the group consisting of an FGF-1 polypeptide, an FGF-2 polypeptide, an FGF-3 polypeptide, an FGF-4 polypeptide, an FGF-5 polypeptide, an FGF-6 polypeptide, an FGF-7 polypeptide, an FGF-8 polypeptide and an FGF-9 polypeptide.

### Claim 30 (depends on 19)

The composition of claim 19, wherein the polypeptide that binds to an FGF receptor is an FGF-2 polypeptide.

### Claim 31 (depends on 30)

The composition of claim 30, wherein FGF-2 of SEQ ID NO:11 has a serine residue at position 96.

### Claim 32 (depends on 19)

The composition of claim 19, wherein the polypeptide that binds to an FGF receptor is an FGF-7 polypeptide.

### Claim 33 (depends on 19)

The composition of claim 19, wherein the cleavable linker is cleavable by a protease.

### Claim 34 (depends on 33)

The composition of claim 33, wherein the protease is selected from the group consisting of cathepsin B, cathepsin D and trypsin.

### Claim 35 (depends on 8)

The composition of claim 8, wherein the prodrug-encoding agent encodes HSV-thymidine kinase or cytosine deaminase.