Method and composition for the treatment of cancer by the enzymatic conversion of soluble radioactive toxic precipitates in the cancer   

This application is a Continuation of U.S. Ser. No. 10/898,585, filed Jul. 23, 2004 which is a Continuation-in-part of U.S. Ser. No. 10/226,288, filed Aug. 22, 2002 which is a Divisional of U.S. Ser. No. 09/314,422, filed May 18, 1999, now U.S. Pat. No. 6,468,503, issued on Oct. 22, 2002 which is a Divisional of U.S. Ser. No. 08/782,219, filed Jan. 13, 1997, now U.S. Pat. No. 6,080,383, issued on Jun. 27, 2000, the contents of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to the treatment of cancer.

BACKGROUND OF THE INVENTION

A considerable portion of worldwide research efforts in the treatment of cancer is currently devoted to killing cancer cells by means of various cell-killing agents. Despite the fact that numerous drugs, including radioactive compounds, have been shown to be capable of killing cancer cells, these agents frequently fail to treat cancer successfully because of their inability to circumvent three universally present obstacles: (1) the agents do not kill all the cancer cells because they do not exhibit cytotoxic specificity for all the cancer cells, (2) the agents also kill normal cells because they do not exhibit cytotoxic specificity exclusively for cancer cells, and (3) the agents are not potent enough at tolerable doses to kill resistant cancer cells or to overcome the ability of cancer cells to adapt and become resistant to the cell-killing agents.

SUMMARY

The invention provides compositions and methods for treating cancer. The methods of the invention are a multi-step therapy process that directs localized supra-lethal doses of radiation called Hot-Spots to virtually any cancer.

In one aspect the invention provides a Step 1 Reagent containing a cell targeting agent linked, e.g., covalently to a platform building material. The platform building material detaches from the cell targeting agent upon uptake of the reagent into a cell, e.g., a cancer cell. The platform building material once detached from the cell targeting agent becomes aqueous insoluble, forming a nano-platform. Optionally, the cell targeting agent is linked to the platform building material by a carrier moiety. In various aspects of the invention, the platform building material has an additional molecular structure that is capable of specifically binding a second reagent, i.e., a Step 3 Reagent.

A cell targeting agent augments cellular uptake of the reagent and is a polypeptide, a cell surface ligand, a peptide, or a small molecule. A polypeptide is, for example, an antibody such as an EGF receptor antibody or a transferrin receptor antibody, epidermal growth factor or a viral protein such as a human immunodeficiency virus (HIV) 1 TAT protein, a functionally effective portion of (HIV) 1 TAT protein, or VP22. A cell surface ligand is for example transferrin, epidermal growth factor or an interleukin.

A peptide is, for example, a peptide hormone such as oxytocin, growth hormone releasing hormone, glucagon, gastrin, secretin, somatostatin, prolactin, follicle stimulating hormone, insulin, growth hormone, or an arginine-glycine-aspartic acid peptide (RGD).

A small molecule is, for example, a hormone such as estrogen, calciferol, or testosterone, a nucleic acid, a peptidomimetic, a carbohydrate, a lipid, a nicotinic acetylcholine receptor agonist or folic acid or analogue or derivative thereof.

The platform building material is, for example, an indoxyl, a porphyrin, a polymer such as a HPMA derivative, a dendrimer, an opio-melanin or a polysaccharide such as dextran, gum Arabic, cellulose or chitin. The indoxyl is, for example, a substituted indoxyl, i.e., a mono-indoxyl, a bis-indoxyl or a poly indoxyl. The indoxyl forms indigo, a linear indigo polymer or a polyindigo lattice.

A carrier moiety is, for example, a protein; a polysaccharide; a polymer, e.g., synthetic polymer or a biopolymer such as polylysine; a dendrimer; a liposome; a nanoparticle; or a polymeric micelle.

Exemplary Step 1 Reagents include the following: An anti-EGF receptor antibody, derivative or fragment thereof linked to a substituted 3-indoxyl phosphate derivative. The antibody is linked to the 3-indoxyl phosphate derivative by a carrier moiety such as dextran. Additionally, a UDP-N-acetylglucosamine enolpyruvoyltransferase inhibitor such as a phosphoenol pyruvate derivative is linked to the 3-indoxyl phosphate derivative.

A transferrin polypeptide or fragment thereof linked to a glycoside, e.g., a galactoside, a glucoside or a glucuronide or derivative thereof. Preferably, the glycoside is a substituted bis-3-indoxyl glycoside derivative. The transferrin polypeptide is linked to the glycoside by a carrier moiety such as an albumin polypeptide or fragment thereof. Additionally, a mutant β-lactamase inhibitor is linked to the bis-3-indoxyl glycoside derivative. The mutant β-lactamase inhibitor is a lactam derivative such as a carbacephem analog. A carbacephem analog is, for example, Loracarbef.

A folate derivative linked to a porphyrin derivative. The folate derivative is linked to the porphyrin derivative by a carrier moiety such as an immunoglobulin polypeptide or fragment thereof. Additionally, an ornithine decarboxylase inhibitor, e.g., an α-difluoromethylornithine or an arginine decarboxylase inhibitor, e.g., an α-difluoromethylarginine is linked to the porphyrin derivative.

A folate derivative linked to a substituted bis-3-indoxyl galactoside derivative. Additionally, a mutant β-lactamase inhibitor is linked to the substituted bis-3-indoxyl galactoside derivative.

An epidermal growth factor polypeptide or fragment thereof linked to HPMA. Additionally, a substituted indoxyl galactoside derivative and a mutant β-lactamase inhibitor are linked to the HPMA.

Another aspect of the invention provides a Step 3 Reagent that is a bi-specific reagent containing a targeting moiety and an isotope trapping moiety. The targeting moiety and the isotope trapping moiety are linked, e.g., covalently. The targeting moiety is capable of binding the nano-platform. For example, the targeting moiety binds to the additional molecular structures on the nano-platform. The isotope trapping moiety is capable of trapping a radio-labeled aqueous soluble Step 4 Reagent.

The targeting moiety or the isotope trapping moiety is an organic functional group such as a hydrazide, a ketone, a mercaptan, or a maleimidyl; a polypeptide; a peptide; or a lectin. The polypeptide is an enzyme such as a β-lactamase, an arginine decarboxylase, an ornithine decarboxylase, a chloramphenicol acetyltransferase, or a UDP-N-acetylglucosamine enolpyruvoyltransferase; a mutant enzyme such as a mutant β-lactamase; or an antibody or a fragment thereof.

Exemplary Step 3 Reagents include the following: A UDP-N-acetylglucosamine enolpyruvoyltransferase linked to Streptavidin. A mutant β-lactamase linked to a β-D-galactosidase. An ornithine decarboxylase or an arginine decarboxylase linked to 4-carboxybenzaldehyde. A mutant β-lactamase linked to an anti-NIP antibody. A mutant β-lactamase linked to an alkaline phosphatase.

Another aspect of the invention provides a kit packaged in one or more containers containing a Step 1 Reagent and a Step 3 Reagent. Optionally, the kit contains a Step 2 cell-killing Reagent and/or a radiolabeled aqueous soluble Step 4 Reagent. Exemplary Step 4 Reagents include, 90Y-biotin-pentyl-DOTA, 131I-5-iodo-3-indoxyl galactoside, 131I-p-iodobenzoic hydrazide, 131I-4-hydroxy-3-iodo-5-nitrophenylacetic acid and 131I-5-iodo-3-indoxyl phosphate.

Cancer is treated or a symptom of cancer is alleviated, by administering to the subject (a) a Step 1 Reagent containing a cell targeting agent linked, e.g., covalently to a platform building material; (b) a Step 3 Reagent containing a targeting moiety and an isotope trapping moiety; and (c) a radiolabeled aqueous soluble Step 4 Reagent. The cell targeting agent augments cellular uptake of the Step 1 Reagent. The platform building material detaches from the cell targeting agent upon uptake of the Step 1 Reagent into the cell and forms an aqueous insoluble nano-platform to which the targeting moiety of the Step 3 Reagent binds. The isotope trapping moiety of the Step 3 Reagent traps the radiolabeled aqueous soluble Step 4 Reagent within the tumor extracellular matrix for the required period of time to create micro-regional radiation fields (Hot Spots) to deliver lethal irradiation to the surrounding tumor cells.

The reagents are administered sequentially. Alternatively, the reagents are administered concurrently. Optionally, a Step 2 cell-killing Reagent is administered to the subject prior to, after or concurrently with the Step 3 Reagent to relocate the nano-platform into the tumor extracellular matrix.

In one aspect, a cancer is treated or a symptom of cancer is alleviated, by administering to the subject (a) a composition containing an anti-EGF receptor antibody, derivative or fragment thereof linked to a substituted 3-indoxyl phosphate derivative with an UDP-N-acetylglucosamine enolpyruvoyltransferase inhibitor linked to the 3-indoxyl phosphate derivative; (b) a composition containing a UDP-N-acetylglucosamine enolpyruvoyltransferase linked to Streptavidin; and (c) a composition containing 90Y-biotin-pentyl-DOTA.

In another aspect, a cancer is treated or a symptom of cancer is alleviated, by administering to the subject (a) a composition containing a transferrin polypeptide or fragment thereof linked to a substituted bis-3-indoxyl glycoside derivative with a mutant β-lactamase inhibitor linked to the bis-3-indoxyl glycoside derivative; (b) a composition containing a mutant β-lactamase linked to a β-D-galactosidase; and (c) a composition containing 131I-5-iodo-3-indoxyl galactoside.

In a further aspect, a cancer is treated or a symptom of cancer is alleviated, by administering to the subject (a) a composition containing a folate derivative linked to a porphyrin derivative with either an ornithine decarboxylase inhibitor or arginine decarboxylase inhibitor linked to the porphyrin derivative; (b) a composition containing an ornithine decarboxylase or arginine decarboxylase linked to 4-carboxybenzaldehyde; and (c) a composition containing 131I-p-iodobenzoic hydrazide.

In yet another aspect, a cancer is treated or a symptom of cancer is alleviated, by administering to the subject (a) a composition containing a folate derivative linked to a substituted bis-3-indoxyl galactoside derivative with a mutant β-lactamase inhibitor linked to the bis-3-indoxyl galactoside derivative; (b) a composition containing a mutant β-lactamase linked to an anti-NIP antibody; and (c) a composition containing 131I-4-hydroxy-3-iodo-5-nitrophenylacetic acid (131I-NIP acid).

In another aspect, a cancer is treated or a symptom of cancer is alleviated, by administering to the subject (a) a composition containing an epidermal growth factor (EGF) polypeptide or fragment thereof linked to HPMA with a substituted indoxyl galactoside derivative linked to the HPMA and a mutant β-lactamase inhibitor linked to the HPMA; (b) a composition containing a β-lactamase linked to an alkaline phosphatase; and (c) a composition containing 131I-5-iodo-3-indoxyl phosphate.

The subject is a mammal such as human, a primate, mouse, rat, dog, cat, cow, horse, pig, and ferret. The subject is suffering from cancer. The cancer is for example breast cancer, skin cancer, prostate cancer, lung cancer, colon cancer, liver cancer, cervical cancer, brain cancer, ovarian cancer, pancreatic cancer, or stomach cancer. A subject suffering from cancer is identified by methods known in the art such as physical examination; blood test for specific cancer antigens such as PSA; MRI; x-ray; or mammography. Symptoms of cancer include fatigue; nausea; frequent urination; weight loss; lump or thickening in the breast or testicles; a change in a wart or mole; a skin sore or a persistent sore throat that doesn\'t heal; a change in bowel or bladder habits; a persistent cough or coughing blood; constant indigestion or trouble swallowing; unusual bleeding or vaginal discharge; flu-like symptoms; bruising; dizziness; drowsiness; abnormal eye movements or changes in vision.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration depicting a cancer cell with receptors.

FIG. 2 is an illustration depicting a Step 1 Reagent.

FIG. 3 is an illustration depicting the accumulation of Step 1 Reagent in cancer cells.

FIG. 4 is an illustration depicting the formation of aqueous insoluble nano-platform in cancer cells.

FIG. 5 is an illustration depicting the continued accumulation of the nano-platform in cancer cells.

FIG. 6 is an illustration depicting the Step 1 Reagent for the first example of a Step 1 Reagent.

FIG. 7 is an illustration depicting the synthesis of Bromo-indoxyl phosphate with linker molecule.

FIG. 8 is an illustration depicting the synthesis of platform building material with irreversible enzyme inhibitor for the first example of a Step 1 Reagent.

FIG. 9 is an illustration depicting conjugating the platform building materials for the first example of a Step 1 Reagent.

FIG. 9b Step 1 Reagent for the first example of a Step 1 Reagent.

FIG. 10 is an illustration depicting the Step 1 Reagent for the second example.

FIG. 11 is an illustration depicting the synthesis of Bis-indoxyl for the platform building materials for the second example of a Step 1 Reagent.

FIG. 12 is an illustration depicting the synthesis of platform building material with irreversible enzyme inhibitor for the second example of a Step 1 Reagent.

FIG. 13 is an illustration depicting conjugating the platform building materials for the second example of a Step 1 Reagent.

FIG. 13b Step 1 Reagent for the second example of a Step 1 Reagent.

FIG. 14 is an illustration depicting the Step 1 Reagent for the third example of a Step 1 Reagent.

FIG. 15 is an illustration depicting the synthesis of a porphyrin-derivative for the platform building materials for the third example of a Step 1 Reagent.

FIG. 16 is an illustration depicting the synthesis of platform building material with irreversible enzyme inhibitor for the third example of a Step 1 Reagent.

FIG. 17 is an illustration depicting the Step 1 Reagent for the third example of a Step 1 Reagent.

FIG. 18 is an illustration depicting the synthesis of irreversible enzyme inhibitor derivative for the third example of a Step 1 Reagent.

FIG. 19 is an illustration depicting the Step 1 Reagent for the fourth example of a Step 1 Reagent.

FIG. 20 is an illustration depicting the synthesis of the platform building materials with cell targeting agent attached for the fourth example of a Step 1 Reagent.

FIG. 21 is an illustration depicting the synthesis of platform building material with cell targeting agent and position for the irreversible enzyme inhibitor for the fourth example of a Step 1 Reagent.

FIG. 22 is an illustration depicting synthesis of the Step 1 Reagent for the fourth example of a Step 1 Reagent.

FIG. 23 is an illustration depicting the Step 1 Reagent for the fifth example of a Step 1 Reagent.

FIG. 24 is an illustration depicting the synthesis of the Step 1 Reagent for the fifth example of a Step 1 Reagent.

FIG. 25 is an illustration depicting the Step 2 cell-killing process.

FIG. 26 is an illustration depicting the Step 3 Bispecific Reagent.

FIG. 27 is an illustration depicting the formation of the hydrazone anchoring the Step 3 Bispecific Reagent to the nano-platform.

FIG. 28 is an illustration depicting the formation of the thioether anchoring the Step 3 Bispecific Reagent to the nano-platform.

FIG. 29 is an illustration depicting the Step 3 Bispecific Reagent covalently bound to irreversible enzyme inhibitor.

FIG. 30 is an illustration depicting the Step 3 Bispecific Reagent bound to the nano-platform via a specific antibody.

FIG. 31 is an illustration depicting the Step 3 Bispecific Reagent binding a Step 4 Reagent that is a hydrazide.

FIG. 32 is an illustration depicting the Step 3 Bispecific Reagent binding a Step 4 Reagent that is an irreversible enzyme inhibitor.

FIG. 33 is an illustration depicting the Step 3 Bispecific Reagent binding a Step 4 Reagent via a high affinity receptor.

FIG. 34 is an illustration depicting the Step 3 Bispecific Reagent which has an enzyme as its isotope trapping moiety that converts an indoxyl galactoside to an indigo derivative

FIG. 35 is an illustration depicting the synthesis of the Step 3 Reagent composed of UDP-N-acetylglucosamine enolpyruvoyltransferase and Streptavidin.

FIG. 36 is an illustration depicting the preparation of plasmid for the β-lactamase mutants.

FIG. 37 is an illustration depicting the preparation of the plasmid for the Step 3 Reagent, mutant β-lactamase-β-D-galactosidase.

FIG. 38 is an illustration depicting the preparation of Step 3 Bispecific Reagent, ornithine decarboxylase with aldehyde sidechains (i.e. ornithine decarboxylase-4-carboxybenzaldehyde).

FIG. 39 is an illustration depicting the preparation of Step 3 Bispecific Reagent, mutant β-lactamase-anti-NIP antibody.

FIG. 40 is an illustration depicting the preparation of Step 3 Bispecific Reagent, mutant β-lactamase-alkaline phosphatase.

FIG. 41 is an illustration depicting the preparation of first example of a Step 4 Reagent.

FIG. 42 is an illustration depicting the preparation of 90Y-biotin-pentyl-DOTA to be used as a Step 4 Reagent.

FIG. 43 is an illustration depicting the Preparation of second example of a Step 4 Reagent.

FIG. 44 is an illustration depicting the preparation of 131I-5-Iodo-3-indoxyl galactoside to be used as a Step 4 Reagent.

FIG. 45 is an illustration depicting the preparation of third example of a Step 4 Reagent.

FIG. 46 is an illustration depicting the preparation of 131I-p-iodobenzoic hydrazide to be used as a Step 4 Reagent.

FIG. 47 is an illustration depicting the preparation of fourth example of a Step 4 Reagent.

FIG. 48 is an illustration depicting the reparation of 131I-4-hydroxy-3-iodo-5-nitrophenylacetic acid (131I-NIP acid) to be used as a Step 4 Reagent.

FIG. 49 is an illustration depicting the preparation of fifth example of a Step 4 Reagent.

FIG. 50 is an illustration depicting the preparation of 131I-5-Iodo-3-indoxyl phosphate to be used as a Step 4 Reagent.

DETAILED DESCRIPTION

The present invention provides compositions and methods for treating a heterogeneous population of cancer cells in a subject by the delivery of local irradiation. The present invention is based in part on the observation of the highly successful treatment of thyroid cancer with radio-iodide. The successful treatment of thyroid cancer is due in part to the fact that many malignant thyroid cells have a unique biological function that allows them to trap iodine. Thus, when a patient with thyroid cancer is treated with radio-iodide, a sufficient fraction of the cancer cells takes up sufficient quantities of the radioisotope and stores the radioisotope long enough to generate overlapping micro-regions of intense radiation (referred to as “Hot-Spots”) in which all the cells in each micro-region are killed. The radiation field in each of these Hot-Spots extends beyond the cells that take up the radioisotope and kills thousands of neighboring cells. Inside these Hot-Spots, the radiation is so intense that all of the cancer cells in the Hot-Spots are killed, including the cells that do not take up the radioisotope, allowing eradication of the entire tumor. No other tissue or group of cells in the body has this same iodine trapping mechanism, thus Hot-Spots are generated exclusively in the normal and malignant thyroid tissue. The method and compositions of the present invention reproduces these radioisotope delivery and trapping conditions for non-thyroid cancers. The generation of “Hot-Spots” in non-thyroid cancers is a multi-step process that generates overlapping Hot-Spots virtually exclusively in the tumors without causing significant systemic toxicity. All cancer cells within these overlapping Hot-Spots are eradicated. The eradicated cells include cancer cells that are not targeted, cancer cells that are resistant and even super-resistant, and cancer cells that would otherwise adapt and become resistant to therapy. Accordingly, the methods of the invention are not defeated by the heterogeneity of cancer cells and the imperfect nature of current cancer targeting agents.

As shown in FIG. 1, cancer contains a population of cancer cells 100 each having internalizing structures 101 which are specific to cancer cells and capable of binding a cell targeting agent. The internalizing structures 101 are capable of internalization when the targeting agent binds to them. Subpopulations of the targeted cancer cells also have a high sensitivity to being killed by the natural system of the subject and/or a high sensitivity to being killed by an administered cell-killing process.

Cancer is treated, or a symptom of cancer is alleviated by administering to a subject multiple reagents in a plurality of steps. All types of cancers are suitable for treatment. Cancers to be treated include for example lung cancer, colon cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, bladder cancer, skin cancer (e.g., melanoma), ovarian cancer, cervical cancer, head and neck cancer, hematological cancers, lung cancer, colon/rectal/anal cancer, cervical cancer, brain cancer, ovarian cancer, stomach cancer, kidney cancer, uterine cancer, bone cancer, esophageal cancer, eye cancer, Kaposi\'s sarcoma, laryngeal cancer, lip cancer, nasopharyngeal cancer, oropharyngeal cancer, oral cavity cancer, testicular cancer, thyroid cancer, sarcomas, lymphomas, adrenocortical cancer, bile duct cancer, bronchial cancer, cancer of unknown primary, gallbladder cancer, germ cell cancer, hypopharyngeal cancer, islet cell cancer, mesothelioma, multiple myeloma, nasal cavity cancer, paranasal sinus cancer, parathyroid cancer, penile cancer, pituitary cancer, salivary gland cancer, small intestine cancer, thymus cancer, ureter cancer, urethral cancer, vaginal cancer, vulvar cancer, and Wilm\'s tumor.

The subject is a mammal. The mammal is, e.g., a human, non-human primate, mouse, rat, dog, cat, horse, or cow. The steps are administered sequentially. Optionally, one or more steps are administered prior to or concurrently with another. Each step is administered at least once. Alternatively, each step is administered 2, 3, 4, 5, 10, 15 or more times or in a continuous infusion. For example, a Step 2 Reagent is administered in multiple doses using standard therapeutic protocols known in the art. The subject is administered a reagent containing a cell targeting agent which augments cellular uptake of the reagent linked to a platform building material (referred to herein as a Step 1 Reagent); an optional cell-killing reagent (referred to herein as a Step 2 Reagent); a bi-specific reagent comprising a targeting moiety capable of binding to the aqueous insoluble nano-platform and an isotope trapping moiety (referred to herein as a Step 3 Reagent); and a radiolabeled aqueous soluble reagent (referred to herein as a Step 4 Reagent).

As shown in FIG. 2, the Step 1 Reagent 1000 comprises cell targeting agent 1100, an optional carrier moiety 1200, and platform building material 1300 with optionally attached additional molecular structures 1400. As shown in FIG. 3, the cell targeting agent portion of the Step 1 Reagent 1100 attaches to the targeted internalizing structure of the cancer cells 101, thereby permitting the Step 1 Reagent 1000 to be transported inside the cancer cells 100. Transport inside the cancer cells results in the Step 1 Reagent being exposed to the intracellular environment. As illustrated in FIG. 4, once inside the targeted cell, the intracellular environment causes the platform building material 1300 with an optionally attached additional molecular structure 1400 to detach from the targeting agent 1100 and the carrier moiety 1200, thereby enabling the platform building material 1300 to be converted into an aqueous insoluble nano-platform 1500 inside the targeted cancer cells. The aqueous insoluble nano-platform 1500 (with or without additional molecular structures 1400) is stable inside the targeted cancer cells and is relatively non-toxic. By stable it is meant that the nano-platform remains trapped in the cancer cell or surrounding extracellular matrix for a 1, 2, 3, 4, 5, 6 or more days to 1, 2, 3, 4 or more weeks. Relatively non-toxic is meant that the nano-platform has no significant deleterious effect on the subject, for example, moderate or minimal inflammation and/or no life threatening effect on the subject. The aqueous insoluble nano-platform with or with out additional molecular structures is referred to herein as the “nano-platform.”

Accumulation of the intracellular nano-platforms is achieved by continuing the administration of the Step 1 Reagent into the subject, resulting in more platform building material transported into the targeted cancer cells (See, FIG. 5). In contrast to soluble chemicals or drugs, the intracellular nano-platform accumulates over time because it is aqueous insoluble and stable and thus does not leave the targeted cancer cell.

As shown in FIG. 25, following the accumulation of the nano-platform in targeted cancer cells, the subject is optionally administered a Step 2 cell-killing Reagent 75. The Step 2 cell-killing Reagent is capable of killing some or all of the targeted cancer cells, causing the nano-platform 1500 to be relocated and retained into the extracellular space of the tumor. Once in the extracellular space the additional molecular structures 1400 on the surface of the nano-platform 1600 are accessible to bind the Step 3 Bispecific Reagent. The Step 2 cell-killing Reagent is optional as the on-going natural killing of cancer cells by the natural immune system of the body or the genetic instability of the cancer cell causing the cells to die spontaneously may be sufficient to relocate enough intracellular nano-platform to the extracellular space of the tumors to ultimately create sufficient numbers of Hot-Spots to destroy the entire tumors. The cancer specificity of the location of the Hot-Spots is enhanced by the application of such very low levels of the Step 2 Reagent that few, if any, normal cells are killed, and systemic toxicity is avoided.

The fourth step includes administering a radiolabeled aqueous soluble Step 4 Reagent that is adapted to carry radioisotopes to the extracellular tumor matrix where they are trapped and retained by the Step 3 Bispecific Reagent. This creates micro-regional radiation fields that deliver lethal irradiation to the surrounding tumor cells.

Although, in many instances, a rest period of 24 to 48 hours between steps will allow for extensive clearance of the previously administered reagent, optionally, prior to administering a reagent of a succeeding step a clearing agent is administered to facilitate the removal of any excess reagent. For example, prior to administering the Step 2 cell-killing Reagent and the Step 3 Bispecific Reagent a clearing agent is administered to facilitate removal of any non-endocytosed Step 1 Reagent. Similarly, prior to administering the Step 4 Reagent, a clearing agent is administered to facilitate removal of any Step 3 Bispecific Reagent that has not bound to the extracellular nano-platform. Clearing agents assist in the recognition of the therapeutic reagents by the subject\'s macrophages or increase processing by hepatocytes. Clearing agents are known in the art. Clearing agents include mannosylated or galactosylated agents that bind to the Step 1 or Step 3 Reagent. Additional clearing agents include antibodies that are generated against a Step 1 or a Step 3 Reagent to augment opsonization of the reagent by macrophages or other lymphoid cells. Alternatively, an extracorporeal circulation is established using an affinity column to remove these reagents.

The Step 1 Reagent is an aqueous soluble compound containing a cell targeting agent linked to a platform building material.

The cell targeting agent is any compound that directs a compound in which it is present to a desired cellular destination. The cell targeting agent is capable of being internalized into a cell. The cell targeting agent binds specifically to an endocytosing receptor or other internalizing unit on a tumor cell. For example, the cell targeting agent is a compound that is not typically endocytosed but is internalized by the process of cross-linking and capping. Thus, the cell targeting agent directs the compound across the plasma membrane, e.g., from outside the cell, through the plasma membrane, and into the cytoplasm. Alternatively, or in addition, the cell targeting agent can direct the compound to a desired location within the cell, e.g., the nucleus, the ribosome, the endoplasmic reticulum, a lysosome, or a peroxisome. Cell targeting agents include, polypeptides such as antibodies; viral proteins such as human immunodeficiency virus (HIV) 1 TAT protein or VP22; cell surface ligands; peptides such as peptide hormones; or small molecules such as hormones or folic acid. Optimally, the receptor for the cell targeting agent is expressed at a higher concentration on a tumor cell compared to a normal cells. For example, the receptor is expressed at a 2, 3, 4, 5, or more-fold higher concentration on a tumor cell compared to a non-tumor cell.

The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. Such antibodies include, polyclonal, monoclonal, chimeric, single chain, Fab and F(ab′)2 fragments, and an Fab expression library or polypeptides engineered therefrom. Suitable antibodies include antibodies to well characterized receptors such as the transferrin receptor (TfR) and the epidermal growth factor receptor (EGFR) as well as antibodies to other receptors, such as for example the interleukin 4 receptor (IL-4R), the insulin receptor, CD30, CD34, and the CCK-A,B, C/Gastrin receptor. Additionally, the antibody is specific for mucin epitopes; glycopeptides and glycolipids, such as the Ley-related epitope (which is present on the majority of human cancers of the breast, colon and lung); the hyaluronan receptor/CD44; the BCG epitope; integrin receptors; the JL-1 receptor; GM1 or other lipid raft-associated molecules; and GD2 on melanomas. Tumor-specific internalizing human antibodies are also selected from phage libraries as described by Poul, et al. (J. Mol. Biol. 301: 1149-1161, 2000).

A cell surface ligand is a natural ligand or some synthetic analog adapted to be specific for an internalizing structure on the targeted cancer cells. Exemplary cell surface ligands include transferrin, epidermal growth factor, interleukins, integrins, angiotensin II, insulin, growth factor antagonist, β-2-adrenergic receptor ligands or dopamine releasing protein. For example, epidermal growth factor (EGF) is used to target the epidermal growth factor receptor (EGFR) or transferrin (Tf) is used to target the transferrin receptor (e.g. TfR and TfR2).

Suitable peptide cell targeting agents include peptide hormones such as oxytocin, growth hormone-releasing hormone, somatostatin, glucagon, gastrin, secretin, growth hormone (somatotropin), insulin, prolactin, follicle stimulating hormone or arginine-glycine-aspartic acid (RGD) peptides. Methods to identify peptides that bind to internalizing receptors and are internalized are known in the art (Hart, et al., J. Biol. Chem. 269: 12468-12474, 1994).

Cell targeting agents include small molecules. A “small molecule” as used herein, is meant to refer to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules are, e.g., nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. For example, a small molecule is a hormone, such as estrogen, testosterone, and calciferol; folic acid or an analogue that binds to the folic acid receptor; nicotinic acetylcholine receptor agonists; or oligonucleotide receptor agonists.

The cell targeting agent is derived from a known membrane-translocating sequence. For example, the trafficking peptide includes the sequences from the human immunodeficiency virus (HIV) 1 TAT protein. This protein is described in, e.g., U.S. Pat. Nos. 5,804,604 and 5,674,980, each incorporated herein by reference. The cell targeting agent is some or all of the entire 86 amino acids that make up the TAT protein. For example, a functionally effective fragment or portion of a TAT protein that has fewer than 86 amino acids, which exhibits uptake into cells, and optionally uptake into the cell nucleus, is used. A TAT peptide that includes the region that mediates entry and uptake into cells can be further defined using known techniques. See, e.g., Franked et al., Proc. Natl. Acad. Sci, USA 86: 7397-7401 (1989).

The amino acid sequence of naturally-occurring HIV TAT protein can be modified, for example, by addition, deletion and/or substitution of at least one amino acid present in the naturally-occurring TAT protein, to produce modified TAT protein (also referred to herein as TAT protein). Modified TAT protein or TAT peptide analogs with increased or decreased stability can be produced using known techniques. In some embodiments TAT proteins or peptides include amino acid sequences that are substantially similar, although not identical, to that of naturally-occurring TAT protein or portions thereof. In addition, cholesterol or other lipid derivatives can be added to TAT protein to produce a modified TAT having increased membrane solubility.

Variants of the TAT protein can be designed to modulate intracellular localization of the Step 1 Reagent. When added exogenously, such variants are designed such that the ability of TAT to enter cells is retained (i.e., the uptake of the variant TAT protein or peptide into the cell is substantially similar to that of naturally-occurring HIV TAT). For example, alteration of the basic region thought to be important for nuclear localization (see, e.g., Dang and Lee, J. Biol. Chem. 264: 18019-18023 (1989); Hauber et al., J. Virol. 63: 1181-1187 (1989); Ruben et al., J. Virol. 63: 1-8 (1989)) can result in a cytoplasmic location or partially cytoplasmic location of TAT, and therefore, of the platform building material. Alternatively, a sequence for binding a cytoplasmic or any other component or compartment (e.g., endoplasmic reticulum, mitochondria, Golgi apparatus, lysosomal vesicles) can be introduced into TAT in order to retain TAT and the platform building material in the cytoplasm or any other compartment to confer regulation upon uptake of TAT and the platform building material.

Other sources for cell targeting moieties include, e.g., VP22 (described in, e.g., WO 97/05265; Elliott and O\'Hare, Cell 88: 223-233 (1997)), or non-viral proteins (Jackson et al, Proc. Natl. Acad. Sci. USA 89: 10691-10695 (1992)).

A platform building material is a compound that when internalized into the cell via the cell targeting agent detaches from the cell targeting agent and becomes aqueous insoluble. By aqueous insoluble it is meant that the concentration of the nano-platform in an aqueous solution is less than 0.01 mM at room temperature. The concentration of an aqueous solution is less than 0.001 mM, 0.0001 mM, 0.00001 mM, or 0.000001 mM at room temperature. The platform building material forms an aqueous insoluble nano-platform spontaneously. Alternatively, the platform building material forms an aqueous insoluble nano-platform following a further chemical reaction. Chemical reactions include reactions facilitated by enzymes or other conditions present within the cellular environment such as, for example, action of an endogenous lysosomal enzyme, the acidic pH of the lysosomes, other intracellular enzymes, other conditions within another appropriate area within the cell, or attachment or intercalation into biological macrostructures inside the cell.

The platform building material once released from the cell targeting agent inside the targeted cell, forms molecular complexes that precipitate, or forms other aqueous insoluble substances such as, an insoluble polymer, a colloid, a wax, an oil, or a material that attaches or intercalates into biological macrostructures. For example, porphyrin complexes with or without appropriate metals chelated within the porphyrins will spontaneously form molecular complexes that precipitate. In addition, indoxyl glycosides produce aqueous insoluble indigo micro-precipitates, bis-indoxyl glycosides produce aqueous insoluble polymeric indigos and poly-indoxyl glycosides produce aqueous insoluble indigoid lattices.

Suitable platform building materials include for example substituted indoxyls; porphyrins; polymers such as HPMA derivatives; polysaccharides such as dextrans, gum Arabic, and chitin; dendrimers; and opio-melanins.

The cell targeting agent is linked directly to the platform building material. Alternatively, the cell targeting agent is attached indirectly to the platform building material, e.g., via a carrier moiety or a cross-linking agent. The linkage is covalent. Alternatively, the linkage is non-covalent. The linkage is such that it permits the platform building material to detach (i.e. separate) from the cell targeting agent after internalization into the cell. For example the linkage: (1) is cleaved by an intracellular enzyme or the acidic environment found within lysosomes inside the targeted cells, (2) is released by enzymatic or other actions in other environments inside targeted cells, and/or (3) attaches or intercalates into biological macrostructures inside targeted cells.

Carrier moieties allow for a higher number of platform building materials to be delivered inside the targeted cancer cells with each cell targeting agent. A carrier moiety includes for example, proteins such as serum albumin; polysaccharides, especially those modified to have functional groups; synthetic polymers and copolymers such as HPMA derivatives; dendrimers; other biopolymers including polypeptides such as polylysine; liposomes; nanoparticles; and polymeric micelles. Any substance that (a) is biologically compatible, (b) has a number of functional groups (e.g., amino groups, carboxyl groups, thiol groups, and the like) to which multiple platform building materials are attached, and (c) has a place for linking a cell targeting agent, is useful as a carrier moiety.

Optionally, the platform building materials contain an additional molecular structure such that the resulting aqueous insoluble nano-platform expresses the additional molecular structures that can bind a subsequently administered Step 3 Bispecific Reagent. Suitable additional molecular structures include for example, antigenic epitopes, neo-antigenic epitopes, ligands that bind proteins, peptides lectins, or organic structures including those prepared by combinatorial chemistry. Preferably, the additional molecular structure enables the formation of a covalent bond between the additional molecular structures on the nano-platform and the targeting moiety of the subsequently administered Step 3 Bispecific Reagent.

An example of an additional-molecular-structure: Step 3 Reagent-targeting-moiety system occurs when the additional molecular structure on the nano-platform is an irreversible inhibitor of an enzyme, and the targeting moiety of the Step 3 Bispecific Reagent is that enzyme, such that the irreversible inhibitor forms a covalent bond with one of the amino acid residues of that enzyme, thus binding the Step 3 Bispecific Reagent covalently to the aqueous insoluble nano-platform.

Alternatively, the additional molecular structure on the nano-platform is an irreversible inhibitor substrate of an enzyme that is the targeting moiety of the Step 3 Bispecific Reagent, because that enzyme is specifically modified or altered such that the enzymatic reaction is not completed and the substrate becomes covalently bound to the modified enzyme as a stable complex. Such methods are known to those skilled in the art. The mutant β-lactamase described is an example of such a modified enzyme.

Optimally, irreversible enzyme inhibitors useful as additional molecular structures on the platform building materials of the Step 1 Reagent have one or more of the following characteristics: (1) a functional group distant to the active binding portion that can be used to attach the irreversible enzyme inhibitor to the platform building material; (2) relative stability in the circulation, intracellularly and extracellularly; (3) stability properties that facilitate the chemical synthesis of the Step 1 Reagent, including the synthesis of the platform building material, as well as during the attachment of the platform building material with additional molecular structures to the carrier moiety and cell targeting agent.

Exemplary enzyme/irreversible enzyme inhibitor pairs include, mutant β-lactamase/penicillin analog or Loracarbef; UDP-N-acetylglucosamine enolpyruvoyltransferase/fosfomycin or phosphoenolpyruvate; ornithine decarboxylase/α-difluoromethyl amino acids; arginine decarboxylase/α-difluoromethyl amino acids; yeast S-adenosylmethionine decarboxylase/1,1′-(methylethanediylididenedinitrilo)-bis(3-aminoguanidine); and β-lactamase PSE-4/clavulanic acid, sulbactam, and tazobactam.

The various components of the Step 1 Reagent are selected from the repertoires of those components to suit a particular type of cancer. Having this versatility in the selection of the various components of the Step 1 Reagent allows this invention to be applied to almost all types of cancer. Exemplary targets for cell targeting agents for particular tumor types are listed in Table 1, wherein “x” denotes that the target has been identified on the particular tumor.

TABLE 1 Target Breast Lung Colon Pancreas Prostate Liver Ovary Bladder Stomach Cervix Uterus Transferrin - 1 & 2 Receptor x x x x x x x x x x x EGF Receptor x x x x x x x x x x x IL-4 Receptor x x x x x x x x x Insulin Receptor x x x x x x x ? x x CD34 x x x x x x ? x CCK-A, B, C/Gastrin Receptor x x x x x x

Download full PDF for full patent description/claims.




You can also Monitor Keywords and Search for tracking patents relating to this Method and composition for the treatment of cancer by the enzymatic conversion of soluble radioactive toxic precipitates in the cancer patent application.
###


Other recent patent applications listed under the agent :