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  Pharmaceutical Patents  

 

Title:  Methods to impair hematologic cancer progenitor cells and compounds related thereto
United States Patent: 
7,651,678
Issued: 
January 26, 2010

Inventors:
 Jordan; Craig (Lexington, KY)
Assignee:
  University of Kentucky Research Foundation (Lexington, KY)
Appl. No.:
 10/830,089
Filed:
 April 23, 2004


 

Woodbury College's Master of Science in Law


Abstract

Primitive or progenitor hematologic cancer cells have been implicated in the early stages and development of leukemia and malignant lymphoproliferative disorders, including acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML) and chronic lymphoid leukemia (CLL). Interleukin-3 receptor alpha chain (IL-3R.alpha. or CD123) is strongly expressed on progenitor hematologic cancer cells, but is virtually undetectable on normal bone marrow cells. The present invention provides methods of impairing progenitor hematologic cancer (e.g., leukemia and lymphomic) cells by selectively targeting cells expressing CD123. These methods are useful in the detection and treatment of leukemias and malignant lymphoproliferative disorders. Also provided are compounds useful for selectively binding to CD123 and impairing progenitor hematologic cancer cells. These compounds may include cytotoxic moieites such as, for example, radioisotopes or chemotherapeutics.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates to a method of using compounds that bind to the human CD123 molecule (CD123 ectopeptide), in the diagnosis and treatment of hematologic cancers (e.g., leukemias and malignant lymphoproliferative disorders). The CD123 specific compounds and mimetics have particular utility as pharmaceuticals and reagents for the therapy of hematologic cancer or malignant disease states and for the diagnosis of hematologic cancer disease states. In one embodiment, the present invention provides a method of impairing a hematologic cancer progenitor cell comprising contacting the cell with a compound that selectively binds to CD123 in an amount effective to impair the progenitor hematologic cancer cell. This contacting step may occur in various environments, including in vitro and in vivo in the body of an animal, including a human.

Throughout this application, reference will be made specifically to leukemia in describing certain embodiments of the present invention. However, it is understood that the present invention is not limited to diagnosis and treatment of leukemia or malignant lymphoproliferative disorders alone, but to any disease in which the cancerous cells selectively express CD123, which includes the genus of hematologic cancer.

In one embodiment, the present invention is directed to a method of detecting the presence of CD123 on, for example, a leukemia progenitor cell. Thus, the invention is also directed to a method of diagnosing leukemia. It is understood that by using a labeled ligand to bind to CD123, it is possible to detect the presence of leukemia progenitor cells. Thus, it is also possible to diagnose the likelihood of the onset of leukemia in patients possessing such leukemic progenitor cells expressing CD123. The CD123 binding ligand may be an antibody to CD123, or it may be any of a variety of molecules that specifically bind to CD123. Furthermore, the label can be chosen from any of a variety of molecules, including, but not limited to, enzymatic compounds, or non-enzymatic compounds that serve as a reporter of the presence of the ligand which has bound to the CD123 molecule. Examples of such labels include those that are, for example, radioactive, fluorescent, chemiluminescent or absorbant-based, or a combination of the foregoing. In one embodiment, an assay is provided for detecting the presence of progenitor leukemia cells in a sample by detecting the presence of CD123 in the sample, which may be accomplished by introducing a compound that selectively binds to CD123 and determining whether the compound binds to a component of the sample.

In another series of embodiments, the present invention also provides compounds or molecules which mimic (mimetics) the three-dimensional structure of part or all of the compounds such as peptides, antibodies, carbohydrates, lipids or nucleic acids that bind to CD123, and in the case of antibodies, of the binding pockets of the antibodies, or of the complementarity determining regions (CDR's).

The present invention also provides pharmaceutical preparations comprising a pharmaceutically acceptable carrier; and any one or more of the CD123 specific compounds and mimetics described above.

In another set of embodiments, the present invention provides a method for the treatment of leukemia, comprising administering to a human subject or other animal in need of such treatment a therapeutically effective amount of the compounds or their mimetic pharmaceutical compositions described above.

In still another set of embodiments, the present invention provides a method of selectively purging leukemic stem cells from bone marrow. These stems cells may give rise to leukemia progenitor cells, or they may be the progenitor cells, which may be impaired by the method of the invention using various compounds or their mimetics and cytotoxic agents that may be contacted to either a bone marrow sample or injected into a bone marrow of an individual, thereby destroying at least some of the leukemic stem cells in the bone marrow.

DETAILED DESCRIPTION OF THE INVENTION

Mimetics that Bind to CD123

Compounds that target CD123 can be found. Phage display libraries can be used to determine the DNA encoding the polypeptide that binds to CD123. The principles of this approach are disclosed in U.S. Pat. No. 5,837,500, which is incorporated by reference herein in its entirety. Other non-peptide molecules that may bind to CD123 include nucleic acids, and liposomes. Carbohydrates may also be used to target CD123. It is possible that the compound may not be a naturally occurring biological molecule. Such chemicals may be made by combinatorial libraries which are well known in the art, with the assay goal being the binding of the chemical compound to CD123. Liposomes may ensconce certain toxins or other cell-impairing substances or cell-imaging compounds may be used to target CD123. Numerous variations and combinations of compounds as targeting agents are contemplated by the method of the invention, so long as CD123 is targeted, with the knowledge that leukemia detection and leukemia treatment is kept in mind.

Mimetics of Anti-CD123 Antibodies

It is also possible to use the anti-idiotype technology to isolate or screen for compounds or mimetics which mimic an epitope. Thus, an anti-idiotypic monoclonal antibody which is the image of the epitope bound by the first monoclonal antibody, since it effectively acts as an antigen, may be used to isolate mimetics from a combinatorial chemical, or other libraries, of chemical or other compounds, such as peptide phage display libraries (Scott and Smith, Science 249: 386-390, 1990; Scott and Craig, Curr. Opin. Biotechnol. 5: 40-48, 1992; Bonnycastle et al., J. Mol. Biol. 258: 747-762, 1996). Hence, peptides or constrained peptides mimicking proteins or other compounds, including those with nucleic acid, lipid, carbohydrate or other moieties, may be cloned (Harris et al., Proc. Natl. Acad. Sci. (USA) 94: 2454-2459, 1997).

Purely synthetic molecules, which may not occur in nature and are therefore more resistant to catabolism, excretion or degradation, may be designed by the three-dimensional placement of atoms, such that similar ionic forces, covalent forces, van der Waal's or other forces, and similar charge complementarity, or electrostatic complementarity, exist between the atoms of the mimetic and the atoms of the antigenic binding site or epitope. These mimetics may then be screened for high affinity binding to CD123 and detect and/or impair the CD123 bearing cell in vitro or in vivo, as described in more detail below.

Diagnostic and Pharmaceutical Preparations

The invention also relates to a method for preparing diagnostic or pharmaceutical compositions comprising the CD123 binding compound and its mimetics. The pharmaceutical preparation includes a pharmaceutically acceptable carrier. Such carriers, as used herein, mean non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials that are well known in the art.

The anti-CD123 antibodies and mimetics may be labeled by a variety of means for use in diagnostic and/or pharmaceutical applications. There are many different labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, and bioluminescent compounds. Those of ordinary skill in the art will know of other suitable labels for binding to the CD123 binding compound, such as monoclonal antibodies, or mimetics thereof, or will be able to ascertain such, using routine experimentation. Furthermore, the binding of these labels to the CD123 specific compounds or their mimetics can be done using standard techniques common to those of ordinary skill in the art.

In the case of antibodies, another labeling technique which may result in greater sensitivity consists of coupling the antibodies or mimetics to low molecular weight haptens. These haptens can then be specifically altered by means of a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, or fluorescein, which can react with specific anti-hapten antibodies.

Diagnostic and Treament Kits

The materials for use in the assay of the invention are ideally suited for the preparation of a kit. Such a kit may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method. For example, one of the container means may comprise a compound that binds to CD123, such as a monoclonal antibody, or a mimetic thereof, which is, or can be, detectably labeled with a label that is suitable for diagnostic purposes or if treatment is desired, a cytotoxic or impairing agent. In the case of a diagnostic kit, the kit may also have containers containing buffer(s) and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic or fluorescent label. In addition to the chemical material, of course a means of instructions for using the kit is included, preferably for either diagnosing leukemia, or treating leukemia. The instruction means may be written on the vial, tube and the like, or written on a separate paper, or on the outside or inside of the container. The instructions may also be in the form of a multi-media format, such as CD, computer disk, video and so on.

Preparation of Immunotoxins

While the preparation of immunotoxins is, in general, well known in the art (see, e.g., U.S. Pat. No. 4,340,535, and EP 44167, both incorporated herein by reference), the inventors are aware that certain advantages may be achieved through the application of certain preferred technology, both in the preparation of the immunotoxins and in their purification for subsequent clinical administration. For example, while IgG based immunotoxins will typically exhibit better binding capability and slower blood clearance than their Fab' counterparts, Fab' fragment-based immunotoxins will generally exhibit better tissue penetrating capability as compared to IgG based immunotoxins.

Additionally, while numerous types of disulfide-bond containing linkers are known which can successfully be employed to conjugate the toxin moiety with the binding agent, certain linkers will generally be preferred over other linkers, based on differing pharmacologic characteristics and capabilities. For example, linkers that contain a disulfide bond that is sterically "hindered" are to be preferred, due to their greater stability in vivo, thus preventing release of the toxin moiety prior to binding at the site of action.

Cross-linking reagents are used to form molecular bridges that tie together functional groups of two different proteins (e.g., a toxin and a binding agent). To link two different proteins in a step-wise manner, hetero bifunctional cross-linkers can be used which eliminate the unwanted homopolymer formation. An exemplary hetero bifunctional cross-linker contains two reactive groups: one reacting with primary amine group (e.g., N-hydroxy succinimide) and the other reacting with a thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the cross-linker may react with the lysine residue(s) of one protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the cross-linker, already tied up to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., dgA).

The spacer arm between these two reactive, groups of any cross-linkers may have various lengths and chemical compositions. A longer spacer arm allows a better flexibility of the conjugate components while some particular components in the bridge (e.g., benzene group) may lend extra stability to the reactive group or an increased resistance of the chemical link to the action of various aspects (e.g., disulfide bond resistant to reducing agents).

The most preferred cross-linking reagent is SMPT, which is a bifunctional cross-linker containing a disulfide bond that is "sterically hindered" by an adjacent benzene ring and methyl groups. It is believed that stearic hindrance of the disulfide bond serves a function of protecting the bond from attack by thiolate anions such as glutathione which can be present in tissues and blood, and thereby help in preventing decoupling of the conjugate prior to its delivery to the site of action by the binding agent. The SMPT cross-linking reagent, as with many other known cross-linking reagents, lends the ability to cross-link functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of cross-linker includes the hetero-bifunctional photoreactive phenylazides containing a cleavable disulfide bond such as sulfosuccinimidyl-2-(p-azido salicylamido) ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group reacts with primary amino groups and the phenylazide (upon photolysis) reacts non-selectively with any amino acid residue.

Although the "hindered" cross-linkers will generally be preferred in the practice of the invention, non-hindered linkers can be employed and advantages in accordance herewith nevertheless realized. Other useful cross-linkers, not considered to contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane. The use of such cross-linkers is well understood in the art.

Once conjugated, it will be important to purify the conjugate so as to remove contaminants such as unconjugated A chain or binding agent. It is important to remove unconjugated A chain because of the possibility of increased toxicity. Moreover, it is important to remove unconjugated binding agent to avoid the possibility of competition for the antigen between conjugated and unconjugated species. In any event, a number of purification techniques are disclosed in the Examples below which have been found to provide conjugates to a sufficient degree of purity to render them clinically useful. In general, the most preferred technique will incorporate the use of Blue-Sepharose with a gel filtration or gel permeation step. Blue-Sepharose is a column matrix composed of Cibacron Blue 3GA and agarose, which has been found to be useful in the purification of immunoconjugates. The use of Blue-Sepharose combines the properties of ion exchange with A chain binding to provide good separation of conjugated from unconjugated binding.

The Blue-Sepharose allows the elimination of the free (non conjugated) binding agent (e.g., the antibody or fragment) from the conjugate preparation. To eliminate the free (unconjugated) toxin (e.g., dgA) a molecular exclusion chromatography step is preferred using either conventional gel filtration procedure or high performance liquid chromatography.

After a sufficiently purified conjugate has been prepared, one will desire to prepare it into a pharmaceutical composition that may be administered parenterally. This is done by using for the last purification step a medium with a suitable pharmaceutical composition.

Suitable pharmaceutical compositions in accordance with the invention will generally comprise from about 10 to about 100 mg of the desired conjugate admixed with an acceptable pharmaceutical diluent or excipient, such as a sterile aqueous solution, to give a final concentration of about 0.25 to about 2.5 mg/ml with respect to the conjugate. Such formulations will typically include buffers such as phosphate buffered saline (PBS), or additional additives such as pharmaceutical excipients, stabilizing agents such as BSA or HSA, or salts such as sodium chloride. For parenteral administration it is generally desirable to further render such compositions pharmaceutically acceptable by insuring their sterility, non-immunogenicity and non-pyrogenicity. Such techniques are generally well known in the art as exemplified by Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980, incorporated herein by reference. It should be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less than 0.5 ng/mg protein. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

A preferred parenteral formulation of the immunotoxins in accordance with the present invention is 0.25 to 2.5 mg conjugate/ml in 0.15M NaCl aqueous solution at pH 7.5 to 9.0. The preparations may be stored frozen at -10.degree. C. to -70.degree. C. for at least 1 year.

It is contemplated that most therapeutic applications of the present invention will involve the targeting of a toxin moiety (cytotoxic agent) to the CD123 leukemia marker. This is due to the much greater ability of most toxins to deliver a cell killing effect as compared to other potential agents.

However, there may be circumstances such as when the target antigen does not internalize by a route consistent with efficient intoxication by immunotoxins, where one will desire to target chemotherapeutic agents such as cytokines, antimetabolites, alkylating agents, hormones, and the like. The advantages of these agents over their non-antibody conjugated counterparts is the added selectivity afforded by the antibody. One might mention by way of example agents such as steroids, cytosine arabinoside, methotrexate, aminopterin, anthracyclines, mitomycin C, vinca alkaloids, demecolcine, etopside, mithramycin, and the like. This list is, of course, merely exemplary in that the technology for attaching pharmaceutical agents to antibodies for specific delivery to tissues is well established.

One preferred cytotoxic moiety for use in the present invention is a radioisotope, which can be coupled to or conjugated with, for example, an anti-CD123 antibody. Preferred radioisotopes include .alpha.-emitters such as, for example, .sup.211Astatine, .sup.212Bismuth and .sup.213Bismuth, as well as .beta.-emitters such as, for example, .sup.131Iodine, .sup.90Yttrium, .sup.177Lutetium, .sup.153Samarium and .sup.109Palladium. Particularly preferred radioisotopes are .sup.211Astatine and .sup.131Iodine.

It is proposed that particular benefits may also be achieved through the application of the invention to cell imaging. Imaging of leukemia cells is believed to provide a major advantage when compared to available imaging techniques, in that the cells are readily accessible.

Moreover, the technology for attaching paramagnetic, radioactive and even fluorogenic ions to antibodies is well established. Many of these methods involve the use of a metal chelate complex employing, for example, an organic chelating agent such a DTPA attached to the antibody (see, e.g., U.S. Pat. No. 4,472,509). In the context of the present invention the selected ion is thus targeted to the cancerous area by the antibody, allowing imaging to proceed by means of the attached ion.

In a preferred embodiment, in the method of the invention, the antibodies may also be fused to a protein effector molecule by recombinant means such as through the use of recombinant DNA techniques to produce a nucleic acid which encodes both the antibody and the effector molecule and expressing the DNA sequence in a host cell such as E. coli. The DNA encoding the chimeric protein may be cloned in cDNA or in genomic form by any cloning procedure known to those skilled in the art. See for example Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, (1989), which is herein incorporated by reference.

Fusion or conjugation of antibodies to various labels produces a highly specific detectable marker that may be used to detect the presence or absence of cells or tissues bearing the particular molecule to which the antibody is detected. Alternatively, the antibodies may be chemically conjugated or fused to an effector molecule that is another specific binding moiety, e.g. a ligand such as those described above. In this form the composition will act as a highly specific bifunctional linker. This linker may act to bind and enhance the interaction between cells or cellular components to which the fusion protein binds. Thus, for example, where the fusion protein is a growth factor joined to an antibody or antibody fragment (e.g. an Fv fragment of an antibody), the antibody may specifically bind antigen positive cancer cells while the growth factor binds receptors on the surface of immune cells. The fusion protein may thus act to enhance and direct an immune response toward target cancer cells.

In Vitro Detection and Diagnostics

The method of using the compounds that bind to CD123 and their mimetics are suited for in vitro use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier. In addition, the monoclonal antibodies in these immunoassays can be detectably labeled in various ways. Examples of types of immunoassays which can utilize the monoclonal antibodies and their mimetics are competitive and non-competitive immunoassays in either a direct or indirect format. Examples of such immunoassays are the radioimmunoassay (RIA) and the sandwich (immunometric) assay. Detection of antigens using the monoclonal antibodies and their mimetics can be done utilizing immunoassays which are run in either the forward, reverse, or simultaneous modes, including immunohistochemical assays on physiological samples. Those of skill in the art will know, or can readily discern, other immunoassay formats without undue experimentation.

The compounds that bind CD123 and mimetics can be bound to many different carriers and used to detect the presence of CD123 bearing leukemia cells, including progenitor cells. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier can be either soluble or insoluble for purposes of the invention. Those skilled in the art will know of other suitable carriers for binding various compounds, or will be able to ascertain such, using routine experimentation.

For purposes of the invention, CD123 may be detected by the compounds and their mimetics when present in biological fluids and tissues. Any sample containing a detectable amount of CD123 ectopeptide can be used. A sample can be a liquid such as urine, saliva, cerebrospinal fluid, blood, serum or the like; a solid or semi-solid such as tissues, feces, or the like; or, alternatively, a solid tissue such as those commonly used in histological diagnosis.

In Vivo Detection of CD123

In using the CD123 binding compounds and mimetics for the in vivo detection of CD123, the detectably labeled compound or its mimetic is given in a dose which is diagnostically effective. The term "diagnostically effective" means that the amount of detectably labeled compound, such as monoclonal antibody or mimetic is administered in sufficient quantity to enable detection of the leukemia cells for which the compounds or mimetics are specific.

The concentration of detectably labeled compound or mimetic which is administered should be sufficient such that the binding to CD123 or CD123-bearing leukemia cells, is detectable compared to the background. Further, it is desirable that the detectably labeled compound or mimetic be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.

As a rule, the dosage of detectably labeled compound or mimetic for in vivo diagnosis will vary depending on such factors as age, sex, and extent of disease of the individual. The dosage of the compound can vary from about 0.01 mg/kg to about 500 mg/kg, preferably about 0.1 mg/kg to about 200 mg/kg, most preferably about 0.1 mg/kg to about 10 mg/kg. Such dosages may vary, for example, depending on whether multiple injections are given, on the tissue being assayed, and other factors known to those of skill in the art.

For in vivo diagnostic imaging, the type of detection instrument available is a major factor in selecting an appropriate radioisotope. The radioisotope chosen must have a type of decay which is detectable for the given type of instrument. Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough such that it is still detectable at the time of maximum uptake by the target, but short enough such that deleterious radiation with respect to the host is acceptable. Ideally, a radioisotope used for in vivo imaging will lack a particle emission but produce a large number of photons in the 140-250 keV range, which may be readily detected by conventional gamma cameras.

For in vivo diagnosis, radioisotopes may be bound to the compound either directly or indirectly by using an intermediate functional group. Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions are the bifunctional chelating agents such as diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetra-acetic acid (EDTA) and similar molecules. Typical examples of metallic ions which can be bound to the monoclonal antibodies and mimetics of the invention are .sup.111In, .sup.97Ru, .sup.67Ga, .sup.68Ga, .sup.72As, .sup.89Zr, .sup.99mTc, .sup.123I and .sup.201Tl.

In the diagnosis method of the invention, the compounds and mimetics can also be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR). In general, any conventional method for visualizing diagnostic imaging can be utilized. Usually gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI. Elements which are particularly useful in such techniques include .sup.157Gd, .sup.55Mn, .sup.167Dy, .sup.52Cr and .sup.56Fe.

In the cell monitoring method of the invention, the compounds and mimetics can be used in vitro and in vivo to monitor the course of leukemia disease therapy. Thus, for example, by measuring the increase or decrease in the biological molecules associated with such a diseases or changes in the concentration of CD123 ectopeptide or CD123 bearing leukemia cells present in the body or in various body fluids, it would be possible to determine whether a particular therapeutic regimen aimed at ameliorating the above leukemia disease is effective.

Prophylaxis and Therapy of Leukemia

The CD123 specific compounds can also be used therapeutically for treatment of leukemia in both humans and other animals. The term, "therapeutically" or "therapy" as used herein in conjunction with the method of the invention is directed to using CD123 binding compounds, such as anti-CD123 monoclonal antibodies and their mimetics, which denotes both prophylactic as well as therapeutic administration and both passive immunization with substantially purified polypeptide products, and mimetics, as well as gene therapy by transfer of polynucleotide sequences encoding the product or part thereof. Thus, the compounds and mimetics can be administered to high-risk subjects in order to lessen the likelihood and/or severity of leukemia relapse, or administered to subjects already evidencing active leukemia disease.

For certain applications, it is envisioned that pharmacologic agents will serve as useful agents for attachment to the compounds, particularly cytotoxic or otherwise anticellular agents having the ability to kill or suppress the growth or cell division of leukemia cells. In general, the invention contemplates the use of any pharmacologic agent that can be conjugated to a CD123 binding compound and delivered in active form to the targeted cell. Exemplary anticellular agents include chemotherapeutic agents, radioisotopes as well as cytotoxins. In the case of chemotherapeutic agents, the inventors propose that agents such as a hormone such as a steroid; an antimetabolite such as cytosine arabinoside, fluorouracil, methotrexate or aminopterin; an anthracycline; mitomycin C; a vinca alkaloid; demecolcine; etoposide; mithramycin; calicheamicin, CC-1065 and derivatives thereof, or an alkylating agent such as chlorambucil or melphalan, will be particularly preferred. Other embodiments may include agents such as a coagulant, a cytokine, growth factor, bacterial endotoxin or the lipid A moiety of bacterial endotoxin. In any event, it is proposed that agents such as these may be successfully conjugated to antibodies in a manner that will allow their targeting, internalization, release or presentation to blood components at the site of the targeted leukemia cells as required using known conjugation technology.

In certain preferred embodiments, cytotoxic agents for therapeutic application will include generally a plant-, fungus- or bacteria-derived toxin, such as an A chain toxins, a ribosome inactivating protein, .alpha.-sarcin, aspergillin, restrictocin, a ribonuclease, diphtheria toxin or pseudomonas exotoxin, to mention just a few examples. The use of toxin-antibody constructs is well known in the art of immunotoxins, as is their attachment to antibodies. Of these, a particularly preferred toxin for attachment to antibodies will be a deglycosylated ricin A chain. Deglycosylated ricin A chain is preferred because of its extreme potency, longer half-life, and because it is economically feasible to manufacture a clinical grade and scale.

In other preferred embodiments, the cytotoxic agent may be a radioisotope. Preferred radioisotopes include .alpha.-emitters such as, for example, .sup.211Astatine, .sup.212Bismuth and .sup.213Bismuth, as well as .beta.-emitters such as, for example, .sup.131Iodine, .sup.90Yttrium, .sup.177Lutetium, .sup.153Samarium and .sup.109Palladium.

As used herein, a "therapeutically effective amount" of a compound is a dosage large enough to produce the desired effect in which the symptoms of leukemia or the likelihood of onset of leukemia is decreased. A therapeutically effective amount is not, however, a dosage so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. Generally, a therapeutically effective amount may vary with the subject's age, condition, and sex, as well as the extent of the disease in the subject and can be determined by one of skill in the art. The dosage may be adjusted by the individual physician or veterinarian in the event of any complication. A therapeutically effective amount may vary from about 0.01 mg/kg to about 500 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days.

In the method of the invention, the compounds and their mimetics can be administered by injection or by gradual infusion over time. The administration of the compounds and their mimetics may be, for example, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions. suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

Depending on the specific clinical status of the disease, administration can be made via any accepted systemic delivery system, for example, via oral route or parenteral route such as intravenous, intramuscular, subcutaneous or percutaneous route, or vaginal, ocular or nasal route, in solid, semi-solid or liquid dosage forms, such as for example, tablets, suppositories, pills, capsules, powders, solutions, suspensions, cream, gel, implant, patch, pessary, aerosols, collyrium, emulsions or the like, preferably in unit dosage forms suitable for easy administration of fixed dosages. The pharmaceutical compositions will include a conventional carrier or vehicle and a CD123 binding compound and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, and so on.

If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and so on.

The compounds of this invention are generally administered as a pharmaceutical composition which comprises a pharmaceutical vehicle in combination with a CD123 binding compound. The amount of the drug in a formulation can vary within the full range employed by those skilled in the art, e.g., from about 0.01 weight percent (wt %) to about 99.99 wt % of the drug based on the total formulation and about 0.01 wt % to 99.99 wt % excipient.

The preferred mode of administration, for the conditions mentioned above, is oral administration using a convenient daily dosage regimen which can be adjusted according to the degree of the complaint. For said oral administration, a pharmaceutically acceptable, non-toxic composition is formed by the incorporation of the selected CD123 binding compound in any of the currently used excipients, such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like. Such compositions take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations and the like. Such compositions may contain between about 0.01 wt % and 99.99 wt % of the active compound according to this invention.

Preferably the compositions will have the form of a sugar coated pill or tablet and thus they will contain, along with the active ingredient, a diluent such as lactose, sucrose, dicalcium phosphate, and the like; a disintegrant such as starch or derivatives thereof; a lubricant such as magnesium stearate and the like; and a binder such as starch, polyvinylpyrrolidone, acacia gum, gelatin, cellulose and derivatives thereof, and the like.

It is understood that by "pharmaceutical composition", it is meant that the CD123 binding compound is formulated into a substance that is to be administered purposefully diagnosing or treating leukemia in the individual. And, by "pharmaceutical composition", it excludes those compositions that are used to administer to individuals as test compounds for a purpose other than as a diagnostic or treatment agent for leukemia.

The invention is described in further detail hereinbelow.

Several recent studies have suggested the presence and importance of stem cells in both the genesis and perpetuation of AML. Phenotypically, cells described as CD34+/CD38- or CD34+/HLA-DR- appear to play a central role in the development of leukemic populations (Bonnet D, et al., Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 1997, 3: 730-737; Blair A, et al., Most acute myeloid leukemia progenitor cells with long-term proliferative ability in vitro and in vivo have the phenotype CD34(+)/CD71(-)/HLA-DR-. Blood 1998, 92: 4325-35). Furthermore, there is evidence suggesting that such cells may be relatively resistant to chemotherapeutic drugs, and consequently contribute to the phenomenon of relapse (Terpstra W, et al., Fluorouracil selectively spares acute myeloid leukemia cells with long-term growth abilities in immunodeficient mice and in culture. Blood 1996, 88: 1944-50). Thus, a better understanding of LSC biology and the characterization of unique LSC antigens are essential to the development of better treatments for AML.

While the various AML subtypes display considerable diversity with respect to developmental characteristics, phenotype, cytokine responsiveness, etc., there appears to be a marked degree of functional conservation at the level of more primitive leukemic cells. This feature has been demonstrated by the work of Bonnet et. al., in which a CD34+/CD38- subpopulation was shown to be sufficient to establish leukemia in NOD/SCID mice (Bonnet D, et al., Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 1997, 3: 730-737). Similar studies by others have corroborated the existence of leukemic stem cells for both AML and CML and confirmed their relatively homogeneous phenotype and functional capacity (Blair A, Hogge e tal., Most acute myeloid leukemia progenitor cells with long-term proliferative ability in vitro and in vivo have the phenotype CD34(+)/CD71(-)/HLA-DR-. Blood 1998, 92: 4325-35; Holyoake T, et al., Isolation of a highly quiescent subpopulation of primitive leukemic cells in chronic myeloid leukemia. Blood 1999, 94: 2056-64). However, to date, no study has identified an antigenic feature of myeloid LSC's that may allow their identification or preferential targeting for ablative therapy. In this report, we have identified an additional commonality among CD34+/CD38- AML stem cells, expression of CD123, which facilitates their discrimination from normal hematopoietic stem cells. While the CD123 antigen was readily detected at high levels on AML cells, the IL-3 receptor .beta. chain, CD131, was not detected.

Our experiments indicate that the transcription factor IRF-1 (Interferon regulatory factor-1) is over-expressed (in 6 of 6 primary AML specimens examined). Previous studies by Korpelainen et. al. have shown that treatment of endothelial cells with IFN-.gamma. results in up-regulation of CD123 (Korpelainen E I, et al., Interferon-gamma upregulates interleukin-3 (IL-3) receptor expression in human endothelial cells and synergizes with IL-3 in stimulating major histocompatibility complex class II expression and cytokine production. Blood 1995, 86: 176-82). Similarly, our own studies have shown that treatment of primary AML cells with IFN-.gamma. increases expression of CD123 (data not shown). Thus, aberrant expression of interferon regulatory molecules might play a role in controlling CD123 expression in AML cells.

Expression of the CD123 antigen formally demonstrates that LSC's are biologically distinct from their normal stem cell counterparts. Because CD123 is not readily found on normal hematopoietic stem cells, it provides a unique marker that can be used to identify malignant tissue. This feature may be useful for research purposes, as well as in minimal residual disease (MRD) studies. Further, the CD123 epitope represents a target to which therapeutic strategies may be directed. Previous clinical trials have used monoclonal antibodies against both the CD33 and CD45 antigens as a means to deliver radioisotopes to AML cells in vivo (Appelbaum F R., Antibody-targeted therapy for myeloid leukemia. Semin Hematol 1999, 36: 2-8.). In addition, several other recent studies have shown exciting results using monoclonal antibodies specific to antigens on malignant cells such as CD20, CD52, and Her-2 (Maloney D G., Advances in immunotherapy of hematologic malignancies. Curr Opin Hematol 1998; 5: 23743; Sikic B I., New approaches in cancer treatment. Ann Oncol 1999, 10 Suppl 6: 149-53). Antibodies to CD123 may be useful in a similar paradigm and will be capable of delivering a cytotoxic hit that specifically targets the leukemic stem cell population.

We have shown that CD123 represents a unique antigenic marker for the identification of primitive leukemic cells from a broad range of human specimens across a broad range of leukemic diseases. Our studies show that CD123 is generally expressed at high levels and may be indicative of previously uncharacterized aspects of leukemia biology.
 

Claim 1 of 11 Claims

1. A method for impairing cancerous progenitor cells, which express CD123, but do not significantly express CD131, in a patient in need thereof, comprising introducing to the patient's bone marrow or peripheral blood a composition comprising an antibody conjugated to a cytotoxic agent, wherein said antibody binds selectively to CD123.
 

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