HK1173970A - Treatment methods utilizing albumin-binding proteins as targets - Google Patents

Description

Therapeutic methods using albumin-binding proteins as targets

The present application is a divisional application of the Chinese patent application having application No. 200580020748.7(PCT/US2005/017174), application date 5/16/2005, and invention name "therapeutic method using albumin-binding protein as a target".

Technical Field

The present invention relates to methods for treating cancer and other diseases, including abnormal proliferation, remodeling, inflammatory activity of tissues and organs. The invention further relates to methods for targeting, imaging and determining the response of mammalian tumors to chemotherapeutic agents using suitable ligands that recognize albumin-binding proteins.

Background

Albumin nanoparticle formulations have been shown to reduce the toxicity of poorly soluble therapeutic agents. For example, U.S. Pat. No. 6,537,579 discloses albumin-nanoparticle paclitaxel formulations without toxic emulsifiers.

By trade markThe anticancer agent, paclitaxel, marketed by Bristol Myers Squibb, is currently approved for the treatment of several cancers, including ovarian, lung, and breast cancers. The main limitation of using paclitaxel is its poor solubility. Therefore, the temperature of the molten metal is controlled,the formulation comprising as a dissolving excipientEL, but in this preparationThe presence of (D) is associated with severe allergic reactions in animals (Lorenz et al, Agents Actions 7, 63-67, 1987) and humans (Weiss et al, J.Clin. Oncol.8, 1263-1268, 1990). Thus, receiveThe patient in need of pre-administration of a corticosteroid (dexamethasone) and an antihistamine to reduce the adverse drug reactionsAllergy and anaphylaxis in the presence of (c).

In contrast, also known as ABI-007For sale by Abraxis Oncology, noneThe paclitaxel albumin-nanoparticle formulation of (1). The use of albumin nanoparticles as an excipient when reconstituted with saline results in a colloidal form. Based on clinical studies, it has been shown thatUse of andcompared to a reduced allergic reaction. Thus, receiveDoes not require prior administration.

Another advantage of the albumin-nanoparticle formulation is that by eliminating toxic emulsifiers, it is possible to use it more than is currently possiblePossibly more frequent intervals, higher doses of paclitaxel are given. There is the potential for enhanced efficacy that can be seen in solid tumors due to: (i) higher tolerated dose (300 mg/m)²) (ii) longer half-life, (iii) prolonged local tumor effectiveness, and/or (iv) sustained release in vivo.Reduce anaphylaxis and maintain or improve the chemotherapy effect of the medicine.

Colloidal nanoparticles or particles < 200nm in size are known to be prone to concentrate at the tumor site due to leaky vasculature. This effect has been described for some liposome formulations (papahadjoulos, et al, proc.natl.acad.sci.u.s.a.88, 11460, 1991); (Gabizon, A., Cancer Res., 52, 891, 1992); (Dvorak, et al, am.J.Pathol.133, 95, 1988); (Dunn, et al, Pharm, Res., 11, 1016-1022, 1994); and (Gref, et al, Science 263, 1600-1603, 1994). It is possible to dissolve the drug in it (contain) The concentration of paclitaxel nanoparticles at the tumor site can result in sustained release of the drug at the tumor site to produce greater efficacy when compared to form administration.

The nanoparticle formulation includes at least about 50% of the active agent in nanoparticle form. Further, the nanoparticle formulation comprises at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the active agent in nanoparticle form. Additionally, the nanoparticle formulation comprises at least about 95% or at least about 98% of the active agent in nanoparticle form.

The acidic, cysteine-rich Secretory Protein (SPARC), also known as osteonectin (osteonectin), is a 281 amino acid glycoprotein. SPARC has affinity for a variety of ligands, including cations (e.g., Ca)²⁺、Cu²⁺、Fe²⁺) Growth factors (e.g., Platelet Derived Growth Factor (PDGF) and Vascular Endothelial Growth Factor (VEGF)), extracellular matrix (ECM) proteins (e.g., collagen I-V and collagen IX, vitronectin (vitronectin) and thrombospondin-1), endothelial cells, platelets, albumin, and hydroxyapatite. SPARC expression is developmentally regulated and is expressed in remodeled tissues primarily during normal development or injury responses (see, e.g., Lane et al, FASEB j., 8, 163-173 (1994)). SPARC protein is expressed at high levels in developing bone and teeth.

SPARC is a cellular matrix protein that is up-regulated in several aggressive cancers, but is absent in normal tissues (Porter et al, j. histochem. cytochem., 43, 791 (1995)). In fact, SPARC expression is induced in various tumors (e.g., bladder, liver, ovary, kidney, gut, and breast). For example, in bladder cancer, SPARC expression is associated with advanced cancer. Aggressive bladder tumors of grade T2 or later have been shown to express higher levels of SPARC than bladder tumors of grade T1 (or a lesser degree superficial tumors) and have a poorer prognosis (see, e.g., Yamanaka et al, j. urology, 166, 2495-2499 (2001)). In meningiomas, SPARC expression is only associated with aggressive tumors (see, e.g., Rempel et al, clinical Cancer res., 5, 237-241 (1999)). SPARC expression was also detected in 74.5% of in situ invasive breast cancer lesions (see, e.g., Bellahcene, et al, am.j.pathol., 146, 95-100(1995)), and in 54.2% of breast infiltrating duct cancers (see, e.g., Kim et al, j.korean med. sci., 13, 652-657 (1998)). SPARC expression is also associated with microcalcifications that are common in breast cancer (see, e.g., Bellahcene et al, supra), suggesting that SPARC expression may be responsible for the affinity of metastatic lesions of the breast for bone. SPARC is also known to bind albumin (see, e.g., Schnitzer, j.biol. chem., 269, 6072 (1994)).

Antibody therapy is an effective method for controlling disease, wherein specific protein markers can be identified. Examples include Avastin-anti-VEGF antibody, Rituxan-anti-CD 20 antibody, and Remicade-anti-TNF antibody. Thus, antibodies to SPARC represent important therapeutic agents for the treatment of human and other mammalian tumors, as well as other proliferative, remodeling and inflammatory disorders that express SPARC proteins. In addition, SPARC antibodies conjugated to imaging or contrast agents would be a tool to detect and diagnose such disorders.

There remains a need for methods of treating tumors, as well as other proliferative, remodeling and inflammatory disorders in humans and other mammals. In addition, there remains a need to determine the tumor response of humans or other mammals to assess the effectiveness of chemotherapeutic agents. Furthermore, suitable methods are needed to detect and diagnose the condition. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

Disclosure of Invention

The present invention provides a method for determining the response of a mammalian tumor to a chemotherapeutic agent, wherein the method comprises the steps of (a) isolating a biological sample from the mammal, (b) detecting the expression of the SPARC protein in the biological sample, and (c) quantifying the amount of the SPARC protein in the biological sample. The invention further provides a method for delivering a chemotherapeutic agent to a disease site in a mammal using albumin-binding protein as a therapy and using SPARC and an antibody to SPARC or SPARC-binding protein as a therapy, wherein the method comprises administering to the mammal a therapeutically effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises a chemotherapeutic agent conjugated to a compound capable of binding albumin-binding protein and a pharmaceutically acceptable carrier. In addition, the invention provides compositions comprising a chemotherapeutic agent conjugated to a compound capable of binding SPARC protein and a pharmaceutically acceptable carrier. In addition, the invention provides a delivery agent comprising a SPARC recognition group and a therapeutic agent, wherein the therapeutic agent is coupled to the SPARC recognition group. In addition, the present invention provides a method for delivering a chemotherapeutic agent to a tumor in a mammal, wherein the method comprises administering to the mammal a therapeutically effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises a chemotherapeutic agent coupled to a SPARC protein that binds albumin and a pharmaceutically acceptable carrier. The compositions of the invention may be small molecules, large molecules or proteins.

Drawings

FIG. 1 illustrates albumin and SPARC staining in MX-1 tumor xenografts.

FIG. 2 is a schematic illustration of transcytosis of paclitaxel by a monolayer of endothelial cells.

Detailed Description

It has now been found that there is an additional localization mechanism for compositions comprising albumin-binding proteins. Albumin-binding proteins, such as SPARC, cubilin, and TGF β, can be used to target therapeutic agents to disease sites characterized by overexpression of albumin-binding proteins.

The invention provides the use of a group conjugated to an agent, wherein the group is capable of binding to an albumin-binding protein, such as SPARC, cubilin or TGF β, and the agent is a therapeutic, imaging or delivery agent for a disease in which albumin-binding protein plays an important role and is overexpressed relative to normal tissues. Preferably, the albumin-binding protein is selected from SPARC, cubilin or TGF β. Most preferably, the albumin-binding protein is SPARC and the group that binds the albumin-binding protein is a SPARC recognition group. Suitable SPARC recognition groups include, but are not limited to, ligands, small molecules, antibodies.

The invention also provides methods for determining the response of a human or other mammalian tumor to a chemotherapeutic agent. The method comprises (a) isolating a biological sample from a human, (b) detecting the expression of the SPARC protein in the biological sample, and (c) quantifying the amount of the SPARC protein in the biological sample. Once the amount of SPARC expressed by the tumor is determined, the effectiveness of the chemotherapeutic agent can be determined, for example, by correlating the expression of SPARC with the dose of the given therapeutic agent. The invention also provides the use of SPARC antibodies as therapeutics or imaging agents for diseases in which SPARC plays an important role and is overexpressed relative to normal tissues.

Hereinafter, all albumin-binding proteins (including SPARC) are referred to as SPARC for simplicity. The SPARC protein is responsible for the accumulation of albumin in certain human tumors. Since albumin is the primary carrier of chemotherapeutic drugs, the expression level of SPARC is an indicator of the amount of chemotherapeutic drug that permeates and remains in the tumor. Thus, the expression level of SPARC is predictive of tumor response to chemotherapy.

Any suitable biological sample may be isolated from the mammal of interest in the context of the methods of the present invention. Preferably, the biological sample is isolated from the tumor, such as by tumor biopsy. Alternatively, the biological sample may be isolated from a bodily fluid of a mammal, including, for example, cerebrospinal fluid, blood, plasma, serum, or urine. Techniques and methods for isolating biological samples are known to those skilled in the art.

Any suitable pharmaceutically active agent may be used in the methods of the invention (e.g., a chemotherapeutic agent conjugated to a SPARC recognition group), so long as albumin is required for transport or binding of the active agent. Suitable active agents include, but are not limited to, tyrosine kinase inhibitors (genistein), biologically active agents (TNF or tTF), radionuclides (e.g.,¹³¹I、⁹⁰Y、¹¹¹In、²¹¹At、³²p and other known therapeutic radionuclides), doxorubicin, an ansamycin antibiotic, asparaginase, bleomycin, busulfan, cisplatin, carboplatin, nitrosurea mechlorethamine, capecitabine, chlorambucil, cytarabine, cyclophosphamide, camptothecin, dacarbazine, actinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide, epothilone, 5-fluorodeoxyuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea, isoxathic, fludarabine, cisplatin, and fludarabineDabigatran, ifosfamide, irinotecan, cyclohexylnitrosurea, mechlorethamine, mercaptopurine, meplhalan, methotrexate, rapamycin (sirolimus) and derivatives, mitomycin, mitotane, mitoxantrone, nitrosourea (nitrourea), paclitaxel, pamidronate (pamidronate), pentostatin, plicamycin, procarbazine, rituximab (rituximab, CD20 human murine chimeric monoclonal antibody), streptozotocin, podophyllotoxin, thioguanine, thiophosphine triamine, taxane, vinblastine, vincristine, vinorelbine, taxel (taxol), combretastatins, discodermolides, inverse platinum (transplatinum), vascular targeting agents, anti-vascular endothelial cell growth factor compounds ("anti-VEGF"), anti-epidermal growth factor receptor compounds ("anti-EGFR"), 5-fluorouracil and derivatives thereof.

Furthermore, the pharmaceutically active agent may be a toxin such as ricin a, a radionuclide, an Fc fragment of an antibody itself, a single chain antibody, a Fab fragment, a diabody, and the like. The selected pharmaceutically active agent may itself recognize and bind SPARC or be suitably attached to a SPARC recognition group that recognizes SPARC, including, for example, a protein or non-protein, an antibody, an Fc fragment of an antibody itself, a single chain antibody, a Fab fragment, a diabody, a peptide, or other non-protein small molecule.

One or more doses of one or more chemotherapeutic agents may be administered according to the methods of the invention. The type and amount of chemotherapeutic agent in the methods of the invention will depend on the standard chemotherapeutic regimen for the particular tumor type targeted. In other words, a particular cancer may be routinely treated with a single chemotherapeutic agent, while another may be routinely treated with a combination of chemotherapeutic agents. Methods for the binding or association of suitable therapeutics, chemotherapeutics, radionuclides, and the like to antibodies or fragments thereof are well described in the art.

Diseases for which the present invention is useful include any abnormal condition of proliferative, tissue remodeling, hyperplastic, exaggerated wound healing in body tissues including soft tissues, connective tissues, bones, solid organs, blood vessels, and the like. Examples of diseases that can be treated or diagnosed by the compositions of the invention include cancer, diabetes or other retinopathy, inflammation, arthritis, restenosis of vascular or artificial vascular grafts or intravascular devices, and the like.

The types of tumors detected and treated according to the present invention are typically those found in humans and other mammals. Tumors can also be the result of vaccination, as in experimental animals. Many types and forms of tumors are encountered in human and other animal settings and are not intended to limit the application of the present methods to any particular tumor type or species. As is known, tumors comprise abnormal tissue masses resulting from uncontrolled and progressive cell division, and are also commonly referred to as "tumors". The methods of the invention are useful, for example, in human tumor cells and associated stromal cells, solid tumors, and tumors associated with soft tissue, such as soft tissue sarcomas. The tumor or cancer may be located in the oral cavity and pharynx, the digestive system, the respiratory system, the bones and joints (e.g., bone metastases), soft tissues, the skin (e.g., melanoma) breast, the reproductive system, the urinary system, the eyes and orbit, the brain and central nervous system (e.g., glioma), or the endocrine system (e.g., thyroid gland) and is not necessarily limited to the primary tumor or cancer. Tissues associated with the oral cavity include, but are not limited to, the tongue and oral tissues. Cancer may arise in digestive system tissues, including, for example, the esophagus, stomach, small intestine, colon, rectum, anus, liver, gall bladder, and pancreas. Cancers of the respiratory system can affect the larynx, lung, and bronchi and include, for example, non-small cell lung cancer. Tumors can arise in the cervix, uterus, ovary vulva, vagina, prostate, testis, and penis, which make up the male and female reproductive system, as well as in the bladder, kidney, renal pelvis, and ureters, which include the urinary system. The tumor or cancer may be located in the head and/or neck (e.g., laryngeal and parathyroid cancers). Tumors or cancers may also be located in the hematopoietic or lymphatic systems and include, for example, lymphomas (e.g., malignant lymphogranulomatous disease and non-malignant lymphogranulomatous lymphoma), multiple myeloma or leukemias (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, and the like). Preferably, the tumor is located in the bladder, liver, ovary, kidney, gut, brain or breast.

SPARC proteins have affinity for a variety of ligands. Thus, the method of the present invention for delivering a therapeutic agent to a disease site is based on the following findings: compounds or ligands with affinity for SPARC, including albumin, can be used to deliver therapeutic agents to the site of disease with little or no delivery to normal tissues.

The invention also provides methods of transporting therapeutic compositions from blood vessels through endothelial barriers into tumor stromal tissue. The major obstacle to antibody therapy and chemotherapy is the migration through the endothelial barrier into the tumor stroma. Albumin passes the endothelial barrier by using the albumin receptor transport mechanism. The transport mechanism is the same as reported in the literature (gp60 and albondin) or other undiscovered mechanism. Therapeutic agents that are piggybacked on albumin have previously been reported to exhibit enhanced tumor uptake (Desai, N. et al incorporated endothielar transduction of nanoparticle album-bound paclitaxel (ABI-007) by endothielar gp60 receptors: a path inhibited by27th Annual Sanantonio Breast Cancer Symposium (SABCS) (2004), Abstract # 1071). In addition, enhanced transfer through the endothelial barrier can be achieved using physiological albumin transport mechanisms (Schnitzer, J.E.; Oh, P.J.biol. chem.269, 6072-6082 (1994)).

For small molecules, modifications can be made to increase drug affinity for albumin. For small molecule preparations, the solvent that prevents the drug from binding to albumin can be removed. Alternatively, the small molecule may be linked to albumin, an albumin antibody, a fragment thereof, or a ligand for an albumin-receptor, as described below.

For biomolecules such as proteins, antibodies and fragments thereof, the biologies can be engineered with albumin binding peptide designs such that they exhibit affinity for albumin. The peptide may be an albumin binding sequence, an antibody or antibody fragment of albumin, an antibody or antibody fragment of an albumin carrier (such as gp 60/albumin/scavenger receptor/or TGF-beta receptor), or an antibody to any protein found in the cell membrane kiln, albumin transporter. The invention also relates to antibodies or suitable fragments thereof prepared as chimeras having a SPARC valence and another effector of transendothelial transport (such as gp 60/albondin/scavenger receptor/or TGF-beta receptor) valence, or any protein found in the endothelial cell membrane kiln.

The invention also provides methods of disrupting SPARC-expressing tissues (e.g., tumor and heart valve restenosis tissues) via complement fixation and/or cell recruitment-mediated immune responses to the SPARC antibody. In this case, like Rituxan (an anti-CD 20 antibody), the effector moiety is an Fc fragment that mediates complement activation with direct destruction of SPARC-expressing cells or tissue destruction of immune cells via a cell-mediated immune response to recruit SPARC-expressing tissues.

The invention also provides methods of inhibiting SPARC activity using neutralizing antibodies to SPARC. Neutralizing antibodies have the ability to block the interaction of SPARC with its effectors in vivo. For example, neutralizing antibodies can block the interaction of SPARC with cell surface components or SPARC binds its natural ligands (e.g., albumin, growth factors, and Ca)²⁺)。

The invention also provides methods for determining the response of a human or other mammalian tumor to anti-SPARC therapy. The method comprises (a) isolating a biological sample from a human, (b) detecting the expression of the SPARC protein in the biological sample, and (c) quantifying the amount of the SPARC protein in the biological sample. Since anti-SPARC therapy relies on the binding of SPARC antibodies to SPARC in the diseased tissue, the presence of SPARC in the diseased tissue is essential for activity.

The invention further provides methods of using one or more diagnostic reagents (such as the antibodies or fragments thereof described above) that bind to the SPARC recognition group. Diagnostic agents include radioisotopes or radionuclides, MRI contrast agents, X-ray contrast agents, ultrasound contrast agents, and PET contrast agents. Methods for conjugation are known in the art.

The expression of SPARC protein in a sample can be detected and quantified by any suitable method known in the art. Suitable methods for protein detection and quantification include western blotting, enzyme-linked immunosorbent assay (ELISA), silver staining, BCA assay (Smith et al, anal. biochem., 150, 76-85(1985)), Lowry protein assay (described, for example, in Lowry et al, j. biol. chem., 193, 265-275 (1951)), which is a protein-copper complex-based colorimetric assay, and Bradford protein assay (described, for example, in Bradford et al, anal. biochem., 72, 248 (1976)), which is based on changes in absorbance of coomassie blue G-250 based on protein binding. Tumor biopsies can be analyzed by any of the previous methods or they can be analyzed by immunohistochemistry using an anti-SPARC antibody (monoclonal or polyclonal) together with a suitable imaging system (i.e., HRP substrate and HRP-conjugated secondary antibody).

Any suitable SPARC antibody can be used in the methods of the invention, so long as the antibody exhibits specific binding to SPARC. The antibody may be monoclonal or polyclonal; and can be produced by immunization of animals or by recombinant DNA techniques such as phage display and in vitro mutagenesis or synthesis of variable regions of antibody heavy and light chain genes. Polyclonal antibodies include, but are not limited to, human antibodies and humanized antibodies derived from, for example, birds (e.g., chickens), rodents (e.g., rats, mice, hamsters, guinea pigs), cows, goats, sheep, rabbits, and the like. Monoclonal antibodies include antibodies derived from a single clone of antibody-producing cells, including but not limited to human cells, and antibodies derived from cells of other animal types (e.g., chicken, rabbit, rat, mouse, hamster, guinea pig, cow, goat, sheep, and the like). Synthetic antibodies include antibodies produced by genetic engineering of the variable regions of the heavy and light chain genes using recombinant DNA techniques. Synthetic antibodies also include chemically synthesized antibody fragments with SPARC binding activity or antibodies derived from phage display or similar techniques.

For human use, in order to avoid immunogenicity and immune responses, it is preferred to use a humanized anti-SPARC antibody or suitable fragment such as Fab', Fab or Fab 2. Humanized antibodies or fragments thereof can be generated, for example, using one of the following established methods: 1) humanized antibodies can be constructed using a human IgG backbone in place of the variable CDR domain of SPARC antibody, where the heavy and light chains are expressed separately under separate promoters or are co-expressed under one promoter with an IRES sequence; 2) the humanized monoclonal antibody can be generated by designing and modifying a mouse with a human immune system; 3) phagemids (M13, lambda coliphage, or any phage system capable of surface display) can be used to generate humanized antibodies to SPARC. To construct full-length antibodies, the variable regions can be transferred to the CDRs of the heavy and light chains. Co-expression of heavy and light chains in mammalian cells such as CHO, 293 or human bone marrow cells produces full-length antibodies. Similarly, Fab', Fab or Fab2 fragments and single chain antibodies can be prepared using well established methods.

Antibodies to SPARC are also not limited to intact antibodies or fragments of antibodies that retain the SPARC binding site (e.g., Fab and Fab 2). The antibodies are also not limited to any type of antibody, such as IgM, IgA, IgG, IgE, IgD and IgY. The antibody is also not limited to bivalent antibodies, monovalent or chimeras with a monovalent for SPARC and another valency for an effector (such as tTF or ricin a). Humanized antibodies are not limited to IgG. The same technique can be used to generate all other classes of antibodies such as IgE, IgA, IgD, IgM, each with different antibody-dependent cellular cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC) activities appropriate for specific disease targeting. Functional fragments of antibodies can be produced by restriction proteolysis. These fragments may be monovalent (e.g., Fab') or bivalent (e.g., Fab 2). Fragments may also be synthesized in E.coli as single chain scfv or diabodies.

The invention further provides compositions comprising a pharmaceutically active agent capable of exerting its pharmacological effect directly or coupled to a compound capable of binding SPARC or other albumin binding moiety, and a pharmaceutically acceptable carrier. A delivery agent, which may be a pharmaceutical composition comprising a pharmaceutically active agent coupled to a SPARC recognition group, is administered to a mammal, such as a human, in an amount such that a therapeutically effective amount of the pharmaceutically active agent is delivered to the mammal.

The invention also provides methods for delivering a chemotherapeutic agent to a tumor in a mammal. The method comprises administering to a human or other mammal a therapeutically effective amount of a pharmaceutical composition comprising a chemotherapeutic agent coupled to a compound or ligand capable of binding SPARC protein and a pharmaceutically acceptable carrier. The descriptions of chemotherapeutic agents, tumors, mammals, and components thereof set forth herein in connection with other embodiments of the invention also apply to those same aspects of the above-described methods of delivering a chemotherapeutic agent to a tumor.

Preferably, the pharmaceutical composition does not contain more than 50% of the therapeutic agent in nanoparticle form. More preferably, the pharmaceutical composition does not contain more than 10% of the therapeutic agent in nanoparticle form. Even more preferably, the pharmaceutical composition does not comprise more than 5%, or more than 4% or more than 3% of the therapeutic agent in nanoparticle form. In a more preferred embodiment, the pharmaceutical composition does not comprise more than 2% or more than 1% of the therapeutic agent in nanoparticle form. Most preferably, the pharmaceutical composition does not contain any therapeutic agent in the form of nanoparticles.

The invention also provides methods for delivering chemotherapeutic agents to tumors in humans or other mammals. The method comprises administering to the human or other mammal a therapeutically effective amount of a delivery agent, such as a pharmaceutical composition, wherein the delivery agent (e.g., pharmaceutical composition) comprises a chemotherapeutic agent coupled to a SPARC recognition group. For example, the chemotherapeutic agent may be conjugated to a SPARC recognition group (e.g., an antibody that recognizes a SPARC protein) or only to a SPARC antibody. The pharmaceutical composition preferably comprises a chemotherapeutic agent coupled to the SPARC recognition group and a pharmaceutically acceptable carrier. The descriptions of chemotherapeutic agents, tumors, mammals, and components thereof set forth herein in connection with other embodiments of the invention also apply to those same aspects of the above-described methods of delivering a chemotherapeutic agent to a tumor.

In other embodiments, the invention provides methods of delivering a pharmaceutically active agent by way of a SPARC recognition group to the site of a disease characterized by overexpression of SPARC or another albumin-binding protein or marker in humans or other animals expressing the protein or marker. The disease includes abnormal conditions of proliferation, tissue remodeling, hyperplasia, enlarged wound healing in body tissues (e.g., soft tissues, connective tissues, bones, solid organs, blood vessels, etc.). Examples of diseases that can be treated or diagnosed by administering a pharmaceutical composition comprising a therapeutic agent coupled to a compound or ligand that binds to SPARC protein or another albumin-binding protein include cancer, diabetes or other retinopathy, inflammation, arthritis, restenosis of vascular or artificial vascular grafts or intravascular devices, and the like. The descriptions of pharmaceutically active agents, tumors, mammals, and components thereof set forth herein in connection with other embodiments of the invention are also applicable to those same aspects of the above-described methods of delivering pharmaceutically active agents.

In still other embodiments, the invention provides methods for delivering a pharmaceutically active agent (e.g., a SPARC antibody alone or a chemotherapeutic agent bound to a SPARC recognition group such as a SPARC antibody, radiolabeled SPARC antibody, and the like) to a site of a disease characterized by overexpression of SPARC in humans or other animals expressing the protein or marker. The disease includes abnormal conditions of proliferation, tissue remodeling, hyperplasia, exaggerated wound healing in body tissues (e.g., soft tissues, connective tissues, bones, solid organs, blood vessels, etc.). Examples of diseases that can be treated or diagnosed by administration of a pharmaceutical composition comprising an anti-SPARC therapy include cancer, diabetes or other retinopathy, inflammation, arthritis, restenosis of vascular or artificial vascular grafts or intravascular devices, and the like. The descriptions of pharmaceutically active agents, tumors, mammals, and components thereof set forth herein in connection with other embodiments of the invention are also applicable to those same aspects of the above-described methods of delivering pharmaceutically active agents.

In other embodiments, the methods of the invention comprise administering to a mammal a therapeutically effective amount of a pharmaceutical composition comprising a chemotherapeutic agent coupled to a compound or ligand capable of binding SPARC protein. The chemotherapeutic agent can be coupled to the compound or ligand capable of binding the SPARC protein using any suitable method. Preferably, the chemotherapeutic agent is chemically coupled to the compound via a covalent bond (including, for example, a disulfide bond).

The invention also provides methods comprising administering to a mammal a therapeutically effective amount of a pharmaceutical composition comprising a chemotherapeutic agent or a radioactive element coupled to a SPARC recognition group. The chemotherapeutic or radioactive agent may be conjugated to an antibody that recognizes SPARC using any suitable method. Preferably, the chemotherapeutic agent can be chemically coupled to the compound via covalent bonds (including, for example, disulfide bonds).

Preferably, the compounds or ligands useful in the methods of the invention are capable of binding to SPARC proteins. In a preferred embodiment of the invention, the compound is a ligand that binds to SPARC protein. Examples of suitable ligands include calcium cation (Ca)²⁺) Copper cation (Cu)²⁺) Iron cation (Fe)²⁺) Platelet Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), collagen (e.g., collagen I, collagen II, collagen III, collagen IV, collagen V, and collagen IX), vitronectin (vitronectin), thrombospondin-1, endothelial cells, platelets, albumin, and hydroxyapatite cations. In another preferred embodiment of the invention, the compound is a small molecule. The term "small molecule" refers to any molecule having a molecular weight of less than about 600. Examples of suitable small molecules include proteins, nucleic acids, carbohydrates, lipids, coenzymes (e.g., vitamins), antigens, hormones, and neurotransmitters. Preferably, the small molecule is a chemical (e.g., organic or inorganic chemical) peptide or peptidomimetic, a protein, or a carbohydrate. In yet another preferred embodiment of the invention, the compound is an antibody directed against the SPARC protein. Suitable antibodies or fragments thereof that bind to SPARC proteins can be used in the methods of the invention.

SPARC expression in tumor tissues has been demonstrated in almost all cancer types. Processing of SPARC on tumor tissue has been shown to result from either tumor expression of SPARC or from stromal cell origin. By administration of exogenous SPARC, the SPARC phenotype of the tumor can be switched from SPARC negative to SPARC positive. Thus, SPARC-positive tumors will become susceptible to chemotherapeutic agents. Alternatively, SPARC may be radiolabeled or conjugated to a different toxin to confer its ability to kill tumors directly or indirectly.

SPARC can be synthesized and purified using known techniques. Cells expressing exogenous SPARC can be generated by placing the SPARC structural gene/cDNA under the control of a strong promoter/translation initiation site and the vector transfected into mammalian cells to drive the expression of SPARC in these cells. Alternatively, SPARC can be expressed using baculovirus or other viruses (e.g., adenovirus). SPARC expressed by these cells can be purified by conventional purification methods such as ion exchange, size exclusion or C18 chromatography. The purified SPARC can be formulated in saline with a preservative and administered intravenously, by aerosol, by subcutaneous injection, or other methods.

The invention also provides methods for delivering chemotherapeutic agents to mammalian tumors. The method comprises administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising a chemotherapeutic agent coupled to a SPARC protein that binds albumin and a pharmaceutically acceptable carrier. The descriptions of chemotherapeutic agents, tumors, mammals, and components thereof set forth herein in connection with other embodiments of the invention also apply to those same aspects of the above-described methods of delivering a chemotherapeutic agent to a tumor.

For in vivo use, the chemotherapeutic agent coupled to a compound or ligand capable of binding the SPARC protein is desirably formulated into a pharmaceutical composition comprising a physiologically acceptable carrier. Any suitable physiologically acceptable carrier may be used in the context of the present invention and such carriers are well known in the art.

For in vivo use, the anti-SPARC therapeutic agent is desirably formulated into a pharmaceutical composition comprising a physiologically acceptable carrier. Any suitable physiologically acceptable carrier may be used in the context of the present invention and such carriers are well known in the art.

The carrier is typically a liquid, but may also be a solid, or a combination of liquid and solid components. As desired, the carrier is a physiologically acceptable (e.g., pharmaceutical or pharmacologically acceptable) carrier (e.g., excipient or diluent). Physiologically acceptable carriers are well known and readily available. The choice of vector is determined, at least in part, by the location of the target tissue and/or cells and the particular method used to administer the composition.

Typically, the compositions may be prepared as injectables (whether as liquid solutions or suspensions); solid forms suitable for solution or suspension preparation by addition of a liquid prior to injection may also be prepared; and may also emulsify the formulation. Pharmaceutical formulations suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing known protein stabilizers and cryoprotectants, formulations containing sesame oil, peanut oil, or propylene glycol in aqueous solution, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the formulation must be sterile and must be liquid to the extent that easy injection is possible. Which must be stable under the conditions of preparation and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions of the active substance as a free base or pharmacologically acceptable salt may suitably be prepared in water in admixture with a surfactant such as hydroxycellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

Chemotherapeutic agents (e.g., anti-SPARC therapies) coupled to compounds or ligands that bind SPARC proteins can be formulated into the compositions in neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids (e.g., hydrochloric or phosphoric acids) or organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like). Salts formed with the free carboxyl groups can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides), as well as organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine, and the like).

The composition may further comprise any other suitable component, particularly for improving the stability of the composition and/or its end use. Thus, there are a variety of suitable formulations of the compositions of the present invention. The following formulations and methods are illustrative only and are in no way limiting.

Formulations suitable for administration by inhalation include aerosol formulations. Aerosol formulations may be placed in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like. It can also be formulated as an atmospheric formulation for delivery by a nebulizer or atomizer.

Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type previously described. In a preferred embodiment of the invention, the chemotherapeutic agent coupled to the compound that binds SPARC protein (e.g., anti-SPARC therapy) is formulated for injection (e.g., parenteral administration). In this regard, the formulations are suitable for intratumoral administration, as desired, but may also be formulated for intravenous injection, intraperitoneal injection, subcutaneous injection, and the like.

Formulations suitable for anal administration may be prepared as suppositories by mixing the active ingredient with various bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration contain, in addition to the active ingredient, suitable carriers known in the art and may be presented in the following manner: pessaries, tampons, creams, gels, pastes, foams or sprays.

In addition, the composition may comprise additional therapeutic or biologically-active agents. For example, therapeutic factors may be present that are effective for the treatment of a particular indication. Factors that control inflammation, such as ibuprofen or steroids, can be part of reducing swelling and inflammation and physiological distress associated with in vivo administration of pharmaceutical compositions.

Detailed Description

The following examples further illustrate the invention but should not be construed as in any way limiting its scope.

Example 1

This example illustrates the co-localization of SPARC with albumin in MX-1 tumor xenografts.

Paclitaxel albumin nanoparticles (Abraxane, ABX or ABI-007) have been shown to have improved response rates (33% versus 19%, p < 0.0001) over Taxol (TAX) in a phase 3 metastatic breast cancer assay (see, e.g., O' shaughanesy, SABCS). Recently, albumin-mediated transendothelial transport of paclitaxel (P) and increased intratumoral accumulation of ABX versus TAX paclitaxel have been demonstrated (see, e.g., Desai, SABCS 2003). Albumin binds SPARC (see, e.g., Schnitzer, j.biol.chem., 269, 6072-82 (1994)).

The MX-1 tumor cell line is derived from human breast cancer. Serial cryosections of human MX-1 tumor xenografts, human primary breast tumor tissue (n ═ 141), and normal human breast tissue (n ═ 115) were immunostained and fractionated for albumin, SPARC (using anti-SPARC antibodies), and caveolin-1 staining (0-4). Cultured MX-1 cells were also immunostained for SPARC. Paclitaxel albumin nanoparticles (Abraxane, ABX or ABI-007) and Taxol (TAX) were prepared with radioactive paclitaxel (P) (20mg/kg IV) and used to determine the biological distribution of paclitaxel in normal tissues of athymic mice.

Albumin stained as aggregated and co-localized with SPARC in MX-1 tumors (fig. 1). Caveolin-1 staining confirmed that the density of blood vessels in the albumin-containing region was not different from that in the albumin-free region. SPARC expression from MX-1 cultured cells was confirmed by positive staining with anti-SPARC antibody. For 7/10 tissues, paclitaxel accumulation in normal tissues (SPARC negative) was significantly lower for ABX than for TAX (p.ltoreq.0.004). Compared to 1% of normal tissue, 46% of human primary breast tumors showed strong SPARC staining (> 2 grade) (p < 0.0001). In a subset of 50 tumor tissues, SPARC expression was independent of grade, ER status or PgR status; however, there is a tendency for high SPARC expression in p 53-negative tumors.

The co-localization of albumin and SPARC suggests that SPARC, through its albumin binding activity, may act as an intratumoral target for albumin binding in breast tumors. Since transport of paclitaxel in ABX is dependent on albumin (see, e.g., Desai SABCS, 2003), this may explain the improved tumor accumulation of ABX over TAX. ABX accumulation was lower in normal tissues than TAX, consistent with the lack of SPARC expression in normal tissues. Screening patients for SPARC allows identification of patients that are more responsive to ABX. The presence of SPARC in these tumors allows for targeting and treatment with anti-SPARC antibodies.

Example 2

This example illustrates SPARC expression in MX-1 tumor cells.

MX-1 cells were cultured on coverslips and stained with an antibody against human SPARC using methods known in the art. Antibody staining was observed, which confirmed that MX-1 expressed SPARC. These results suggest that SPARC expression detected in MX-1 tumor cells is a result of SPARC secretion by MX-1 tumor cells. For MX-1 tumor cells, staining was stronger than normal primary cells such as HUVEC (human umbilical vein endothelial cells), HLMVEC (human lung microvascular endothelial cells) and HMEC (human mammary epithelial cells). While most SPARC staining was internal SPARC, significant levels of surface SPARC were detected as confirmed by confocal microscopy and staining of non-permeabilized cells.

Example 3

This example illustrates the overexpression of the SPARC protein in human breast cancer cells.

SPARC expression in human breast cancer cells was determined using a tumor array of Cybrdi Inc. (Gaithersburg, Md.). The results of the analysis are shown in table 1. Staining intensity was graded as "negative" to 4+, with higher numbers corresponding to greater overexpression intensity. SPARC staining was positive (2+ and above) in 49% of breast cancers compared to 1% of normal tissues (p < 0.0001).

Example 4

This example illustrates endothelial receptor (gp60) -mediated caveolar transcytosis of paclitaxel albumin nanoparticles (ABI-007).

Paclitaxel (P) albumin nanoparticles (Abraxane, ABX or ABI-007) have improved response rates (33% vs 19%, P < 0.0001) over Taxol in a breast cancer assay demonstrating phase III metastasis (SABCS, O' Shaughessy et al, 2003). The cremophors in Taxol (TAX) entrap P micelles in plasma, reducing the paclitaxel available to cellular partitions (see, e.g., Sparreboom et al, cancer. Res., 59, 1454 (1999)). Studies in athymic mice showed that ABX had a 30-40% higher intratumoral paclitaxel concentration compared to an equivalent dose of TAX (SABCS, Desai et al, 2003). Albumin is transported through Endothelial Cells (ECs) via specific receptor (gp60) -mediated pocket transport (see, e.g., John et al, am.j. physiol., 284, L187 (2001)). It is hypothesized that albumin-bound paclitaxel in ABX may be transported through tumor microvascular EC via gp60, and this mechanism may be particularly active for ABX as compared to TAX.

A series of experiments were performed to evaluate ABX and TAX for the binding and transport of paclitaxel by Human Umbilical Vein Endothelial Cells (HUVEC) and human pulmonary microvascular endothelial cells (HLMVEC). Fluorescent Paclitaxel (FP) was used as a probe and fluorescent ABX and TAX were formulated with FP to detect binding and transport of paclitaxel through EC monolayers cultured on transwell devices.

For ABX, paclitaxel-bound cells (HUVECs) were 10-fold higher than TAX. Paclitaxel transport from ABX through the EC monolayer was increased 2-3 fold and 2-4 fold, respectively, for HUVEC and HMVEC compared to TAX. Transport is dependent on albumin. The transport of paclitaxel from ABX is inhibited by the presence of anti-SPARC antibodies, which are known to bind gp60, a receptor essential for caveolar albumin transcytosis. Known inhibitors of caveolar transcytosis, NEM and β -methyl cyclodextrin (BMC), also inhibit paclitaxel transport from ABX through the endothelial monolayer (figure 2). Inhibition of foveal transport reduces P transport from ABX to levels of TAX transport.

These results demonstrate that paclitaxel is actively transported from ABX through EC via gp 60-mediated caveolar transcytosis, whereas P appears to be transported from TAX at a 2-4 fold lower rate, primarily through a paracellular (non-caveolar) mechanism. This pathway may result in some degree of the observed intratumoral concentration of paclitaxel with increased ABX relative to TAX. Cremophor in TAX inhibits transcytosis of paclitaxel by endothelial cells.

Example 5

This example demonstrates the correlation of SPARC overexpression with high response rates in head and neck squamous carcinoma using nanoparticle albumin-bound paclitaxel (ABI-007).

Head and neck (H)&N) and squamous cell carcinoma of the anal canal (SCC) patients, response rates of 78% and 64%, respectively, were observed for intra-arterially delivered nanoparticle albumin-bound paclitaxel (Abraxane, ABX or ABI-007) (see, e.g., damascolli et al, Cancer, 92(10), 2592-2602(2001), and damascolli et al, AJR, 181, 253-260 (2003)). In comparing the in vitro cytotoxicity of ABX and Taxol (TAX), it was observed that the squamous neck (A431) line showed an improved IC for ABX (0.004. mu.g/ml) over TAX (0.012. mu.g/ml)₅₀. Recently, albumin-mediated transendothelial pit transport of paclitaxel (P) and increased intratumoral accumulation of P for ABX compared to TAX has been demonstrated (see, e.g., Desai, SABCS 2003).

Human H & N tumor tissue (N ═ 119) and normal human H & N tissue (N ═ 15) were immunostained and SPARC staining was graded using tumor and normal tissue arrays (0-4). Immunostaining was performed using polyclonal rabbit anti-SPARC antibody. In a new phase I dose escalation study (ABX IV given for more than 30 minutes q3w), head and neck cancer patients (n ═ 3) were analyzed for their subgroup response to ABX.

Comparison to 0% (0/15), H in Normal tissue&SPARC was overexpressed (grade. gtoreq.2) in 60% (72/119) of N tumors (p < 0.0001). In phase I study, 2/3H&The patient with N is 135mg/m²(1pt) and 225mg/m²(1pt) dose level Partial Response (PR) was achieved after 2 rounds of treatment. At 260mg/m²The next 1/3 patients continued to develop.

SPARC was found to be overexpressed in 60% of squamous H & N tumors. This may explain the high ABX single-agent activity previously seen in squamous H & N cancer due to albumin-bound paclitaxel binding to SPARC expressed in these tumors. Patients with 2/3 squamous H & N tumors in a new phase I study reached PR.

Human H & N tumor tissue (N ═ 119) and normal human H & N tissue (N ═ 15) were immunostained and SPARC staining was graded using tumor and normal tissue arrays (0-4). Immunostaining was performed using polyclonal rabbit anti-SPARC antibody. In contrast to 0% (0/15) in normal tissue, 60% (72/119) of H & N tumors over-expressed SPARC (grade. gtoreq.2) (p < 0.0001). This could explain the high ABX mono-agent activity seen in d squamous H & N carcinoma due to albumin-bound paclitaxel binding to SPARC expressed in these tumors before.

In a new phase I dose escalation study (ABX IV given for more than 30 minutes q3w), head and neck cancer patients (n ═ 3) were analyzed for their subgroup response to ABX. In phase I study, 2/3H&The patient with N is 135mg/m²(1pt) and 225mg/m²(1pt) dose level Partial Response (PR) was achieved after 2 rounds of treatment. At 260mg/m²The next 1/3 patients continued to develop. Tumor tissue from these patients stained for SPARC and 1 responding patient showed strong over-expression of SPARC.

In another phase II clinical study of head and neck (H & N) Squamous Cell Carcinoma (SCC) patients treated with intra-arterial Abraxane, an overall response rate of 68% was noted. Of 10 responding patients whose tissues were analyzed for SPARC staining, 70% were found to strongly overexpress SPARC.

Example 6

This example illustrates internalization of labeled albumin into MX-1 tumor cells and co-localization with intracellular SPARC expression within MX-1 cells.

MX-1 cells were cultured on coverslips and permeabilized with the appropriate agent. Cells were exposed to fluorescent albumin and after washing were exposed to SPARC antibody. And thereafter exposed to a secondary antibody having a different fluorescent label than albumin. Co-localization of labeled albumin with the presence of SPARC within the cell was surprisingly observed, indicating that albumin is rapidly internalized and targets intracellular SPARC.

Example 7

This example demonstrates the improved endothelial transcytosis of a pharmaceutical composition comprising paclitaxel and albumin via gp60 (albumin receptor) compared to Taxol.

Human lung microvascular endothelial cells (HLMVEC) were grown to confluence on a transwell. A pharmaceutical composition of the invention comprising paclitaxel and albumin at a concentration of 20 μ g/mL or Taxol comprising fluorescent paclitaxel (Flutax) is added to the chamber above the transwell.

The transport of paclitaxel from the upper compartment to the lower compartment by transcytosis was continuously monitored using a fluorometer. A control containing Flutax alone without albumin was also used. The control with flutmax showed no transport, confirming the integrity of the confluent HLMVEC monolayer. Paclitaxel transport from the albumin-paclitaxel composition was faster in the presence of 5% HSA (physiological concentration) than paclitaxel from Taxol. The transport rate constants (Kt) of the albumin-paclitaxel composition and Taxol are 1.396h respectively^-1And 0.03h^-1. For the albumin-paclitaxel composition, the total amount of paclitaxel transported through the monolayer is three times higher than Taxol. Therefore, the temperature of the molten metal is controlled,the use of albumin or other suitable mimetics comprising antibodies or fragments against gp60receptor or other endothelial cell receptors can aid in the transport of a desired therapeutic agent across the endothelial barrier into the tumor stromal tissue.

Example 8

This example demonstrates that anti-SPARC antibodies specifically bind to SPARC.

Whole cell extracts were prepared from HUVEC cells by sonication. Proteins were separated on 5-15% SDS-PAGE, transferred to PVDF membrane and visualized with polyclonal antibodies to SPARC and monoclonal antibodies to SPARC. Both antibodies reacted with a single band of 38kDa, the correct molecular weight for SPARC being 38 kDa. When MX-1 was analyzed by the same method, SPARC was detected in either the clarified cell lysate or the membrane-rich membrane fraction.

Example 9

This example demonstrates the lack of SPARC expression in normal tissues.

Normal human and mouse tissues were immunostained and SPARC staining was graded using tumor and normal tissue arrays (0-4). Immunostaining was performed using polyclonal rabbit anti-SPARC antibody. SPARC was not expressed in any normal tissue except the esophagus. Similarly, SPARC is not expressed in any normal mouse tissue except the kidney of female mice. However, it is likely that this expression is due to follistatin (follistatin) which is homologous to SPARC.

SPARC expression in human normal tissues

Mouse normal tissue

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary terms (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims (9)

1. Use of a therapeutic agent conjugated to a compound capable of binding to a 281 amino acid SPARC glycoprotein in the manufacture of a medicament for treating a tumor in a mammal, comprising:

(1) isolating a biological sample from the mammal and,

(2) detecting the expression of the SPARC protein in the biological sample,

(3) quantifying the amount of SPARC protein in a biological sample, and

(4) administering to the mammal a therapeutically effective amount of a therapeutic agent conjugated to a compound capable of binding to a 281 amino acid SPARC glycoprotein if the amount of SPARC protein in the biological sample.

2. The use of claim 1, wherein the biological sample is isolated from a tumor.

3. The use of claim 1, wherein the biological sample is isolated from a bodily fluid.

4. The use of claim 3, wherein the body fluid is selected from the group consisting of cerebrospinal fluid, blood, plasma, serum and urine.

5. The use of claim 1, wherein the tumor is located in the head, neck, bladder, liver, ovary, kidney, gut, brain or breast.

6. The use of claim 1, wherein the mammal is a human.

7. The use of claim 1, wherein the detection of SPARC with a SPARC binding compound.

8. The use of claim 7, the SPARC binding compound is an antibody that binds to a 281 amino acid SPARC glycoprotein.

9. The use of claim 1, wherein the therapeutic agent is selected from the group consisting of tyrosine kinase inhibitors, bioactive agents, biomolecules, radionuclides, doxorubicin, ansamycin antibiotics, asparaginase, bleomycin, busulfan, cisplatin, carboplatin, nitrosurea mustard, capecitabine, chlorambucil, cytarabine, cyclophosphamide, camptothecin, dacarbazine, actinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide, epothilone, 5-fluorodeoxyuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, mercaptopurine, meplhalan, methotrexate, rapamycin (sirolimus), mitomycin, mitotane, mitoxantrone, and combinations thereof, Nitrosoureas, paclitaxel, pamidronate, pentostatin, plicamycin, procarbazine, rituximab, streptozotocin, etoposide, thioguanine, thiophosphine triamide, taxanes, vinblastine, vincristine, vinorelbine, taxotere, discodermolides, platinations, anti-vascular endothelial growth factor compounds ("anti-VEGF"), anti-epidermal growth factor receptor compounds ("anti-EGFR"), 5-fluorouracil, and derivatives and combinations thereof.