Title:  Conjugates targeted to the interleukin-2 receptor

United States Patent:  6,251,866

Assignee:  Watson Laboratories, Inc. (Salt Lake City, UT)

Filed:  August 4, 1998

A composition for intracellular delivery of a chemical agent into an interleukin-2-receptor-bearing cell, e.g. an activated T cell, includes a chemical agent and at least one copy of an interleukin-2-receptor-binding and endocytosis-inducing ligand coupled to a water soluble polymer. The ligand binds to a receptor on the interleukin-2-receptor-bearing cell and elicits endocytosis of the composition. The composition also preferably includes a spacer for coupling the chemical agent and the ligand to the polymer. Chemical agents can include cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like. A preferred water soluble polymer is a polyalkylene oxide, such as polyethlene glycol and polyethylene oxide, and activated derivatives thereof. The composition can further comprise a carrier such as another water soluble polymer, liposome, or particulate. Methods of using these compositions for delivering a chemical agent in vivo or in vitro are also disclosed. A method of detecting a disease, such as T-cell lymphocytic leukemia, T-cell acute lymphoblastic leukemia, peripheral T-cell lymphoma, Hodgkin's disease, or non-Hodgkin's lymphoma, associated with elevated levels of soluble IL-2 receptor is also disclosed.

BRIEF SUMMARY OF THE INVENTION

It is an object of the present invention to provide compositions for intracellular delivery of selected chemical agents to a specific cell type, i.e. IL-2-receptor-bearing cells.

It is also an object of the invention to provide methods of making and methods of using compositions for delivery of selected chemical agents into IL-2-receptor-bearing cells.

It is another object of the invention to provide compositions and methods for delivering selected chemical agents into IL2-receptor-bearing cells using water soluble polymers that are inexpensive, FDA-approved, and resistant to development of an antibody response.

It is yet another object of the invention to provide compositions and methods of use thereof for delivery of selected chemical agents into activated T cells.

It is still another object of the invention to providing compositions and methods of use thereof for detecting a disease associated with elevated levels of soluble IL-2 receptor in body fluids, such as serum.

These and other objects are achieved by providing a composition for delivery of a chemical agent into an IL-2-receptor bearing cell, the composition comprising (a) a water-soluble, biocompatible polymer, (b) the chemical agent covalently, releasably coupled to the polymer, and (c) a ligand comprising an IL-2-receptor-binding peptide covalently coupled to the polymer.

In a preferred embodiment of the invention, the composition has a formula selected from the group consisting of P--[T_a --L--S--A]_c and [A--S]_d --P--[T_a --L]_c, wherein L is the ligand; A is the chemical agent; S and T are spacers, wherein at least S is biodegradable and S and T can be the same or different; P is the water soluble polymer having functional groups compatible with forming covalent bonds with the ligand; a is 0 or 1; and c and d are integers of at least 1.

Preferably, P is a polyalkylene oxide. Preferred polyalkylene oxides are selected from the group consisting of alpha-substituted polyalkylene oxide derivatives, polyethylene glycol (PEG) homopolymers and derivatives thereof, polypropylene glycol homopolymers and derivatives thereof, alkyl-capped polyethylene oxides, bis-polyethylene oxides, copolymers of poly(alkylene oxides), and block copolymers of poly(alkylene oxides) or activated derivatives thereof. Preferably, the polyalkylene oxide has a molecular weight of about 200 to about 50,000. More preferably, the polyalkylene oxide has a molecular weight of about 2,000 to about 20,000. Most preferably, the polyalkylene oxide has a molecular weight of about 20,000. Especially preferred polyalkylene oxides are polyethylene glycol and polyethylene oxide.

The IL-2-receptor-binding peptide is preferably a member selected from the group consisting of SEQ ID NO:1 and biologically functional equivalents thereof. More preferably, the IL-2-receptor-binding peptide is a member selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 11, and SEQ ID NO:24 through SEQ ID NO:47.

The chemical agent is preferably selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, and drugs.

Preferably, the spacer comprises a peptide. A preferred peptide spacer comprises Gly-Phe-Leu-Gly (SEQ ID NO:21).

In one preferred embodiment, the composition further comprises a carrier selected from the group consisting of other water soluble polymers, liposomes, and particulates. Preferably, such water soluble polymers are selected from the group consisting of dextran, inulin, poly(L-lysine) with modified epsilon amino groups, poly(L-glutamic acid), and N-substituted methacrylamide-containing polymers and copolymers.

A method of delivering a chemical agent in vitro into an IL-2-receptor-bearing cell in a population of cells comprises the steps of:

(a) providing a composition comprising (i) a water-soluble, biocompatible polymer, (ii) the chemical agent covalently, releasably coupled to the polymer, and (iii) a ligand comprising an IL-2-receptor-binding peptide covalently coupled to the polymer; and

(b) contacting the population of cells with an effective amount of the composition under conditions wherein the ligand binds to an IL-2 receptor on the IL-2-receptor-bearing cell and elicits endocytosis of the composition.

A method of delivering a chemical agent into an IL-2-receptor-bearing cell in a warm-blooded animal, comprises the steps of:

(a) providing a composition comprising (i) a water-soluble, biocompatible polymer, (ii) the chemical agent covalently, releasably coupled to the polymer, and (iii) a ligand comprising an IL-2-receptor-binding peptide covalently coupled to the polymer; and

(b) systemically administering to the warm-blooded animal an effective amount of the composition under conditions wherein the ligand contacts and binds to an IL-2 receptor on the IL-2-receptor-bearing cell and elicits endocytosis of the composition.

Another aspect of the invention relates to a composition comprising a peptide selected from the group consisting of SEQ ID NO:2 through SEQ ID NO: 11 and SEQ ID NO:22 through SEQ ID NO:47 and amides thereof.

A method for detecting a disease associated with elevated levels of soluble interleukin-2 receptor in circulation comprises the steps of:

(a) providing a composition comprising an IL-2-receptor-binding peptide;

(b) mixing the composition with a body fluid to be tested under conditions suitable for binding of the composition to said soluble interleukin-2 receptor in the body fluid to form a complex; and

(c) detecting the complex and determining whether the complex is present at elevated levels as compared to normal individuals.

Diseases that can be detected according to this method include T-cell lymphocytic leukemia, T-cell acute lymphoblastic leukemia, peripheral T-cell lymphoma, Hodgkin's disease, and non-Hodgkin's lymphoma. Preferably, the body fluid that is tested is serum. Detection of the complex of peptide and soluble interleukin-2 receptor preferably comprises an enzyme-linked or radio-linked sorbent assay. IL-2 receptor-binding peptides that are suitable for this process include SEQ ID NO: 1 through SEQ ID NO: 11 and SEQ ID NO:22 through SEQ ID NO:47 and amides thereof. Especially preferred peptides are SEQ ID NO:27 and the amide thereof. As is well known in the art, such an amide is generally formed by reaction of an acid chloride of the peptide with ammonia, resulting in replacement of the -OH group of the C-terminal carboxylic acid with -NH₂.

Claim 1 of 27 Claims

We claim:

1. A composition for delivery of a chemical agent into an IL2-receptor bearing cell, the composition comprising (a) a water-soluble, biocompatible polymer, (b) the chemical agent covalently, releasably coupled to the polymer, and (c) at least one copy of a ligand comprising an IL-2-receptor-binding peptide selected from the group consisting of SEQ ID NO:22 through SEQ ID NO:47, covalently coupled to the polymer.

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