United States Patent:  6,037,329

Assignee:  Selective Genetics, Inc. (La Jolla, CA)

Filed:  September 24, 1996

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.

SUMMARY OF THE INVENTION

The present invention generally provides therapeutic compositions. In one aspect, the composition has the formula: receptor-binding internalized ligand-nucleic acid binding domain-cytocide-encoding agent. The receptor-binding internalized ligand is a polypeptide reactive with a cell surface receptor, the nucleic acid binding domain binds to a nucleic acid, the cytocide-encoding agent is a nucleic acid molecule encoding a cytocide and which binds to the nucleic acid binding domain, and the composition binds to the cell surface receptor and internalizes the cytocide-encoding agent in cells bearing the receptor. In another aspect, the composition has the formula: receptor-binding internalized ligand-nucleic acid binding domain-prodrug-encoding agent.

In certain embodiments, the receptor-binding internalized ligand is a polypeptide reactive with an FGF receptor, VEGF receptor, HBEGF receptor, or a cytokine. In other embodiments, the cytocide-encoding agent encodes a protein that inhibits protein synthesis and is preferably a ribosome inactivating protein, most preferably saporin. The protein is gelonin or diphtheria toxin in other embodiments. In other embodiments, the prodrug-encoding agent encodes HSV-thymidine kinase.

The nucleic acid binding domain is poly-L-lysine in one embodiment. In other embodiments, the nucleic acid binding domain is a transcription factor selected from the group consisting of helix-turn-helix motif proteins, homeodomain proteins, zinc finger motif proteins, steroid receptor proteins, leucine zipper motif proteins, helix-loop-helix motif proteins, and .beta.-sheet motif proteins. In other embodiments, the nucleic acid binding domain binds nonspecifically to nucleic acids and is selected from the group consisting of poly-L-lysine, protamine, histone and spermine. In a preferred embodiment, the nucleic acid binding domain binds the coding region of a ribosome inactivating protein such as saporin. In another preferred embodiment, FGF is conjugated to poly-L-lysine.

In yet other embodiments, the cytocide-encoding agent contains a tissue-specific promoter, such as alpha-crystalline, gamma-crystalline, .alpha.-fetoprotein, CEA, prostate-specific antigen, erbB-2, tyrosinase, .alpha.-actin, c-myc, VEGF receptor, FGF receptor or cyclin D.

In another aspect, the composition also contains a linker. In various embodiments, the linker increases the flexibility of the conjugate and is (Gly_m Ser_p)_n, (Ala Ala Pro Ala)_n, wherein n is 1 to 6, m is 1 to 6 and p is 1 to 4, or the linker is a disulfide bond.

In yet another aspect, the composition has the formula: receptor-binding internalized ligand-cytocide encoding agent-nucleic acid binding domain, wherein the receptor-binding internalized ligand is conjugated to the cytocide-encoding agent, which is bound to the nucleic acid binding domain to form a complex.

In other aspects, the invention provides methods for preventing excessive cell proliferation in the anterior eye following surgery, treating corneal clouding following excimer laser surgery, preventing closure of a trabeculectomy, preventing pterygii recurrence, treating hyperproliferative diseases in the back of the eye, such as macular degeneration, diabetic retinopathy and proliferative virtreal retinopathy, treating smooth muscle cell hyperplasia after a wound healing response to a procedure, e.g., vein grafting, endarterectomies and arteriovenous shunts and treating cancer. In these aspects, an effective amount of the compositions described above are administered.

Claim 1 of 35 Claims

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.

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