JP2007515924A - Vimentin-oriented diagnostic methods and treatment methods for neoplastic diseases exhibiting multidrug resistance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect multidrug resistance in neoplastic or damaged cells, or multidrug resistant (MDR) neoplastic or damaged cells.
A method for detecting an increase in cell surface expression of vimentin protein in the cells as compared to the cell surface expression level of vimentin protein in normal cells or non-MDR neoplastic cells.
[Selection figure] None

Description

  The present invention relates to the field of diagnostic and therapeutic methods. In particular, the present invention relates to the detection and treatment of neoplastic and / or damaged cells, as well as the detection and treatment of neoplastic and / or damaged cells exhibiting multidrug resistance.

  Diseases such as cancer or those caused by pathogen infections are often treated with drugs (eg, chemotherapeutic agents and antibiotics). In order to kill cancer or diseased cells, the drug (s) must deliver effective doses to enter the cells and interfere with essential biochemical pathways. However, some cells may avoid killing by developing resistance to drugs (called “drug resistance”). In addition, in some cases, including cancer cells (also called tumor cells or neoplastic cells), damaged cells (eg, pathogen-infected cells), or drugs that were not originally included in the treatment. It may also develop resistance to a broad drug spectrum. This phenomenon is called “multidrug resistance” (MDR). There are different types of MDR, each associated with a different biological mechanism, and there are biological “markers” that are specific for different types of MDR, It is clinically useful in detecting and diagnosing MDR.

  MDR can be associated with infectious agents such as cancer cells or viruses, bacteria, parasites, and other microorganisms that may be present in cells and body fluids. The appearance of the MDR phenotype is a major cause of failure in the treatment of infectious diseases (see Non-Patent Documents 1 and 2). Patient cross-resistance to various antibacterial and anticancer agents, which are structurally and functionally distinct, can be a significant problem for pathogen-infected patients. Similarly, the development of cancer cells with multidrug resistance is a major reason for the poor treatment of cancer patients (see Non-Patent Document 3).

  Multidrug resistance is multifactorial. The classical MDR mechanism involves genetic alterations in cell membrane proteins (P-glycoprotein or MDR1) that are evolutionarily highly conserved. This protein actively transports or pumps drugs out of cells or microorganisms (see Non-Patent Documents 4 and 5). Both human cancer cells and infectious bacterial pathogens appear to develop classical MDR through a mechanism involving overexpression of P-glycoprotein by amplification of the gene encoding P-glycoprotein. Overexpression of P-glycoprotein mRNA or protein in MDR cancer cells or pathogen-infected cells is a biological marker for MDR. In order to diagnose and treat MDR cancer and pathogen infections, diagnostic tests and treatment methods that utilize overexpression of P-glycoprotein have been conventionally developed (Non-patent Document 6). However, since various normal tissues express various amounts of P-glycoprotein, even if a therapeutic method targeting P-glycoprotein on the surface of MDR cancer cells can be made, If so, it can also affect normal tissues that exhibit relatively high levels of P-glycoprotein expression, such as liver, kidney, stem cells, and blood brain barrier epithelium.

  “Atypical MDR” is used to describe an MDR cancer cell or pathogen whose mechanism of multidrug resistance is unknown, novel, or different from the classical mechanism involving P-glycoprotein Is the term. For example, human lung tumors have multidrug resistance but do not show changes in P-glycoprotein (see Non-Patent Document 7). Rather, the tumor in question expresses another drug transport factor (multidrug resistance associated protein or “MRP1”). A new MDR mechanism has recently been described, including lung resistance-related proteins. This protein is a marker for this type of atypical MDR (Patent Document 1). Another example of an atypical marker for MDR is MRP5. This is a novel mammalian efflux pump for nucleoside-like drugs (Patent Document 2) and certain glycosphingolipids (see Patent Document 3). It should be noted that MDR cells may simultaneously express more than one MDR marker (classical P-glycoprotein marker and atypical marker) on the same cell, and multiple markers It is expressed independently (Non-patent Document 8). Thus, it is possible to combine multiple therapies directed against more than one cell surface MDR marker, possibly blocking more than one MDR mechanism.

  Intermediate filaments are a major component of the higher eukaryotic cytoskeleton. Intermediate filaments are composed of several separate structurally related proteins, and various intermediate filament protein genes are expressed in various tissues. Studies involving spontaneously and experimentally induced mutations in intermediate filament genes have shown that intermediate filaments work to enhance the mechanical stability of epithelial and myocytes (non-patent literature). 9). Vimentin (gi / 4507895) is an intracellular homodimeric protein found in class III intermediate filaments in Z-disks of mesenchymal and other non-epithelial cells and skeletal and cardiomyocytes ( For example, refer nonpatent literature 9,10,11). Vimentin is a phosphoprotein whose phosphorylation is enhanced during cell division. Both human and rodent vimentin genes have been elucidated (see, for example, Non-Patent Documents 12 and 13). Purified eukaryotic vimentin has a molecular weight of 54 kDa and is soluble in the natural form, but becomes insoluble when denatured. Regulation of the vimentin gene appears to participate in several steps of viral infection (Non-patent Document 12).

  Various cytoskeletal modifications are associated with malignant cell deformation and are used as prognostic factors. Vimentin expression is characteristic of cells of mesenchymal origin In contrast to the internal context, vimentin synthesis is characteristic of all proliferating cells in vitro, regardless of mesenchymal origin, and some progenitor cells The switch is turned off as soon as it is differentiated. Vimentin gene expression is up-regulated in certain metastatic tumor cells and is therefore a marker for the progression of tumor formation (see Non-Patent Documents 12 and 14).

  Vimentin and other IF proteins have been conventionally used in the histological classification of human tumors (for non-patent references 15, 16). In particular, vimentin has been used as a marker for dedifferentiation in several types of tumors.

  It describes tumor markers that co-exist with vimentin and other intermediate filaments. For example, De Bernard and Marina (Patent Document 4) are a novel neuroblastoma marker called VIP54, for proteins closely associated with vimentin and desmin filaments, and as a marker for tumor development and / or progression. Describes the detection of intermediate filaments (especially class III vimentin or desmin filaments) and their use as internal markers for cell imaging.

  Finally, there are several examples of various types of human solid tumors and solid tumor cell lines that show changes in vimentin expression levels and subcellular distribution, often associated with the emergence of multidrug resistance. 17, 18).

Regardless of the mechanism of MDR development (whether naturally occurring or drug-induced) in both humans and animals, classical and atypical in cancer cells and damaged cells that are not cancer There remains a need for means to detect, treat, prevent, and / or reverse the development of a dynamic MDR phenotype. In addition, the ability to identify multi-drug resistant cells and the ability to utilize identifying reagents has led to the treatment, monitoring, diagnosis and medical imaging of multi-drug resistant cancers and multi-drug resistant damaged cells. Has clinical potential to improve
Rome LH et al., PCT Publication WO9962547 Friedland and Schutz, PCT Publication WO0058471 U.S. Patent No. 6,090,565 PCT Gazette WO0127269 Davis J., Science 264: 375-382, 1994 Poole, K., Cur. Opin. Microbiol. 4: 500-5008, 2001 Gottesman, MM, Ann. Rev. Med. 53: 615-627, 2000 Volm M., et al., Cancer 71: 3981-3987, 1993 Bradley and Ling, Cancer Metastasis Rev. 13: 223-233, 1994 Szakacs G. et al., Pathol. Oncol. Res. 4: 251-257, 1998 Cole SP et al., Science 258: 1650-1654, 1992 Grandjean F. et al., Anticancer Drugs, 12: 247-258, 2001 Evans RM, BioEssays, 20: 79-86, 1998 Hermann, H., Aebi, U. (2000) Curr. Opin. Cell Biol. 12: 79-90 ibid. (1998) Subcell Biochem. 31: 319-62 Kryszke et al. (1998) Pathol. Biol. 46: 39-45 Paulin, D. (1989) Pathol. Biol. 37: 277-82 Osborn et al. (1989) Curr. Comm. In Mol. Biol., Cold Spring Harbor Press Ramaekers et al. (1982) Cold Spring Harb. Symp. Quant. Biol. 46: 331-339 Thomas et al. (1999) Clin. Cancer Res. 5: 2698-2703 Conforti G., et al., Br. J. Cancer, 3: 505-511, 1995 Moran E. et al., Eur. J. Cancer, 33: 652-660, 1997

  The present invention is based in part on the following discoveries. That is, the full length of vimentin, which is usually an intracellular protein, is expressed on the surface of neoplastic cells and damaged cells in its full length, and is expressed in large amounts on multidrug resistant (MDR) neoplastic cells and MDR damaged cells. It is a discovery that will be done. In contrast to other cell surface MDR markers (eg P-glycoprotein), vimentin expressed on the surface of drug-sensitive neoplastic cells is low, whereas vimentin is negligible on the normal cell surface of living organisms Only moderate amounts are expressed. “Negligible amount” means that cell surface vimentin is less than 100 molecules. Thus, the present invention allows the use of binding agents to which toxins or other therapeutic or diagnostic agents are bound. This drug binds specifically to vimentin without harmful side effects. This is because the non-vimentin cells that are killed are drug-sensitive neoplastic or damaged cells and normal cells remain intact.

  In one aspect, the invention is a method of detecting multidrug resistance or potential for multidrug resistance in a test neoplastic cell, wherein the method is expressed on the cell surface in a test neoplastic cell of any origin or cell type. Provides a method of detection by measuring the level of vimentin protein and comparing the results to the level of vimentin expressed on the cell surface in non-resistant neoplastic cells of the same origin or cell type. If the vimentin level expressed on the cell surface of the test neoplastic cell is higher than the vimentin level expressed on the cell surface in a non-resistant neoplastic cell of the same origin or cell type, the test neoplastic cell is multidrug Has resistance or potential for multidrug resistance. In some embodiments, the level of cell surface expressed vimentin in the test neoplastic cell is determined by isolating the plasma membrane fraction from the cell and measuring the vimentin level in the plasma membrane fraction. taking measurement. In another embodiment, the level of cell surface expressed vimentin in the test neoplastic cell is measured by contacting the cell with an anti-vimentin antibody and measuring the level of antibody bound to the cell surface vimentin. For example, the level of antibody bound to cell surface vimentin may be measured by immunofluorescent radiation or radiolabel.

  In certain embodiments of this aspect of the invention, the test neoplastic cell is a promyelocytic leukemia cell, a T lymphoblastoid cell, a breast epithelial cell, or an ovarian cell. In another embodiment, the test neoplastic cell is a lymphoma cell, melanoma cell, sarcoma cell, leukemia cell, retinoblastoma cell, hepatoma cell, myeloma cell, glioma cell, mesothelioma cell, or epithelial cancer cell. . In yet another embodiment of the invention, the test neoplastic cell is a tissue, such as blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, or ovary. Obtained from such organizations. In yet another embodiment, the non-resistant neoplastic (control) cell is a drug sensitive neoplastic cell line such as HL60, NB4, CEM, HSB2, Molt4, MCF-7, MDA, SKOV-3, or 2008.

  In another aspect, the invention provides a method for detecting one or more multidrug resistant cells in a patient, wherein the vimentin binding agent is operably linked to a detectable label for the patient. The detection method performed by administering is provided. The label is then operatively linked to a vimentin-binding agent that specifically binds to vimentin expressed on the cell surface of the patient's multidrug resistant cell (s) so that when the label is subsequently detected The presence of multidrug resistant cell (s) in the patient (if any) will be located. In certain embodiments, the vimentin binding agent used is an antibody, or fragment thereof. In another embodiment, the vimentin-binding agent is a vimentin ligand, such as a modified LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein or peripherin, fimbrin, RhoA-binding kinase alpha, or protein phosphatase 2A. It is. In certain embodiments, the vimentin binding agent is a natural ligand, a synthetic small molecule, a drug, a nucleic acid, a peptide, a protein, or an antibody. In another embodiment, the detectable label is a fluorescent dye, chemical dye, radioactive compound, chemiluminescent compound, magnetic compound, paramagnetic compound, parent magnetic compound, enzyme that produces a colored product, enzyme that produces a chemiluminescent product Or an enzyme that produces a magnetic product.

  In certain embodiments of this aspect, the multidrug resistant cell is a neoplastic cell. In certain embodiments, the neoplastic cell is a breast cancer cell, ovarian cancer cell, myeloma cell, lymphoma cell, melanoma cell, sarcoma cell, leukemia cell, retinoblastoma cell, hepatoma cell, glioma cell, mesothelioma cell, or , Epithelial cancer cells. In certain embodiments, the neoplastic cells are promyelocytic leukemia cells, T lymphoblastoid cells, breast epithelial cells, or ovarian cells. In certain embodiments, the patient is a human, eg, a human patient suffering from a disease or disorder caused by the presence of multidrug resistant cells.

  In another aspect, the present invention provides a kit for diagnosing or detecting multidrug resistance of a test neoplastic cell. The kit includes one probe for vimentin detection and another probe for detection of another multidrug resistance marker, such as nucleophosmin or HSC70. In certain embodiments, the kit of the invention includes a first probe for vimentin detection and another multidrug resistance marker, eg, a second probe for detection of MDR1, MDR3, MRP1, MRP5, or LRP. In a particular embodiment, the kit includes an anti-vimentin antibody as a probe for vimentin detection. In another embodiment, the kit comprises a vimentin ligand, such as LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein and peripherin, fimbrin, RhoA-binding kinase alpha, or protein phosphatase 2A. In yet another embodiment, the kit includes a nucleophosmin antibody or HSC70 antibody as a second probe for detecting a non-vimentin multidrug resistance marker. In another embodiment, the second probe is a nucleophosmin ligand (eg, protein kinase R (PKR), RNA, retinoblastoma protein, IRF-1, or nuclear localization signal (NLS), eg, Rex protein N-terminal nuclear localization signal) or HSC70 ligand (eg Alzheimer's tau protein, BAG-1, small glutamine-rich tetratricopeptide repeat containing protein (SGT), rotavirus VP5 protein (aa642-658), auxililine Or the immunosuppressant 5-deoxyspergualin (DSG)).

  In another embodiment of this aspect of the invention, the kit includes a probe that detects vimentin present on the surface of the test neoplastic cell. In another embodiment, the kit includes a second probe that detects another (non-vimentin) marker present on the surface of the test MDR neoplastic cell. In certain embodiments, the kit includes a second probe that is an MDR1, MDR3, MRP1, MRP3, or LRP antibody.

  In another aspect, the present invention provides an in-cell surface vimentin detection probe for detecting cell surface vimentin in a patient. This cell surface vimentin probe has a vimentin binding component and a detectable label (eg, a technetium label) for in vivo detection. In certain embodiments, the vimentin binding component is an antibody.

  In yet another aspect, the present invention provides cell surface vimentin targeting agents for treating or preventing multidrug resistant neoplasms. This vimentin-targeted agent is both a vimentin-binding component and a therapeutic component. Contains two components that work together to treat drug-resistant neoplasms. In certain embodiments, the vimentin binding component is an anti-vimentin antibody. In another embodiment, the vimentin binding component is a vimentin ligand, such as LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein, peripherin, fimbrin, RhoA-binding kinase alpha, or protein phosphatase 2A. . In certain embodiments, the vimentin binding component is a natural ligand, a synthetic small molecule, a drug, a nucleic acid, a peptide, or a protein.

  In certain useful embodiments of this aspect of the invention, the therapeutic component is a chemotherapeutic agent such as actinomycin, adriamycin, altretamine, asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphine Famide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epoetin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, lomustremto, melostamine, tomeltamine Mitomycin, Mitotein, Mitoxantrone, Paclitaxel, Pent Statins, procarbazine, taxol, teniposide, topotecan, vinblastine, vincristine, or a chemotherapeutic agent such as vinorelbine. In certain embodiments, the therapeutic ingredient is present as a liposomal formulation.

In another embodiment, the therapeutic component is a radioisotope, such as ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ²¹¹ At, or ²¹³ Bi.

  In yet another embodiment, the therapeutic ingredient is a toxin capable of killing or inducing killing targeted multi-drug resistant neoplastic cells. Such toxins used in the present invention include Pseudomonas exotoxins, diphtheria toxins, plant ricin toxins, plant abrin toxins, plant saponin toxins, plant gelonin toxins, and pokeweed antiviral proteins.

  In certain useful embodiments, the vimentin binding component of the cell surface vimentin-targeted therapeutic agent binds to the target cell surface and the therapeutic element is incorporated internally, preventing cell growth and jeopardizing cell viability. Or kill the cells.

  In another aspect, the present invention provides a method for treating or preventing a multidrug resistant neoplasm in a subject by administering the aforementioned cell surface vimentin targeted therapeutic agent. In certain embodiments of this aspect, the neoplasm to be treated is breast cancer, ovarian cancer, myeloma, lymphoma, melanoma, sarcoma, leukemia, retinoblastoma, hepatoma, glioma, mesothelioma, or epithelial cancer. In yet another embodiment, the neoplasm is a tissue, such as blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, or ovary. Obtained from. In certain embodiments, the subject is a human patient, eg, a human patient suffering from a disease or disorder caused by the presence of multidrug resistant cells.

  In yet another aspect, the present invention provides a vaccine for treating or preventing multidrug resistant neoplasms. This vaccine of the present invention comprises vimentin polypeptide, or a partial sequence of vimentin polypeptide, and at least one pharmaceutically acceptable vaccine component. In certain embodiments, the vimentin polypeptide, or polypeptide subsequence, is a human vimentin polypeptide sequence having the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the vimentin polypeptide subsequence is at least 8 amino acids long and in one embodiment functions as a hapten.

  In certain embodiments, the vaccine formulation includes an adjuvant, or other pharmaceutically acceptable vaccine component. In certain embodiments, the adjuvant is aluminum hydroxide, aluminum phosphate, calcium phosphate, oil suspension, bacterial product, whole inactivated bacteria, endotoxin, cholesterol, fatty acid, aliphatic amine, paraffin-like compound, vegetable oil, mono Phosphoryl lipid A, saponin, or squalene.

  In another aspect, the present invention provides a method for treating or preventing a multidrug resistant neoplasm in a subject by administering any one of the aforementioned vimentin vaccines. In certain embodiments of this aspect, the neoplasm to be treated is breast cancer, ovarian cancer, myeloma, lymphoma, melanoma, sarcoma, leukemia, retinoblastoma, hepatoma, glioma, mesothelioma, or epithelial cancer. In yet another embodiment, the neoplasm is a tissue, such as blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, or ovary. Obtained from. In certain embodiments, the subject is a human patient, eg, a human patient suffering from a disease or disorder caused by the presence of multidrug resistant cells.

  In yet another aspect, the invention is a method for detecting whether a neoplasm, measuring the level of vimentin protein expressed on the cell surface in a test cell of any origin or cell type, and Provide a detection method that is performed by comparing the results to the level of vimentin expressed on the cell surface in non-neoplastic cells of the same origin or cell type. A test cell is neoplastic if the vimentin level expressed on the cell surface of the test cell is higher than the vimentin level expressed on the cell surface of a non-neoplastic cell of the same origin or cell type.

  In certain embodiments, the level of cell surface expressed vimentin in a test cell is measured by isolating a plasma membrane fraction from the cell and measuring the vimentin level in the plasma membrane fraction. In another embodiment, the level of cell surface expressed vimentin in the test cell is measured by contacting the cell with an anti-vimentin antibody and measuring the level of antibody bound to the cell surface vimentin. For example, the level of antibody bound to cell surface vimentin may be measured by immunofluorescent radiation or radiolabel.

  In certain embodiments of this aspect of the invention, the test cells are promyelocytic leukemia cells, T lymphoblastoid cells, breast epithelial cells, or ovarian cells. In another embodiment, the test cell is a lymphoma cell, melanoma cell, sarcoma cell, leukemia cell, retinoblastoma cell, hepatoma cell, myeloma cell, glioma cell, mesothelioma cell, or epithelial cancer cell. In yet another embodiment of the invention, the test cell is of tissue, such as blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, or ovary. Obtained from such organizations.

  In another aspect, the invention provides a method for detecting one or more neoplastic cells in a patient, wherein the patient is administered a vimentin binding agent operably linked to a detectable label. The detection method performed by doing is provided. The label is operatively linked to a vimentin binding agent that specifically binds to vimentin expressed on the cell surface of the patient's neoplastic cell (s), so that the next time the label is detected, the neoplasia in that patient The presence of the cell (s) will be located (if any). In certain embodiments, the vimentin binding agent used is an antibody, or fragment thereof. In another embodiment, the vimentin-binding agent is a vimentin ligand, such as a modified LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein or peripherin, fimbrin, RhoA-binding kinase alpha, or protein phosphatase 2A. It is. In certain embodiments, the vimentin binding agent is a natural ligand, synthetic small molecule, drug, nucleic acid, peptide, protein, or antibody. In another embodiment, the detectable label is a fluorescent dye, chemical dye, radioactive compound, chemiluminescent compound, magnetic compound, paramagnetic compound, parent magnetic compound, enzyme that produces a colored product, enzyme that produces a chemiluminescent product Or an enzyme that produces a magnetic product.

  In one embodiment of this aspect, the neoplastic cell is a breast cancer cell, ovarian cancer cell, myeloma cell, lymphoma cell, melanoma cell, sarcoma cell, leukemia cell, retinoblastoma cell, hepatoma cell, glioma cell, mesothelioma cell Or an epithelial cancer cell. In certain embodiments, the neoplastic cells are promyelocytic leukemia cells, T lymphoblastoid cells, breast epithelial cells, or ovarian cells. In certain embodiments, the patient is a human, eg, a human patient suffering from a disease or disorder caused by the presence of neoplastic cell (s).

  In another aspect, the present invention provides a kit for diagnosing or detecting a neoplasm. The kit includes at least one probe for vimentin detection and at least one other probe for detection of another neoplastic marker, such as nucleophosmin or HSC70. In one embodiment, the vimentin detection probe is an anti-vimentin antibody, or a fragment thereof. In another embodiment, the vimentin detection probe comprises a vimentin ligand, such as modified LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein and peripherin, fimbrin, RhoA-binding kinase alpha, or protein phosphatase Includes 2A.

  In certain embodiments, the second probe for detecting a non-vimentin neoplastic marker is a nucleophosmin antibody or an HSC70 antibody. In another embodiment, the second probe for detecting a non-vimentin neoplastic marker is a nucleophosmin ligand, such as protein kinase R (PKR), RNA, retinoblastoma protein, IRF-1, or nuclear localization signal ( NLS) For example, N-terminal nuclear localization signal of Rex protein and HSC70 ligand. In yet another embodiment, the second probe for detecting a non-vimentin neoplastic marker is an HSC70 ligand, such as Alzheimer's tau protein, BAG-1, small glutamine-rich tetratricopeptide repeat-containing protein (SGT), rotavirus VP5 protein (Aa642-658), auxillin, or the immunosuppressant 5-deoxyspergualin (DSG).

  In a particularly useful embodiment, the kit comprises a first probe that detects vimentin present on the surface if the test cell is neoplastic, and another (non- Vimentin) and a second probe for detecting the marker.

  In yet another aspect, the present invention provides a cell surface vimentin targeting agent for treating cancerous neoplastic cell growth. This cell surface vimentin targeting agent generally comprises a vimentin binding component and a therapeutic component. This vimentin binding component targets the therapeutic component towards neoplastic cell growth and thereby treats the cancer. Thus, the vimentin binding component and the therapeutic component cooperate with each other so that the vimentin binding component targets the therapeutic component toward the neoplasm and treats the neoplasm.

  In certain embodiments, the vimentin binding component is an anti-vimentin antibody. In another embodiment, the vimentin binding component is a vimentin ligand, such as LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein, peripherin, fimbrin, RhoA-binding kinase alpha, or protein phosphatase 2A. . In certain embodiments, the vimentin binding component is a natural ligand, a synthetic small molecule, a drug, a nucleic acid, a peptide, or a protein.

  In certain useful embodiments of this aspect of the invention, the therapeutic component is a chemotherapeutic agent such as actinomycin, adriamycin, altretamine, asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphine Famide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epoetin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, lomustremto, melostamine, tomeltamine Mitomycin, Mitotein, Mitoxantrone, Paclitaxel, Pent Statins, procarbazine, taxol, teniposide, topotecan, vinblastine, vincristine, or a chemotherapeutic agent such as vinorelbine. In certain embodiments, the therapeutic ingredient is present as a liposomal formulation.

In another embodiment, the therapeutic component is a radioisotope, such as ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ²¹¹ At, or ²¹³ Bi.

  In yet another embodiment, the therapeutic ingredient is a toxin capable of killing or inducing killing targeted multi-drug resistant neoplastic cells. Such toxins used in the present invention include Pseudomonas exotoxins, diphtheria toxins, plant ricin toxins, plant abrin toxins, plant saponin toxins, plant gelonin toxins, and pokeweed antiviral proteins.

  In certain useful embodiments, the vimentin binding component of the cell surface vimentin-targeted therapeutic agent binds to the target cell surface and the therapeutic element is incorporated internally, preventing cell growth and jeopardizing cell viability. Or kill the cells.

  In another aspect, the present invention provides a method of treating a neoplasm in a subject by administering any of the aforementioned cell surface vimentin targeted therapeutic agents. In certain embodiments of this aspect, the neoplasm is breast cancer, ovarian cancer, myeloma, lymphoma, melanoma, sarcoma, leukemia, retinoblastoma, hepatoma, glioma, mesothelioma, or epithelial cancer. In yet another embodiment, the neoplasm is a tissue, such as blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, or ovary. Obtained from. In certain embodiments, the subject is a human patient, eg, a human patient suffering from a disease or disorder caused by the presence of a neoplasm.

  In yet another aspect, the present invention provides a vaccine for treating or preventing a neoplasm. This vaccine of the present invention comprises vimentin polypeptide, or a partial sequence of vimentin polypeptide, and at least one pharmaceutically acceptable vaccine component. In certain embodiments, the vimentin polypeptide, or polypeptide subsequence, is a human vimentin polypeptide sequence having the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the vimentin polypeptide subsequence is at least 8 amino acids long and in one embodiment functions as a hapten.

  In certain embodiments, the vaccine formulation includes an adjuvant, or other pharmaceutically acceptable vaccine component. In certain embodiments, the adjuvant is aluminum hydroxide, aluminum phosphate, calcium phosphate, oil suspension, bacterial product, whole inactivated bacteria, endotoxin, cholesterol, fatty acid, aliphatic amine, paraffin-like compound, vegetable oil, mono Phosphoryl lipid A, saponin, or squalene.

  In another aspect, the present invention provides a method for treating or preventing a neoplasm in a subject by administering any one of the aforementioned vimentin vaccines. In certain embodiments of this aspect, the neoplasm to be treated is breast cancer, ovarian cancer, myeloma, lymphoma, melanoma, sarcoma, leukemia, retinoblastoma, hepatoma, glioma, mesothelioma, or epithelial cancer. In yet another embodiment, the neoplasm is a tissue, such as blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, or ovary. Obtained from. In certain embodiments, the subject is a human patient, eg, a human patient suffering from a disease or disorder caused by the presence of multidrug resistant cells.

  In yet another aspect, the present invention is a method for detecting damage (eg, pathogen infection) in a test cell, measuring the level of vimentin protein expressed on the cell surface in a test cell of any origin or cell type. And a method of detection performed by comparing the results to the level of vimentin expressed on the cell surface in undamaged cells of the same origin or cell type. If the vimentin level expressed on the cell surface of the test cell is higher than the vimentin level expressed on the cell surface of an undamaged cell of the same origin or cell type, the test cell is damaged (eg , Is infected).

  In certain embodiments, the damaged cell is infected with a pathogen. In certain embodiments, the level of cell surface expressed vimentin in a test cell is measured by isolating a plasma membrane fraction from the cell and measuring the vimentin level in the plasma membrane fraction. . In certain embodiments, the level of cell surface expressed vimentin in the test cell is measured by an anti-vimentin antibody. In certain embodiments, the anti-vimentin antibody measures the level of cell surface vimentin present in intact test cells. For example, the level of antibody bound to cell surface vimentin may be measured by immunofluorescent radiation or radiolabel.

  In certain embodiments, the damaged cell is infected by a pathogen that is a virus, bacterium, or parasite. In certain embodiments, the pathogen is a virus, such as HIV, West Nile virus, or dengue virus. In another embodiment, the pathogen is a bacterium, such as mycobacteria, rickettsia, or chlamydia. In yet another embodiment, the pathogen is a parasite, such as plasmodium, leishmania, or taxoplasma.

  In certain other embodiments, the test cell is a tissue, eg, a tissue such as blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, or ovary. Obtained from. In certain embodiments, the test cell is from a human. In certain embodiments, the human patient suffers from a disease or disorder caused by the presence of pathogen-infected cells.

  In another aspect, the invention provides a method of detecting one or more damaged (eg, pathogen-infected) cells in a patient, operably linked to the patient with a detectable label. Methods of detection performed by administering a vimentin binding agent are provided. The label is then operatively linked to a vimentin-binding agent that specifically binds to vimentin expressed on the cell surface of the patient's damaged (eg, pathogen-infected) cell (s) so that the label is then detected The presence (if any) of damaged (eg pathogen-infected) cells in the patient. In certain embodiments, the vimentin binding agent used is an antibody, or fragment thereof. In another embodiment, the vimentin-binding agent is a vimentin ligand, such as a modified LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein or peripherin, fimbrin, RhoA-binding kinase alpha, or protein phosphatase 2A. It is. In certain embodiments, the vimentin binding agent is a natural ligand, synthetic small molecule, drug, nucleic acid, peptide, protein, or antibody. In another embodiment, the detectable label is a fluorescent dye, chemical dye, radioactive compound, chemiluminescent compound, magnetic compound, paramagnetic compound, parent magnetic compound, enzyme that produces a colored product, enzyme that produces a chemiluminescent product Or an enzyme that produces a magnetic product.

  In another aspect, the present invention provides a kit for diagnosing or detecting a pathogen infection in a test cell. The kit includes one probe for vimentin detection and another marker for damage (eg, pathogen infection), eg, a second probe for nucleophosmin or HSC70 detection. In a particular embodiment, the kit includes an anti-vimentin antibody as a probe for detecting vimentin. In another embodiment, the kit comprises a vimentin ligand, such as LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein and peripherin, fimbrin, RhoA-binding kinase alpha, or protein phosphatase 2A. In yet another embodiment, the kit includes a nucleophosmin antibody or HSC70 antibody as a second marker for detecting non-vimentin damage (eg, pathogen infection). In another embodiment, the second probe is a nucleophosmin ligand (eg, protein kinase R (PKR), RNA, retinoblastoma protein, IRF-1, or nuclear localization signal (NLS), eg, Rex protein N-terminal nuclear localization signal) or HSC70 ligand (eg Alzheimer's tau protein, BAG-1, small glutamine-rich tetratricopeptide repeat-containing protein (SGT), rotavirus VP5 protein (aa642-658), auxililine Or the immunosuppressant 5-deoxyspergualin (DSG)). In certain embodiments, the vimentin binding moiety is a natural ligand, a synthetic small molecule, a drug, a nucleic acid, a peptide, a protein, or an antibody or fragment thereof.

  In yet another aspect, the present invention provides a cell surface vimentin targeting agent that treats infection by pathogens. The vimentin targeting agent includes a vimentin binding component and a therapeutic component. This vimentin binding component targets the therapeutic component towards the pathogen-infected cells and thereby treats the infection. In certain embodiments, the vimentin binding agent is an anti-vimentin antibody. In another embodiment, the vimentin binding moiety is a vimentin ligand such as modified LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein and peripherin, fimbrin, RhoA-binding kinase alpha, or protein phosphatase 2A. It is. In certain embodiments, the vimentin binding moiety is a natural ligand, a synthetic small molecule, a drug, a nucleic acid, a peptide, a protein, or an antibody or fragment thereof.

  In certain embodiments, the therapeutic component is an antibacterial agent, antiviral agent, or antiparasitic agent. In certain embodiments, the vimentin binding agent binds to the surface of the target cell and the therapeutic element is taken up internally, preventing pathogen growth, jeopardizing pathogen viability, or killing the pathogen infected cell.

  In yet another aspect, the present invention provides a vaccine for treating or preventing infection by a pathogen. The vaccine comprises a vimentin polypeptide, or a partial sequence of a vimentin polypeptide, and at least one pharmaceutically acceptable vaccine component. In certain embodiments, the vimentin polypeptide, or polypeptide subsequence, is a human vimentin polypeptide sequence having the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the vimentin polypeptide subsequence is at least 8 amino acids long and in one embodiment functions as a hapten.

  In certain embodiments, the vaccine formulation includes an adjuvant, or other pharmaceutically acceptable vaccine component. In certain embodiments, the adjuvant is aluminum hydroxide, aluminum phosphate, calcium phosphate, oil suspension, bacterial product, whole inactivated bacteria, endotoxin, cholesterol, fatty acid, aliphatic amine, paraffin-like compound, vegetable oil, mono Phosphoryl lipid A, saponin, or squalene.

  In another aspect, the present invention provides a method for treating or preventing an infection in a subject by administering any one of the aforementioned vimentin vaccines. In certain embodiments of this aspect, the subject is a human patient. In certain embodiments, the human patient suffers from a disease or disorder caused by the presence of an infection. In certain embodiments, the infection occurs in a tissue, eg, a tissue such as blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, or ovary.

  The patent and scientific literature cited herein ensures knowledge that is available to those with skill in the art. Reference materials, including published US patents, allowed applications, published foreign applications, and GenBank database sequences, cited herein are each specifically and individually incorporated by reference. To the extent they are intended to be incorporated herein by reference.

In particular, this application includes the entirety of the following patent applications by reference. No. 60 / 433,480 filed Dec. 13, 2002, entitled “Methods for Diagnosing and Treating Damaged Cells, Neoplastic Cells and Multidrug Resistance Based on Vimentin Detection”; December 15, 2003, including US Application No. 60 / 433,351, filed 13th May, entitled "Method for Diagnosing and Treating Damaged Cells, Neoplastic Cells and Multidrug Resistance Based on Nucleophosmin Detection" Submitted, US Application No. YY / XXXXX, entitled “Nucleophosmin-Directed Diagnosis and Treatment of Multidrug-resistant Neoplastic Diseases”; filed January 1, 2003, entitled “Injured Cells, Neoplastic Cells and Multidrugs” U.S. Application No. 60 / 438,012 of "Methods for Diagnosing and Treating Resistance Based on HSC70 Detection"; and filed December 15, 2003, entitled "HSC70-directed treatment of multidrug resistant neoplastic disease and U.S. Application No. YY / XXXXX for "Diagnostic Methods".
Overview The present invention relates to the development of both naturally occurring, drug-induced MDR phenotypes for diagnosing, preventing, and / or treating cancer and for both non-cancerous and cancerous cells. Methods and reagents for diagnosis, prevention, and / or treatment are provided. The present invention allows for improved clinical management of multidrug resistant tumors and pathogen infections. Furthermore, the present invention allows the identification of patients with neoplastic or damaged cells, including MDR cells, thus improving the treatment, monitoring, diagnosis and medical imaging of multidrug resistant cancers and pathogen infections. Reagents that can be provided are provided.

  Accordingly, certain embodiments of the present invention provide methods for detecting multidrug resistance in test damaged cells. The method measures the cell surface expression level of vimentin protein in damaged cells of a particular cell type; measures the level of vimentin protein expressed on the cell surface in drug-sensitive damaged cells of the same cell type; and If there is an increase in the level of cell surface expressed vimentin relative to the level of cell surface expressed vimentin in the drug sensitive damaged cell, this includes determining that the test damaged cell is multidrug resistant. In certain embodiments, the level of cell surface-expressed vimentin is a cell comprising separating the cellular components of the test damaged cells and drug-sensitive damaged cells into fractions, and the plasma membrane or cell membrane of those cells. It is measured by measuring vimentin levels in the component fractions.

  In certain embodiments, the test damaged cell is infected by a pathogen. In certain embodiments, the pathogen is a virus, bacterium, or parasite. Exemplary viruses include, but are not limited to, HIV, West Nile virus, and dengue virus. Exemplary bacteria include, but are not limited to, mycobacteria, rickettsia, and chlamydia. Exemplary parasites include, but are not limited to, plasmodium, leishmania, and taxoplasma. In certain embodiments, the test damaged cell is from a tissue selected from the group consisting of blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, and ovary. It is. In certain embodiments, the test damaged cell is from a human.

  The present invention also provides a method of detecting multidrug resistance in a test neoplastic cell. The method measures the cell surface expression level of vimentin protein in test neoplastic cells of a particular cell type; measures the level of vimentin protein expressed on the cell surface in drug-sensitive neoplastic cells of the same cell type; and If there is an increase in the cell surface expression level of vimentin compared to the cell surface expression level of the drug-sensitive neoplastic cell, then determining that the test neoplastic cell is multidrug resistant. In certain embodiments, the cell surface expression level of vimentin is a cell comprising separating the cellular components of the test neoplastic cells and drug-sensitive neoplastic cells into fractions, and the plasma membrane or cell membrane of those cells. It is measured by measuring vimentin levels in the component fractions.

  Exemplary neoplastic cells include lymphoma cells, melanoma cells, sarcoma cells, leukemia cells, retinoblastoma cells, hepatoma cells, myeloma cells, glioma cells, mesothelioma cells, and epithelial cancer cells, but It is not limited to them. In certain embodiments, the test neoplastic cell is obtained from a tissue selected from the group consisting of blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, and ovary. It is done. In certain embodiments, the test neoplastic cell is obtained from a human.

  The present invention provides a method of detecting multidrug resistant cells in a patient. This method involves administering a binding agent that specifically binds to vimentin protein operably linked to a detectable label and a binding agent that specifically binds to vimentin protein on a patient's multidrug resistant cell surface. Detection of an increase relative to a binding agent that binds to vimentin protein on the surface of drug-sensitive cells. In one embodiment of the invention, the medical imaging device or system detects a binding agent that specifically binds to the surface of the patient's multidrug resistant cells. Exemplary binding agents include, but are not limited to, natural ligands, synthetic small molecules, drugs, nucleic acids, peptides, proteins, antibodies or fragments thereof. In certain embodiments, the binding agent is an antibody.

  Exemplary detectable labels include fluorescent dyes, chemical dyes, radioactive compounds, chemiluminescent compounds, magnetic compounds, paramagnetic compounds, parent magnetic compounds, enzymes that produce colored products, enzymes that produce chemiluminescent products, and magnetism Examples include, but are not limited to, enzymes that produce products. In certain embodiments, the patient is a human. In certain embodiments, the multidrug resistant cell is a damaged cell or a neoplastic cell. In certain embodiments, the damaged cell is infected by a pathogen. Exemplary pathogens include, but are not limited to, viruses, bacteria, and parasites. Exemplary viruses include, but are not limited to, HIV, West Nile virus, and dengue virus. Exemplary bacteria include, but are not limited to, mycobacteria, rickettsia, and chlamydia. Exemplary parasites include, but are not limited to, plasmodium, leishmania, and taxoplasma. In certain embodiments, the neoplastic cell is a breast cancer cell, ovarian cancer cell, myeloma cell, lymphoma cell, melanoma cell, sarcoma cell, leukemia cell, retinoblastoma cell, hepatoma cell, glioma cell, mesothelioma cell, or , Selected from the group consisting of epithelial cancer cells. In certain embodiments, the patient is a human. In certain embodiments, the patient suffers from a disease or disorder caused by the presence of multidrug resistant cells.

  The present invention also provides a method for detecting neoplastic cells. This method measures cell surface-expressed vimentin protein in suspected neoplastic cells and increases the level of cell surface-expressed vimentin protein relative to the level of cell surface-expressed vimentin in normal cells of the same cell type. If present, includes determining that the cell is a neoplasm. In some embodiments, the cell surface expressed vimentin separates the suspected test cell and the cellular components of normal cells into fractions, and vimentin in the cellular component fraction comprising the plasma membrane or cell membrane of those cells. Measured by measuring the level. In certain embodiments, the cellular components of the test and normal cells are contacted with a detectable binding agent, which is then detected to quantify the level of cell surface expressed vimentin protein in each cell. In another embodiment, the cell surface-expressed vimentin specifically detects untreated, suspected neoplastic cells of certain cell types and untreated normal cells of the same cell type specifically binding to vimentin protein. It is measured by contacting with a possible binding agent and detecting the binding of the agent to quantify the level of cell surface expressed vimentin protein in the untreated cells.

  In certain embodiments, the test cell is from a tissue selected from the group consisting of blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, and ovary. is there. In certain embodiments, the neoplastic suspect cell is from a human. In one embodiment, the human suffers from cancer caused by the presence of neoplastic cells.

  As used herein, a “neoplastic cell” is a cell that exhibits abnormal cell growth, eg, proliferating cell growth. Neoplastic cells are hyperplastic cells, cells that show lack of contact growth inhibition in vitro, tumor cells that cannot metastasize in vivo, or cancer cells that can metastasize in vivo. Non-limiting examples of neoplastic cells include lymphoma cells, melanoma cells, breast cancer cells, ovarian cancer cells, prostate cancer cells, sarcoma cells, leukemia cells, retinoblastoma cells, hepatoma cells, myeloma cells, glioma cells, mesothelial cells Tumor cells and epithelial cancer cells.

  As used in accordance with the present invention, a “damaged cell” means a cell that is non-neoplastic but has been otherwise damaged. For example, a non-neoplastic damaged cell may be a cell that is infected by a pathogen, such as a virus, bacterium, or parasite. In one non-limiting example, the cell may be damaged by a multicellular parasite or may be damaged by the action of infection by the parasite. Such non-limiting parasites include cystosomes, plasmodium, trypanosomes, leishmania, and taxoplasma.

  As used herein, the terms “multi-drug resistant” and “multi-drug resistant” include in neoplastic cells or damaged cells, agents that have never been exposed to the neoplastic or damaged cells, Refers to the development of resistance to several different drugs. For example, if a leukemia patient being treated with vincristine has resistance to vincristine and other chemotherapeutic agents that the patient has never taken before (eg, methotrexate or mercaptopurine) When leukemia cells are trained, the patient's leukemia cells are multidrug resistant. Similarly, if a tuberculosis patient being treated with penicillin is resistant to other drugs (eg, erythromycin) that the patient has never taken before, such as penicillin. When the cells are trained, the patient's tuberculosis infected cells are multidrug resistant. It should be noted that multidrug resistance (MDR) is acquired for a broad spectrum of drugs with little or no functional similarity to the original drug (s). Tolerance, which may include resistance that results in reduced efficacy of all drugs involved.

  The terms “multi-drug resistant” and “multi-drug resistant” are atypical by conventional mechanisms (ie, including P-glycoprotein, or other MDR proteins) or without P-glycoprotein Note that it is used to describe neoplastic or damaged cells with multidrug resistance by mechanism (non-conventional mechanism) (eg, an atypical mechanism that includes the MRP1 multidrug resistance marker).

  As used herein, “MDR protein” includes any of a number of ABC-type transmembrane integrated glycoproteins that are (multiple) involved in drug resistance. Such includes MDR1 (P-glycoprotein or P-glycoprotein 1), an energy-dependent pumping pump that reduces drug retention in multidrug resistant cells. Examples of MDR1 include human MDR1 (see, for example, database code MDR1_HUMAN, GenBank accession number P08183, 1280 amino acids (141.34 kDa)). Other MDR proteins include MDR3 (or P-glycoprotein 3). This is an energy-dependent pump that reduces drug retention but cannot itself confer drug resistance. Examples of MDR3 include MDR3 (eg, database code MDR3_HUMAN, GenBank accession number P21439, 1279 amino acids (140.52 kDa). Other MDR-related proteins participate in active transport of drugs to intracellular organelles. Examples obtained from humans include MRP1, ie, multidrug resistance-related protein 1, database code MRP_HUMAN, GenBank accession number P33527, 1531 amino acids (171.49 kDa).

  According to the present invention, cells that are multidrug resistant (eg, neoplastic or damaged cells) are exposed to drugs (eg, chemotherapeutic agents or antibiotics) or spontaneously cause such MDR. By developing (ie, the cell is not exposed to an agent that develops resistance to it), it is possible to develop an MDR condition. In this regard, the present invention allows detection of a potential multidrug resistance of a neoplasm even before the neoplasm is treated. Similarly, the present invention allows, for example, a potentially multi-drug resistant neoplasm to be effectively treated even before the neoplasm has been treated and revealed to be drug resistant.

  The present invention also allows for early identification of patients with such MDR neoplastic or damaged cells. For example, a patient identified as having such cells is an asymptomatic patient who has been treated with an infection or has been treated with an infection (eg, hepatitis B). In some cases, the present invention not only allows identification of these patients prior to reversal of symptoms, but also allows monitoring of these patients during drug treatment, for which such MDR cells were detected. If so, it becomes possible to change the treatment schedule. Similarly, patients identified as having such cells are patients in cancer remission or are being treated for cancer (eg, breast cancer, ovarian cancer, prostate cancer, leukemia, etc.). The present invention not only allows for the identification of such patients prior to their return and / or progression of the cancer, but also allows such patients to be monitored during treatment with the drug. The schedule can be changed.

  The present invention shows that vimentin expressed on the cell surface is expressed at a significantly higher level on the surface of multidrug resistant cells than that found on other cells, and thus the multidrug resistant of the cell. It starts with the discovery that it is partially useful as a marker. An important advantage of vimentin protein cell surface markers is that vimentin is present at negligible levels on the surface of normal cells in the body. This expression is in contrast to the situation seen with other known MDR markers, such as P-glycoprotein and multidrug resistance protein (MRP). In the latter case, levels vary, including high levels on the surface of various normal tissues and on the surface of liver, kidney, stem cells, and blood brain barrier epithelial cells (Cordon-Cardo C. et al. , J. Histochem. Cytochem., 38: 1277-1287, 1990; Nakamura T. et al., Drug Metabolism & Disposition, 30: 4-6, 2002). Therefore, the use of drugs directed against MDR cancer cell markers such as P-glycoprotein and MRP, as well as side effects caused by killing normal cells expressing high levels of cell surface P-glycoprotein and MRP (See, for example, FitzGerald, DJ et al., Proc. Natl. Acad. Sci. 84: 4288-4292, 1987). The present invention overcomes this harmful side effect. Because vimentin MDR markers are also expressed at a moderate level on the surface of drug-sensitive cancer cells (when compared to very low to negligible levels in drug-sensitive, non-cancerous normal cells), but MDR cancer This is because it is expressed at a higher level in cells and in non-MDR cancerous cells (for example, virus-infected cells). Accordingly, the present invention is a drug that is emitted toward cell surface-expressed vimentin, which kills drug-sensitive neoplastic cells, including MDR-damaged or MDR-neoplastic cells, while leaving normal cells intact I will provide a.

  Note that the same MDR neoplastic or damaged cells may simultaneously express one or more markers (eg, both P-glycoprotein and vimentin) or may independently express these MDR markers It must be noted that there is sex. This co-expression in the same MDR cells offers the possibility of combining binding agents that are emitted towards one or more cell surface MDR markers. For example, a sublethal amount of a binding agent that specifically binds vimentin can be combined with a sublethal amount of a binding agent that specifically binds to P-glycoprotein. Since normal cells do not express vimentin on their surface, they are not damaged by binding agents that specifically bind vimentin. Rather, only MDR cells that express both P-glycoprotein and vimentin on their cell surface will be killed by this combination treatment.

  Therefore, vimentin expressed on the cell surface is an excellent marker used for therapy (eg, immunotoxin therapy) that kills MDR neoplastic cells and MDR damaged cells that accompany the vimentin marker on the cell surface. This is because normal cells are free from cell killing and thus delete or eliminate harmful side effects of treatment. Similarly, diagnosis and imaging of MDR neoplastic or damaged cells with cell surface vimentin markers can be used to detect MDR neoplastic and damaged cells using other MDR markers that are also expressed in normal tissues, such as P-glycoprotein or MRP. Is more sensitive, accurate, and has fewer false positives to present. In addition, cell surface vimentin is useful as an anti-MDR cancer vaccine antigen or as an anti-MDR damaged cell vaccine antigen in immunizing a patient against cancer or damaged cell tissue that expresses vimentin on the cell surface.

  The present invention also allows the identification of patients whose damaged or neoplastic cells have acquired multidrug resistance. In certain situations, patients are identified when they no longer respond to drugs used in therapy. For example, a patient in remission breast cancer treated with a chemotherapeutic agent (eg, vincristine) may suddenly escape from remission despite being steadily treated with the chemotherapeutic agent. Unfortunately, such patients often do not respond to other chemotherapeutic agents, including those that have never been exposed. Of course, once such patients have become multi-drug resistant, it is difficult to treat such patients to control the cancer that has now returned, or the disease caused by damaged cells, For example, radiation therapy, or surgery (eg, bone marrow transplantation or removal of necrotic tissue) may be required.

  The present invention enables early diagnosis of multidrug resistance by detecting increased cell surface expression of vimentin in neoplastic or damaged cells of a patient. Such an early diagnosis makes it possible to distinguish a patient who is initially a drug treatment responder and sensitive to the drug treatment from a patient who has not responded to the drug treatment from the beginning. Furthermore, the diagnostic process by vimentin cell surface expression is also resistant to drug treatment and can be used to follow the development and appearance of MDR damage or new cells that appear during the course of drug treatment. For example, this process treats AIDS patients who are first treated with AZT and then reported to develop multidrug resistance to a broad spectrum of antiviral, antibacterial, and anticancer agents. (See Gollapudi et al., Biochem. Biophys. Res. Commun. 171: 1002-1007, 1990; Antonelli et al., AIDS Research and Human Retroviruses 8: 1839-1844, 1992).

  Furthermore, diagnostic assays for vimentin cell surface expression are useful for selecting patients in clinical experiments involving the treatment of damaged or neoplastic cells. The presence of vimentin on the patient's cell surface determines whether the patient is eligible for inclusion in any clinical experiment.

  Accordingly, in one aspect, the present invention provides a method for detecting multidrug resistance in test damaged cells suspected of having multidrug resistance. This method measures the level of vimentin protein expressed on the cell surface in test damaged cells of a particular cell type; the level of vimentin protein expressed on the cell surface in drug-sensitive damaged cells of the same cell type. And if there is an increase in the cell surface expression level of vimentin protein compared to the level of cell surface expression vimentin protein in non-MDR damaged cells (eg, damaged cells sensitive to drugs) Determining that the test damaged cells are multi-drug resistant.

  In another aspect, the present invention provides a method of detecting multidrug resistance in a test neoplastic cell suspected of having multidrug resistance. This method measures the level of vimentin protein expressed on the cell surface in test neoplastic cells of a particular cell type; measures the level of vimentin expressed on the cell surface in drug-sensitive neoplastic cells of the same cell type And if there is an increase in the cell surface expression level of vimentin protein compared to the level of cell surface expression vimentin protein of drug-sensitive neoplastic cells, the test cell is determined to be multidrug resistant Including that.

  In one embodiment, the test neoplastic cell or test damaged cell has at least twice as much vimentin on its cell surface as the cell surface expression level of vimentin protein in normal cells or non-MDR neoplastic cells or non-MDR damaged cells, respectively. To express. Such quantification of cell surface expressed vimentin levels can be performed by any number selected from known methods, including but not limited to the methods described below.

  As described later, all normal cells express vimentin protein intracellularly, but it was unexpectedly discovered that neoplastic cells and damaged cells express full-length vimentin protein on their cell surfaces. Furthermore, as these neoplastic or damaged cells become multidrug resistant, they increase the expression of full-length vimentin protein on their cell surface. Drug-sensitive normal cells contain vimentin protein mainly in the nucleus, nucleolus, and protoplast. A moderate amount of vimentin protein is also expressed on the surface of drug-sensitive neoplastic cells. Drug sensitive cells that do not express vimentin on their cell surface (or that express a small amount of vimentin on their surface) are killed by treatment with the drug. In contrast, acquired neoplastic or damaged cells are recognizable whether multidrug resistance occurs naturally or after treatment with a drug. This is because the expression level of vimentin protein is increased on the surface of such cells. Thus, antibodies or other binding agents that specifically bind to cell surface vimentin protein will develop diagnostic, therapeutic, screening, and imaging methods for MDR damaged and neoplastic cells that express vimentin on their surface. Useful for.

  In certain embodiments, the test damaged cells are obtained from a tissue, eg, from a biopsy of a damaged tissue (eg, necrotic tissue) or from a cell type infected with a pathogen. For example, hepatitis B virus usually only infects liver cells. Thus, damaged cells (eg, liver cells infected with hepatitis B virus) are obtained from a single tissue (eg, liver). Similarly, human immunodeficiency virus (HIV) usually infects only CD4 + T cells and macrophages. Thus, damaged cells (eg, CD4 + T cells infected with HIV) are obtained from a single tissue (ie, blood or bone marrow).

  It should be noted that in certain restricted situations, infection with a virus can make the cell neoplastic. For example, some of the B cells become neoplasms when infected with Epstein Barr Virus (EBV). Such neoplastic B cells, although damaged by viral infection, are included herein as “neoplastic cells” rather than damaged cells.

  In certain embodiments, the test neoplastic cell is obtained from a biopsy of a tissue, eg, a hyperplastic tissue (eg, a breast lump). Non-limiting examples of tissues from which test neoplastic cells can be obtained include blood, bone marrow, spleen, lymph nodes, liver, thymus, kidney, brain, skin, gastrointestinal tract, eye, breast, prostate, and ovary.

  According to the present invention, neoplastic cells may be obtained from a patient, eg, a human suffering from a disease or disorder caused by the presence of neoplastic cells. For example, if the neoplastic cell is a neoplastic melanoma cell, the disease is cancer of the melanoma cell (ie, the cancer caused by cell proliferation and neoplastic cell metastasis is melanoma).

  According to the present invention, damaged cells may be obtained from a patient, eg, a human suffering from a disease or disorder caused by the presence of damaged cells. For example, if the damaged cell is infected with a pathogen, the disease is an infection caused by the presence or absence of the damaged cell infected by the pathogen (eg, CD4 + T cells infected by the HIV virus Lack of AIDS).

  As used herein, a “patient suffering from a disease or disorder” means a patient who presents clinical signs and / or symptoms of the disease or disorder. In certain situations, a patient with a disease or disorder may be asymptomatic but present with clinical signs of the disease or disorder. For example, a patient with leukemia may be asymptomatic (eg, not wandered or weakened), but has clinical signs in that it has more white blood cells than a healthy individual of the same age and weight. Show. In another non-limiting example, a patient who is infected with a virus (eg, HIV) may be asymptomatic (eg, may not show a decrease in the number of CD4 + T cells), Presents clinical signs in having anti-HIV antibodies.

  According to the present invention, damaged or neoplastic cells with multidrug resistance can be distinguished from cells without multidrug resistance by the increased expression of full-length vimentin protein on the surface of multidrug resistant cells. is there. Representative nucleotide and amino acid sequences of vimentin are set forth in FIG. Thus, when cell components are separated into fractions, cells with multidrug resistance contain vimentin in their plasma membrane fraction. Cell surface expression of vimentin protein is typically determined by non-limiting methods such as FACS analysis, cell surface biotinylation followed by 2-D gels, immunoprecipitation (see Examples), or clinical fixed specimens. Detection can be by immunofluorescence analysis and other routine methods performed by those skilled in the art.

  In one embodiment, measuring vimentin protein expression levels on the surface of a test damaged cell or test neoplastic cell is by separating the cellular components of the test cell into fractions and then examining the fraction containing the plasma membrane or cell membrane for vimentin. Including that.

  Alternatively, the measuring step includes separating the enzymatic digestion product of the cell surface expressed vimentin protein from the test cell. Peptides obtained from digested surface exposed proteins are isolated by rapidly rotating the cells and leaving the digested peptides in the supernatant. The digested peptides are then analyzed by various methods (eg, immunological methods or mass spectrometry) to determine if they are vimentin peptides.

  Separation of cellular components can be achieved by any standard separation process, ie thin layer chromatography, gas chromatography, high performance liquid chromatography, paper chromatography, affinity chromatography, supercritical flow chromatography, gel electrophoresis, and It can be performed by processes including but not limited to the processes described in the Examples section. Separation processes are generally known (see, eg, Scopes and Scopes, “Protein Purification: Principles and Practice”, Springer Verlag, 1994).

  In certain embodiments, measuring vimentin protein expression levels on the test damaged cell surface comprises contacting an intact test damaged cell or test neoplastic cell with a detectable binding agent that specifically binds to vimentin protein. In one embodiment, the cells are then separated into fractions and the amount of labeled vimentin in the fraction containing the plasma or plasma membrane is measured.

  The binding agent useful in the present invention may be any as long as it specifically binds to vimentin protein or a fragment thereof. The binding agent specifically binds to any part of the vimentin protein. This is because the entire protein is expressed on the surface of multidrug resistant lesions or neoplastic cells. The binding agents of the invention specifically bind to neoplastic or damaged cells that express vimentin at a higher level compared to the amount expressed on the surface of drug sensitive neoplastic or drug sensitive damaged cells. To identify that the cell is multidrug resistant.

  Of course, vimentin is also expressed inside normal cells, drug-sensitive neoplastic cells, and drug-sensitive damaged cells, so if normal cells, drug-sensitive neoplastic or damaged cells are first degraded, or if If the membrane was made permeable before addition of the binder, the binder would also bind to normal cells and intracellular vimentin in drug-sensitive neoplastic or damaged cells.

  In certain embodiments, MDR neoplastic or MDR damaged cells that express vimentin on their cell surface can be distinguished from other types of cells in the following ways: That is, MDR neoplastic or damaged cells have vimentin on their surface at a level at least two times higher than normal cells obtained from tissues of the same origin as the neoplastic or damaged cells, or from tissues of the same origin. It is expressed at a level that is at least twice as high as drug-sensitive neoplastic or damaged cells. For example, leukemia T cells express more vimentin on their surface than normal cells. Furthermore, as described later, MDR leukemia T cells express at least twice the amount of vimentin on the surface of leukemia T cells that do not have multidrug resistance. Similarly, as described below in the Examples, MDR breast cancer cells express at least twice as much vimentin on their cell surface as compared to drug-sensitive breast cancer cells (ie, drug-sensitive breast cancer cells are not multidrug resistant) . Thus, when cell components are separated (eg, plasma membrane fraction and cytosol fraction), MDR neoplastic or damaged cells that express vimentin on their cell surface are derived from the same tissue and are multidrug resistant The plasma membrane fraction contains vimentin at more than twice the level, whether normal, neoplastic, or damaged, compared to other cells that do not have that multidrug resistance .

In another aspect, the present invention provides a binding agent that specifically binds to vimentin protein or a fragment thereof. As used herein, “specifically binds” means that the binding agent recognizes and binds vimentin protein or fragments thereof, but does not substantially recognize and bind other molecules in the sample. Means. Therefore, the binding agent of the present invention specifically binds to the surface of MDR cells that express vimentin on the cell surface. Useful binders are at least 10 ⁶ M ⁻¹ , or at least 10 ⁷ M ⁻¹ in water, physiological conditions, or conditions similar to physiological conditions with respect to ionic strength, eg, 140 mM NaCl, 5 mM MgCl ₂ . Or associate with vimentin protein with an affinity of at least 10 ⁸ M ⁻¹ , or at least 10 ⁹ M ⁻¹ .

  As used herein, a “binding agent” is a molecule that specifically binds or attaches to a vimentin protein or fragment thereof. The binding agent need not have any specific size or structure as long as it specifically binds to vimentin protein or fragments thereof. Thus, a “binding agent” specifically binds to any region (eg, three-dimensional structure, amino acid sequence, or specific small chemical group) as long as it specifically binds to vimentin protein or fragments thereof. A molecule that attaches. Non-limiting examples of binding agents include natural ligands (eg, hormones or GTP), as well as synthetic small molecules, drugs, nucleic acids, peptides, and proteins such as hormones, antibodies or portions thereof. Usually, the ability of a binding agent to specifically bind to an epitope is a highly complementary structure. That is, the structure of the binding agent includes a structure that is complementary to the antigen moiety to which the binding agent specifically binds. The antigen portion to which the antibody binds is called the “epitope”.

In certain embodiments, the binding agent is an antibody. When the binding agent that specifically binds to vimentin protein is an antibody, the antibody is not intended to be limited, but is a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a genetically engineered antibody, Bispecific antibodies (one of the bispecific antibodies specifically binds to vimentin protein), antibody fragments (“Fv,” “F (ab ') ₂ ,” “F (ab), "And" Dab "), and may be a single chain (" SC-Mab ") representing the reactive portion of an antibody. Methods for producing antibodies and other binding agents are well known (eg, Coligan et al., “Current Protocols in Immunology”, John Wiley and Sons, New York, NY). 1991; Jones et al., Nature 321: 522-525, 1986; Marx, Science 229: 455-456, 1985; Rodwell, Nature 342: 99-100, 1989; Clackson, Br. J. Rheumatol., 3052: 36-39, 1991; Reichman et al., Nature 332: 323-327, 1988; Verhoeyen, et al., Science 239: 1534-1536, 1988).

  As used herein, “detectably labeled” means that the binding agent of the invention is operably linked to a detectable functional group. “Operatively linked” means that the functional group is attached to the binder by a covalent bond or a non-covalent bond (eg, an ionic bond). Methods for forming covalent bonds are known (eg, Wong, SS, “Chemistry of Protein Conjugation and Cross-Linking”, CRC Press, 1991; Burkhart et al., “ Amino crosslinking agents, aminoplast chemistry and applications (see "The Chemistry and Application of Amino Crosslinking Agents or Aminoplasts"), John Wiley & Sons Inc., New York, NY, 1999, see the entire protocol).

  According to the present invention, the detectably labeled binding agent of the present invention includes a binding agent that is conjugated to a detectable functional group. Another detectably labeled binding agent of the present invention is a fusion protein. In this case, one of the binding partners is a binding agent and the other is a detectable label. Yet another non-limiting example of a detectably labeled binding agent includes a binding agent, a first fusion protein comprising a first functional group with high affinity for the second functional group, and a second function A second fusion protein containing a group and a detectable label. For example, a binding agent that specifically binds to vimentin protein may be operatively linked to a streptavidin functional group. A second fusion protein comprising a biotin functional group operatively linked to a fluorescein functional group may be added to the binder-streptavidin fusion protein. In that case, binding of the second fusion protein to the binder-streptavidin fusion protein will result in a detectably labeled binder (ie, a binder operably linked to the detectable label). .

According to the present invention, a detectable label is a functional group that can be traced and is not intended to be limiting, but includes fluorescent dyes (eg, fluorescein (FITC), phycoerythrin, rhodamine), chemical dyes, radioactive compounds , Chemiluminescent compounds, magnetic compounds, paramagnetic compounds, parent magnetic compounds, or enzymes that produce colored products, chemiluminescent products, or magnetic products. In certain embodiments, the detectable label is detectable to the medical imaging device or system. For example, if the medical imaging system is an X-ray device, the detectable label detected by the X-ray device is a radioactive label (eg, ³² P). Note that the binding agent need not be directly conjugated to the detectable functional group. For example, a binding agent (eg, a mouse anti-human vimentin antibody) that is itself specifically bound by a second detectable binding agent (eg, a FITC-labeled goat anti-mouse secondary antibody) has a detectable function. Operably linked to a group (ie, a FITC functional group).

  In certain embodiments, measuring the expression level of vimentin protein on the surface of the test damaged cell comprises contacting an intact test damaged cell with a detectable binding agent that specifically binds the vimentin protein. For example, if a detectable binding agent is detectably labeled by being operably linked to a fluorescent dye, cells that are stained with the fluorescent dye (ie, specifically bound by the binding agent) Can be identified by fluorescence activated cell sorter analysis (see Examples) or routine fluorescence microscopy of clinical specimens prepared on slides.

  In addition to the detectable functional group, other non-limiting functional groups that may be operatively linked to the binding agents of the present invention include, without limitation, toxins (eg, radioisotopes), enzymes, An antibody (or part thereof), a cytotoxic agent, or a conjugate thereof. When a toxin is operably linked to a binding agent of the invention, non-limiting examples of toxins that are operably linked to a binding agent of the invention include radioisotopes, diphtheria toxins, nucleases (eg, DN RNase), protease, degrading enzyme, Pseudomonas exotoxin (PE), ricin A or B chain, and ribonuclease A (Fizgerald D., Semin. Cancer Biol., 7: 87-95, 1996) It is done.

  In certain embodiments, the binding agent is an immunotoxin (eg, an antibody-toxin conjugate or an antibody-drug conjugate). Non-limiting examples of immunotoxins include antibody-anthracycline conjugates (Braslawsky GR et al., European Patent No. EP0398305), antibody-cytokine conjugates (Gilles SD, PCT publication WO9953958), and monoclonal antibodies- PE conjugate (Roffler SR et al., Cancer Res. 51: 4001-4007, 1991).

  In yet another aspect, the present invention provides a therapeutic composition comprising a cytotoxic agent, a binding agent that specifically binds to vimentin protein or a fragment thereof, and a pharmaceutically acceptable carrier. Non-limiting examples of such pharmaceutically acceptable carriers are described in more detail in the standard textbook Remington “The Science and Practice of Pharmacy”, Gennaro et al. 20th edition, Lippincott Williams & Wilkins, Philadelphia, Pennsylvania, 2001 (ISBN 0-683-306472). In certain embodiments, binding of the binding agent is toxic to damaged cells, whether the cells are drug sensitive or multidrug resistant. In certain embodiments, binding of the binding agent is toxic to neoplastic cells, whether the cells are drug sensitive or multidrug resistant. In certain embodiments, the binding agent of the composition is operably linked to a toxin.

  Actual methods for preparing therapeutic compositions are known or will be apparent to those skilled in the art and are also described in Reminton's “Pharmaceutical Science and Practice” 2001 (above) and “Formulations and Drug Delivery Systems (“ Pharmaceutical Dosage ”). Forms and Drug Delivery Systems ")", 6th edition, Williams & Wilkins (1995). The therapeutic composition of the present invention may exist in any dosage form suitable for administration, including without limitation, tablets, capsules, powders, solutions, or elixirs.

  It should be noted that the cytotoxic agent of the therapeutic composition of the present invention need not be toxic to all cells. In certain embodiments, when the therapeutic composition is being administered to a patient suffering from a disease caused by the presence of damaged cells, the cytotoxic agent of the therapeutic composition is an anti-pathological or antimicrobial agent . In certain embodiments, when the damaged cell is infected with a pathogen (eg, a virus, bacterium, or multicellular parasite) and the infection is causing a disease, the cytotoxic agent of the therapeutic composition is anti-pathological. It is an agent. If the damaged cell is infected with a pathogen, non-limiting examples of different drugs depending on the pathogen include acyclovir, amphotericin, ampicillin, anthracycline, b-lactam antibiotics, cephalotin, chloramphenicol, chloroquine (CQ), cidofovir (CDV), ciprofloxacin, erythromycin, fluconazole, 5 flucytosine, fluoroquinolone, foscarnet, ganciclovir, halophanthrin, itraconazole, lamivudine, macrolides, mefloquine, mesicillin, metronidazole, miconazole, Nelfinavir, ofloxacin, penicillin, primaquine, quinoline, streptomycin, sulfonamides, teicoplanin, terbinafine, tetracycline, vancomycin, voricona Include Lumpur, but without limitation thereto. Such therapeutically effective amounts of drugs are known to physicians and pharmacists with ordinary skill. In addition, such information can be obtained from drug manufacturers or “Physician's Desk Reference”, Medical Economics Co. (published annually).

  In certain embodiments, when the therapeutic composition is being administered to a patient suffering from cancer caused by the presence of neoplastic cells, the cytotoxic agent of the therapeutic composition is an anti-cancer agent. Such anti-cancer agents include, without limitation, chemotherapeutic agents and radiotherapeutic agents. Non-limiting examples of such anticancer agents include actinomycin, adriamycin (AR), altretamine, asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine , Dactinomycin, daunorubicin, docetaxel, doxorubicin (DOX), epoetin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, lomustine, mechloretamine, melphalametreto, melchlortomelamine ), Mitothein, mitoxantrone, paclitaxel, pentostatin, procarbazine Taxol, teniposide, topotecan, vinblastine (VLB), vincristine, and include vinorelbine. Such therapeutically effective amounts of drugs are known to physicians and pharmacists with ordinary skill. In addition, such information can be obtained from drug manufacturers or “Physician's Desk Reference”, Medical Economics Co. (published annually).

  In another aspect, the present invention provides a method for treating a patient suffering from a disease caused by the presence of damaged cells. The method includes administering to the patient a therapeutically effective amount of the agent and a therapeutically effective amount of a binding agent that specifically binds to vimentin protein or a fragment thereof. In certain embodiments, the binding agent kills damaged cells that are multi-drug resistant, while the agent kills damaged cells that are drug sensitive. Of course, since vimentin protein is also expressed at moderate levels in drug-sensitive damaged cells, in certain embodiments, a binding agent that is different from the drug also kills the drug-sensitive damaged cells. This method improves the patient's prognosis in the disease compared to an untreated patient. In some embodiments, naïve patients do not take a binder, but do take a drug. An “naïve patient” or “control patient” is a patient who does not take a binding agent but who takes a drug, or who does not receive any treatment (ie, does not take a binding agent or a drug). The drug and binder may be administered separately in any order at different times, or may be administered together. In certain embodiments, the patient is a human.

  In some embodiments, the patient's damaged cells are infected with a pathogen. In certain embodiments, the pathogen is a virus, bacterium, or parasite (HIV, West Nile virus, and Dengue virus; Mycobacteria, Rickettsia, and Chlamydia; Plasmodium, Leishmania, and Taxoplasma).

  In another aspect, the present invention provides a method for treating a patient suffering from a disease (eg, cancer) caused by the presence of neoplastic cells. The method includes administering to the patient a therapeutically effective amount of the agent and a therapeutically effective amount of a binding agent that specifically binds to vimentin protein or a fragment thereof. In certain embodiments, the binding agent kills neoplastic cells that are multi-drug resistant, while the agent kills neoplastic cells that are drug sensitive. Since vimentin protein is also expressed at moderate levels in drug-sensitive neoplastic cells, in certain embodiments, a binding agent that is different from the drug also kills the drug-sensitive neoplastic cells. This method improves the patient's prognosis in the disease compared to an untreated patient. In certain embodiments, an untreated patient receives no therapy (ie, does not take a binder or drug). The agent and binding agent (eg, antibody) may be administered separately at different times in any order, or may be administered together. In certain embodiments, the patient is a human.

  In certain embodiments, the patient's neoplastic cells are breast cancer cells, ovarian cancer cells, myeloma cells, lymphoma cells, melanoma cells, sarcoma cells, leukemia cells, retinoblastoma cells, hepatoma cells, glioma cells, mesothelioma cells, Or it is an epithelial cancer cell.

  In certain embodiments, the binding agent is operably linked to a toxin. Non-limiting examples of such toxins are as described above.

  As used herein, “therapeutically effective amount” is used to describe a known treatment of a drug that is performed at a dose and for a period of time effective to kill damaged cells. Administration is without limitation, intravenous, parenteral, oral, sublingual, transdermal, topical, intranasal, intraocular, intravaginal, rectal, intraarterial, intramuscular, subcutaneous, and It may be done through any route including intraperitoneal.

  The dose and schedule of administration of the binding agent, agent, and / or therapeutic composition according to the present invention determines the extent of the disease or cancer symptoms, the type of agent used (eg, chemotherapeutic agent, radiotherapeutic agent, or antibiotic). ), The patient (eg, the patient's gender, age, and / or weight), the patient's medical history, and the patient's response to treatment. The dose of the binding agent, agent, and / or therapeutic composition may be a single dose or multiple doses. If multiple doses are used, the frequency of administration (schedule) will depend on, for example, the patient, the type of response, and the type of drug used. Some patients are effective once a week, while others are effective daily, every other day, and once every three days. A professional practitioner will be able to ascertain which route of administration and which dose is most effective in a particular case by routine experimentation.

  In yet another aspect, the invention features a method for detecting multidrug resistance in a patient. This method is intended for patients suspected of having multidrug resistant cells and is a binding agent that specifically binds to vimentin protein or a fragment thereof and is operatively linked to a label detectable by a medical imaging device. Administering a binding agent and examining the patient with the medical imaging device or system. According to this method, a medical imaging device or system detects a binding agent (eg, an antibody) that specifically binds to the surface of a patient's multidrug resistant cells.

  Medical imaging devices and systems are known, as are labels detectable by such systems. As previously mentioned, one non-limiting example of such a system and label is an x-ray device capable of detecting a radiolabeled binding agent. Other non-limiting examples of medical imaging systems include: (a) X-ray computed tomography (CT), positron emission tomography (PET), and new mergers and improvements based on these technologies [( PET + CT, spiral CT, single photon emission CT (SPECT), high resolution PET (microPET), and immunoscintigraphy (using radiolabeled antibodies (Czernin, J. and ME Phelps, Ann. Rev. Med. 53: 89-112, 2002; Goldenberg, DM, Cancer 80 (12): 2431-2435, 1997; Langer, SG et al. (2001) World J. Surg. 25: 1428-1437; Middleton, ML., Shell EG., Postgrad Med. 111 (5): 89-90, 93-6, 2002); (b) Magnetic Resonance Imaging (MRI) (Helbich, TH, J. Radiol., 34: 208-219, 2000; Langer, SG et al., World Journal of Surgery 25: 1428-1437, 2001; Nabi, HA and Zubeldia, JM, Oncology J. Nuclear Med. Technol., 30 (1): 3-9, 2002); Ultrasound Imaging device (US) (Harvey, CJ, et al. Advances in Ultrasound Clin. Radiol., 57: 157- 177, 2002; Langer, SG et al., WJ Surg. 25: 1428-1437, 2001); (c) Optical fiber endoscope (Shelbase DE, Curr. Opin. Pediatr. 14: 327-33, 2002); d) Gun machine scintillation detector (detects gamma emitters, eg 192-Ir) and beta scintillation detector (detects beta emitters, eg 90-Sr / Y) (Hanefeld C, Amirie, S et al., Circulation 105: 2493-6, 2002).

  In certain embodiments, the patient is a mammal, eg, a human. The patient may be, for example, a human suffering from a disease caused by the presence of multidrug resistant cells. For example, a patient may suffer from cancer caused by multidrug resistant neoplastic cells. Such multi-drug resistant neoplastic cells include, without limitation, ovarian cancer cells, myeloma cells, lymphoma cells, melanoma cells, sarcoma cells, leukemia cells, retinoblastoma cells, hepatoma cells, glioma cells, mesothelial cells Tumor cells or epithelial cancer cells.

  In certain embodiments, the multidrug resistant cell is a damaged cell and the patient suffers from a disease caused by the presence of such multidrug resistant damaged cell. Non-limiting examples where cells are damaged include infection by pathogens (eg, viruses, bacteria, or parasites), or cells may be damaged by necrosis. In certain embodiments, the damaged cell is infected by a pathogen (eg, a virus, parasite, or bacterium). For example, a patient may suffer from tuberculosis caused by a multidrug resistant strain of Mycobacterium tuberculosis.

The practice of the present invention, unless otherwise indicated, is included in the conventional art of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology. Technology is used. Such techniques are explained fully in the literature. For example, "Molecular Cloning A Laboratory Manual", 2nd edition, edited by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); "DNA Cloning" , I and II (DN Glover, 1985); “Oligonucleotide Synthesis”, (MJ Gait, 1984); Mullis et al. US Pat. No. 4,683,195; “Nucleic hybridization (” Nucleic Acid Hybridization ”), (BD Hames & SJ Higgins, 1984);“ Transcription and Translation ”, (BD Hames & SJ Higgins, 1984);“ Animal cell culture (“Culture Of Animal Cells ”)” (RL Freshney, Alan R. Liss, Inc., 1987); “Immobilized Cells and Enzymes” ”, (IRL Press, 1986); B. Perbel,“ Molecules “A Practical Guide to Molecular Cloning” (1984); “Methods In Enzymology” ”(Academic Press, Inc., New York);“ Gene Transfer Vectors For Mammalian Cells ”, (JH Miller and MP Calos, 1987, Cold Spring Harbor Laboratory "Methods In Enzymology", Volumes 154 and 155 (Edited by Wu et al.); "Immunochemical Methods In Cell and Molecular Biology" ”(Mayer and Walker, Academic Press, London, 1987);“ Handbook Of Experimental Immunology ”, Volume I-IV (DM Weir and CC Blackwell, 1986); See "Manipulating the Mouse Embryo", (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1986).
4.2 Vimentin antibodies The present invention provides antibodies directed against vimentin for use in the detection, imaging, and treatment of cancer and damaged (eg, pathogen-infected) cells. Anti-vimentin antibodies used in the present invention are available from several vendors. For example, CHEMICON (Temecula, CA) and ABR-Affinity BioReagents (Golden, CO) both produce anti-human vimentin mouse monoclonal and / or rabbit polyclonal antibodies.

  The term “antibody” is used in the broadest sense and includes single anti-vimentin monoclonal and polyclonal antibodies, anti-vimentin antibody fragments (eg, Fab, F (ab) 2, and Fv), and polyepitope Specifically covers specific anti-vimentin antibody compositions (including binding and non-binding antibodies). As used herein, “monoclonal antibody” refers to a substantially homogeneous antibody population, ie, individual antibodies that contain the population, except for naturally occurring mutations that may be present in small amounts. Refers to antibodies that are identical. Monoclonal antibodies are highly specific and are directed to a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody specimens, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. It is done. Novel monoclonal antibodies or fragments thereof can in principle represent all classes of immunoglobulins, e.g. IgM, IgG, IgD, IgE, IgA, or their subclasses, e.g. IgG subclasses or mixtures thereof. means. Also included are IgG and its subclasses, eg, IgG1, IgG2, IgG2a, IgG2b, IgG3, or IgGM. IgG subtypes, IgG1 / kappa and IgG2b / kappa are also included as embodiments.

  Monoclonal antibodies herein can be anti-vimentin antibodies (eg, humanized antibodies) with constant domains, light chains with heavy chains, species of origin, immunoglobulin class, or subclass designations. Variable in antibody chains (eg, Fab, F (ab) 2, and Fv), such as chains of one species, chains with another species, or fusion with heterologous proteins Hybrids and recombinant antibodies produced by splicing domains (including hypervariables) are included as long as they exhibit the desired biological activity (eg, US Pat. No. 4,816,567 and Mage & Lamoyi, “ See Monoclonal Antibody Production Techniques and Applications, pages 79-97 (Marcel Dekker, Inc., New York (1987)). Therefore, the modified “monoclonal” displays the nature of the antibody as being derived from a substantially homogeneous population of antibodies and should not be considered as requiring production of the antibody by any particular method. Don't be. For example, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described by Kohler and Milstein, Nature 265: 495 (1975), or may be recombinant DNA methods (US Pat. No. 4,816,567). ). “Monoclonal antibodies” may also be isolated from phage libraries generated using, for example, the techniques described in McCafferty et al., Nature 348: 552-554 (1990).

  “Humanized” forms of non-human (eg, rodent) antibodies are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, those containing minimal sequence derived from non-human immunoglobulin) Fv, Fab, Fab ′, F (ab) 2, or other antigen binding sequences of antibodies). Humanized antibodies are mostly human immunoglobulins (recipient antibodies), but the complementarity determining region (CDR) residues of the recipient antibody have the desired specificity, affinity, and capacity. Substituted by the residue (donor antibody) of the CDRs of a non-human species such as mouse, rat, or rabbit. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human FR residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or FR sequences. These modifications are performed to further fine tune and optimize antibody performance. Generally, a humanized antibody comprises substantially all of at least one, typically two variable domains, wherein all or substantially all of the CDR regions are non-human immunoglobulin. All or substantially all of the FR residues become residues of the human immunoglobulin consensus sequence. Humanized antibodies also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

  Vimentin or an anti-vimentin monoclonal antibody or fragment thereof is in principle all immunoglobulin classes, such as IgM, IgG, IgD, IgE, IgA, or subclasses thereof, such as IgG subclasses or their It means a mixture. IgG and its subclass are, for example, IgG1, IgG2, IgG2a, IgG3, or IgGM. IgG subtypes, IgG1 / kappa and IgG2b / kappa are also included as embodiments. All fragments that may be mentioned are cleaved or modified antibody fragments with one or two antigen-complementary binding sites. This complementarity-determining site corresponds to the antibody and has a binding site formed by the light and heavy chains, such as an antibody portion, e.g., an Fv, Fab, or F (ab ') 2 fragment, or a single chain fragment. Thus, it shows a high degree of binding and binding activity to mammalian vimentin. The cleaved double-stranded fragment is, for example, Fv, Fab, or F (ab ') 2. These fragments can be obtained, for example, by enzymatic means, by removing the Fc portion of an antibody by an enzyme such as papain or pepsin, by chemical oxidation, or by genetic manipulation of antibody genes. . It is also possible and advantageous to use fragments that have not been cut by genetic engineering. Anti-vimentin antibodies or fragments thereof may be used alone or in admixture.

Conveniently, the novel antibody, antibody fragment, mixture or derivative thereof has a dissociation constant value from about 1 × 10 ⁻¹¹ M (0.01 nM) to about 1 × 10 ⁻⁸ M (10 nM), or from about 1 × ^{10 −10} M (0.1 nM). Has binding affinity for vimentin over a long range of about 3x10 ^-9 M (3 nM).

  The antibody gene for gene manipulation can be isolated from hybridoma cells in a manner known to skilled researchers, for example. For this purpose, antibody-producing cells are cultured and once the optical density of the cells is sufficient, the mRNA is separated from the cells in a known manner, i.e. the cells are degraded by guanidinium thiocyanate and washed with sodium acetate. Acidify, extract with phenol, chloroform / isoamyl alcohol, precipitate with isopropanol and separate by washing with ethanol. Next, cDNA is synthesized from this mRNA using reverse transcriptase. Synthesized cDNA can be introduced into appropriate animal, fungal, bacterial, or viral vectors, either directly or after genetic manipulation, eg, by introducing site-directed mutations, insertions, inversions, deletions, or base exchanges. It can be inserted later and expressed in a suitable host organism. Preferred are bacteria or yeast vectors for gene cloning, such as pBR322, pUC18 / 19, pACYC184, lambda, or yeast mu vectors, for expression bacteria such as E. coli, or It is a yeast like Saccharomyces cerevisiae.

  The invention further relates to cells that synthesize vimentin antibodies. Such include animal cells, fungal cells, bacterial cells, or yeast cells after transformation as described above. Hybridoma cells or trioma cells are preferred, but hybridoma cells are preferred. This hybridoma cell can be produced, for example, in a known manner. That is, an animal is immunized with vimentin, antibody-producing B cells are isolated, and vimentin-binding antibodies are selected from these cells, after which these cells are treated with, for example, human or animal, eg, mouse myeloma By fusing cells, human lymphoma-like cells or heterohybridoma cells (see eg Koeler et al., (1975) Nature 256: 496) or infecting these cells with the appropriate virus Produce. Hybridoma cell lines produced by fusion are particularly useful and murine hybridoma cell lines are very useful. The hybridoma cell line of the present invention secretes IgG type antibodies. The binding of the mAb antibody of the present invention binds to vimentin with high affinity.

The present invention further includes derivatives of these anti-vimentins. These derivatives preferably maintain their vimentin binding activity while altering one or more properties associated with their use as a formulation, such as serum stability or production efficiency. Examples of such anti-vimentin antibody derivatives include peptides obtained from antigen-binding regions of antibodies, peptide-like substances; solid phase or liquid phase carriers such as polyethylene glycol, glass, synthetic polymers such as polyacrylamide, polystyrene, and polypropylene. Antibodies, fragments or peptides bound to carriers such as polyethylene, or natural polymers such as cellulose, sepharose or agarose; or enzymes, toxins, or radioactive or non-radioactive markers such as ³ H, ¹²³ I, ¹²⁵ I ¹³¹ I, ³² P, ³⁵ S, ¹⁴ C, ⁵¹ Cr, ³⁶ Cl, ⁵⁷ Co, ⁵⁵ Fe, ⁵⁹ Fe, ⁹⁰ Y, ⁹⁹ mTc (technetium transferable isotope), ⁷⁵ Se, Or fluorescent / chemiluminescent labels such as rhodamine, fluorescein, isothiocyanate, phycoerythrin, There are antibodies, fragments or peptides covalently attached to labels such as icocyanin, fluorescamine, metal chelators, avidin, streptavidin, or biotin.

  The novel antibodies, antibody fragments, mixtures and derivatives thereof can be used directly or other pharmaceutical activities after drying, eg after lyophilization, after adherence to the aforementioned carriers, or as required for pharmaceutical production And can be used after prescription with auxiliary substances. Examples of activities and auxiliary substances that may be mentioned include other antibodies, antimicrobial or antimicrobial active substances with antimicrobial activity, for example antibiotics in general or sulphonamides, antitumor agents, water, buffers, saline, Alcohols, oils, waxes, inert vehicles, or other substances associated with parenteral formulations such as amino acids, thickeners, or sugars. These formulations are used to control disease control, preferably joint damage, preferably joint cartilage damage.

  The anti-vimentin antibody of the present invention can be administered orally or parenterally, subcutaneously, intramuscularly, intravenously, or intraperitoneally.

  The antibody, antibody fragment, mixture or derivative thereof can be directly or after conjugation with a solid or liquid phase carrier, enzyme, toxin, radioactive or non-radioactive label, or with a fluorescent / chemiluminescent label as described above. It can be used after the coupling. Vimentin can be detected in a wide variety of cell types, particularly in neoplastic cells. The human vimentin monoclonal antibody of the present invention may be obtained as follows. One skilled in the art will recognize that other equivalent processes are available for obtaining vimentin and are also included in the present invention.

  First, a mammal is immunized with human vimentin. Purified human vimentin can be purchased from other vendors, including Sigma (St. Louis, MO, catalog A6152). Human vimentin can be easily purified from human placental tissue. Furthermore, immunoaffinity purification methods are known to obtain high purity vimentin immunogens (see, eg, Vladutiu et al., (1975) 5: 147-59 Prep. Biochem.). Mammals used to produce anti-human vimentin antibodies are not limited and may be primates, rodents such as mice, rats, or rabbits, cows, sheep, goats, or dogs.

  Next, antibody producing cells such as spleen cells are removed from the immunized animal and fused with myeloma cells. This myeloma cell is known in the art (eg, p3x63-Ag8-653, NS-0, NS-1 or P3U1 cells may be used). The cell fusion operation may be performed by a well-known conventional method.

  The cells are subjected to a cell fusion operation and then cultured in a HAT selection medium to select hybridomas. Next, screening is performed for hybridomas that produce anti-human monoclonal antibodies. This screening can be performed by, for example, a sandwich ELISA (enzyme immunoassay). At this time, the produced monoclonal antibody is bound to the well in which human vimentin is immobilized. In this case, as the secondary antibody, an antibody specific to the immunoglobulin of the immunized animal and an antibody labeled with an enzyme such as peroxidase, alkaline phosphatase, glucose oxidase, beta-D-galactosidase, etc. is used. May be. This label may be detected by reacting the labeled enzyme with its substrate and measuring the color development. As a substrate, 3,3-diaminobenzidine, 2,2-diaminobis-o-dianiside, 4-chloronaphthol, 4-aminoantipyrine, o-phenylenediamine and the like may be produced.

  By the above-described operation, it is possible to select a hybridoma that produces an anti-human vimentin antibody. The selected hybridoma is then cloned by conventional limiting dilution or soft agar methods. If necessary, the cloned hybridoma can be cultured on a large scale using a serum-containing or serum-free medium, or it can be inoculated into the peritoneal cavity of a mouse and recovered from ascites to obtain a large number of clones. A hybridoma may be obtained.

  Next, from the selected anti-human vimentin monoclonal antibodies, those having the ability to bind to cell surface vimentin are selected and used for subsequent analysis and manipulation.

  The monoclonal antibody obtained here can be an anti-vimentin antibody (eg, a humanized antibody) with a constant domain, a light chain with a heavy chain, a species of origin, an immunoglobulin class, or a subclass designation. First, a chain of one species, a chain with another species, or a fusion with a heterologous protein, such as antibody fragments (for example, Fab, F (ab) 2, and Fv) Hybrids and recombinant antibodies produced by splicing variable (including hypervariable) domains are included as long as they exhibit the desired biological activity (eg, US Pat. No. 4,816,567 and Mage & Lamoyi, See “Monoclonal Antibody Production Techniques and Applications”, pages 79-97 (Marcel Dekker, Inc.), New York (1987)).

  Thus, the term “monoclonal” indicates that the nature of the antibody is derived from a substantially homogeneous antibody population and should not be considered as requiring production of the antibody by any particular method. . For example, monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described by Kohler and Milstein, Nature 265: 495 (1975), or may be recombinant DNA methods (US Pat. No. 4,816,567). ). “Monoclonal antibodies” may also be isolated from phage libraries generated using, for example, the techniques described in McCafferty et al., Nature 348: 552-554 (1990).

  “Humanized” forms of non-human (eg, rodent) antibodies are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, those containing minimal sequence derived from non-human immunoglobulin) , Fv, Fab, Fab ′, F (ab) 2, or other antigen binding subsequences of antibodies). Humanized antibodies are mostly human immunoglobulins (recipient antibodies), but the complementarity determining region (CDR) residues of the recipient antibody have the desired specificity, affinity, and capacity. Substituted by the residue (donor antibody) of the CDRs of a non-human species such as mouse, rat, or rabbit. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human FR residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or FR sequences. These modifications are performed to further fine tune and optimize antibody performance. Generally, a humanized antibody comprises substantially all of at least one, typically two variable domains, wherein all or substantially all of the CDR regions are non-human immunoglobulin. All or substantially all of the FR residues become residues of the human immunoglobulin consensus sequence. Humanized antibodies also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

  Methods for humanizing non-human antibodies are well known in the prior art. Generally, one or more amino acid residues are introduced into a humanized antibody from a non-human source. This non-human amino acid residue is often referred to as an “import” residue. This residue is usually removed from the “importing” variable domain. Humanization is in effect according to the method of Winter and co-workers (Jones et al., (1986) Nature 321: 522-525; Riechmann et al., (1988) Nature, 332: 323-327; and Verhoeyen et al., (1988) Science 239: 1534-1536), carried out by replacing the corresponding sequence of a human antibody by a rodent CDR or CDR sequence. Accordingly, such “humanized” antibodies are chimeric antibodies in which substantially less than the original human variable domain has been replaced by the corresponding sequence from a non-human animal species. In practice, humanized antibodies are usually human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

  The choice of human variable domains used to produce humanized antibodies, whether light or heavy, is extremely important in reducing antigenicity. In the so-called “optimal fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that most closely resembles that of rodents is then accepted as the human framework (FR) of humanized antibodies (Sims et al., (1993) J. Immunol., 151: 2296; and Chothia and Lesk (1987) J. Mol. Biol., 196: 901). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework can be used for several separate humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. (USA), 89: 4285; and Presta et al., ( 1993) J. Immunol., 151: 2623).

  It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, a humanized antibody is analyzed by analyzing the parent sequence and various conceptual humanized products using a three-dimensional model of the parent sequence and the humanized sequence. Is prepared. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs that illustrate and display the expected three-dimensional structure of the selected candidate immunoglobulin sequences are also available. By scrutinizing these displays, it is possible to analyze the possible role of residues in the function of a candidate immunoglobulin sequence, i.e., analyze the residues that affect the ability of the candidate immunoglobulin to bind to the antigen. Become. In this way, FR residues can be selected and removed from the common / import sequence and bound to achieve the desired antibody properties, eg, increased affinity for the target antigen (s). . In general, the CDR residues are most substantially involved directly in antigen binding.

  Human antibodies directed against vimentin are also included in the present invention. Such an antibody can be produced, for example, by the hybridoma method. For human myeloma cells and mouse-human heteromyeloma cell lines for human monoclonal antibody production, see, for example, Kozbor (1984) J. Immunol., 133, 3001; Brodeur et al., “Monoclonal Antibody Production Techniques and Applications (“Monoclonal Antibody Production Technique and Applications”), pp. 51-63, (Marcel Dekker, Inc., New York, 1987); and Boemer et al., (1991) J. Immunol., 147: 86. -95. Specific methods for producing such human antibodies, such as phage display, transgenic mouse engineering, and / or in vitro display methods such as ribosome display or covalent display have also been described (Osbourn et al. ( 2003) Drug Discov. Today 8: 845-51; Maynard and Georgiou (2000) Ann. Rev. Biomed. Eng. 2: 339-76; and U.S. Pat. reference).

  Currently, it is possible by immunization to produce transgenic animals (eg, mice) that are capable of producing the entire repertoire of human antibodies while lacking endogenous immunoglobulin production. For example, it has been described that in chimeric, germline mutant mice, homozygous deletion of the antibody heavy chain joining region (JH) gene results in complete suppression of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays to such germline mutant mice results in the production of human antibodies against antigen challenge (see, eg, Jakobovits et al., (1993) Proc. Natl. Acad. Sci. (USA), 90: 2551; Jakobovits et al., (1993) Nature, 362: 255-258; and Bruggermann et al., (1993) Year in Immuno., 7:33).

  Separately, humans obtained from a gene repertoire of immunoglobulin variable (V) domains from non-immunized donors using the phage display method (McCafferty et al., (1990) Nature, 348: 552-553). Antibodies and antibody fragments can be produced in vitro. According to this method, the antibody V domain gene repertoire is cloned to match the reading frame to a filamentous bacteriophage, eg, M13 or fd large or small coat protein gene, and a functional antibody fragment on the surface of the phage particle. As displayed. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody allows selection of the gene encoding the antibody exhibiting that property. Thus, phage mimics some of the properties of B cells. Phage display can be performed in a variety of ways (see, eg, Johnson et al., (1993) Curr. Opin. In Struct. Bio., 3: 564-571 for a review). Several supplies of V gene segments can be used for phage display. For example, Clackson et al. ((1991) Nature, 352: 624-628) developed a diverse array of anti-oxazolone antibodies from a small library of random combinations of V genes obtained from the spleens of immunized mice. Isolated. Building a repertoire of V genes from non-immunized human donors and isolating antibodies against diverse antigen arrays (including self-antigens) are also described in Marks et al. ((1991) J. Mol. Biol., 222: 581-597, or Griffith et al., (1993) EMBO J., 12: 725-734).

  In a natural immune response, antibody genes accumulate mutations at a high rate (somatic fast mutation). Some of the changes introduced give higher affinity, and B cells presenting high affinity surface immunoglobulins are preferably replicated and differentiate upon subsequent antigen challenge. This natural process can be mimicked using a technique known as “chain shuffling” (see Marks et al., (1992) Bio / Technol., 10: 779-783). In this method, the affinity of the “primary” human antibody obtained by phage display, the heavy and light chain V region genes, and the naturally occurring variant of the V domain gene obtained from a non-immunized donor. It is possible to improve by sequentially replacing the repertoire (repertoire). This technique allows the production of antibodies and antibody fragments with affinities in the nM range. A strategy for producing a large phage antibody repertoire has been described by Waterhouse et al. ((1993) Nucl. Acids Res., 21: 2265-2266).

  Gene shuffling can also be used to derive a human antibody from a rodent antibody, in which case the human antibody has similar affinity and specificity as the starting rodent antibody. According to this method, also called “epitope imprinting”, the heavy chain or light chain V domain gene of a rodent antibody obtained by phage display method is replaced by a repertoire of human V domain genes to provide rodents. Forms a human chimera. By selecting on the basis of the antigen, a human variable region is isolated that makes it possible to restore a functional antigen binding site, ie, the epitope governs the choice of binding partner. By repeating this process and replacing the remaining rodent V domain, a human antibody can be obtained (see PCT WO93 / 06213 published on April 1, 1993). Unlike conventional humanization of rodent antibodies by CDR grafting, this technique yields fully human antibodies that do not have any rodent framework or CDR residues.

  By using the aforementioned monoclonal antibody of the present invention, human vimentin in a sample can be detected and quantified. Detection or quantification of human vimentin in a sample can be performed by an immunoassay utilizing a specific binding reaction between the monoclonal antibody of the present invention and human vimentin. Various immunoassays are well known in the prior art, and any one selected from them can be used. Examples of immunoassays include a sandwich method using a monoclonal antibody and another monoclonal antibody as primary and secondary antibodies, a sandwich method using a monoclonal antibody and a polyclonal antibody as primary and secondary antibodies, and a gold colloid, respectively. Examples include dyeing, aggregation, latex, and chemiluminescence. Of these, a sandwich ELISA is particularly preferred. As is well known, in this method, the primary antibody is immobilized on, for example, the inner wall of a well, and then the sample is reacted with the immobilized primary antibody. After washing, the secondary antibody is reacted with the antigen-antibody complex immobilized in the well. After washing, the immobilized secondary antibody is quantified. As the primary antibody, an antibody that specifically reacts with human vimentin is preferably used.

The quantification of the secondary antibody is performed by reacting a labeled antibody specific to the immunoglobulin of the animal from which the secondary antibody was obtained (for example, an enzyme-labeled antibody) with the secondary antibody, and then measuring the label. To be executed. Alternatively, a labeled (eg, enzyme labeled) antibody may be used as the secondary antibody and quantification of the secondary antibody may be performed by measuring the label on the secondary antibody.
4.3 Vimentin Binding Agent In another aspect, the present invention provides a binding agent that specifically binds to vimentin protein or a fragment thereof. As used herein, “specifically binds” means that the binding agent recognizes and binds vimentin protein or a fragment thereof, but does not substantially recognize and bind other molecules in the sample. To do. Therefore, the binding agent of the present invention specifically binds to the surface of MDR cells that express vimentin on its surface. Useful binders are at least about 10 ⁶ M-1 in water, under physiological conditions, or conditions that approximate physiological conditions with respect to ionic strength, for example, in 140 mM NaCl, 5 mM MgCl ₂ , or It forms an association with the vimentin protein with an affinity of at least about 10 ⁷ M-1, or at least about 10 ⁸ M-1, or at least 10 ⁹ M-1. As used herein, a “binding agent” is a molecule that specifically binds or attaches to a vimentin protein or fragment thereof.

  The binding agent need not be of any particular size or have any particular structure as long as it specifically binds to vimentin protein or fragments thereof. Thus, a “binding agent” specifically binds to any region (eg, three-dimensional structure, amino acid sequence, or specific small chemical group), as long as it specifically binds to vimentin protein or fragments thereof. A molecule that attaches. Non-limiting examples of binding agents include natural ligands (eg, hormones or GTP), synthetic small molecules, drugs, nucleic acids, peptides, and proteins such as hormones and antibodies, and portions thereof. It is done.

Some examples of non-antibody vimentin binding agents are known in the prior art. For example, modified LDL has been shown to bind specifically to vimentin with an affinity of Kd of about 1.7 × 10 ⁻⁷ M (Ka equals about 5.9 × 10 ⁶ M-1) (Heidenthal et al., (2000) Biochem. Biophys. Res. Comm. 267: 49-53). The modified LDL may optionally be oxidized / acetylated LDL if desired. Such modified LDL production methods are known in the prior art. For example, oxidized LDL can be prepared by incubating LDL with 5 uM CuSO ₄ dissolved in O ₂ -saturated PBS without EDTA (Hrboticky et al., (1999) Arterioscler. Throm. Basc Biol. 19: 1267-75). Acetylation and iodination of oxidized LDL can be performed by methods known in the prior art (Basu et al. (1976) Proc. Natl. Acad. Sci. USA 73: 3178-82) (acetylation) and Bilheimer et al. (See Biochem. Biophys. Acta 260: 212-21 (iodination)). Other examples of non-antibody vimentin binding agents include Nlk1 protein (US Pat. No. 6,476,193) and SNAP23 protein (see Faigle et al. (2000) Mol. Biol. Cell. 11: 3485-3494), desmin , Glial fibrillary acidic protein and peripherin, fimbrin, RhoA-binding kinase alpha, and protein phosphatase 2A. Vimentin itself is also known to become a multimer by self-association and is therefore also a non-antibody vimentin binding agent.
4.4 Vimentin Targeted Diagnostic Method The present invention further allows for early identification of patients with MDR neoplastic or damaged cells. For example, if a patient identified as having such cells is a patient in remission of cancer, or is currently being treated for cancer (eg breast cancer, ovarian cancer, prostate cancer, leukemia, etc.) The present invention not only allows the identification of these patients prior to their return and / or progression of the cancer, but also allows these patients to be monitored during treatment with the drug and thereby to change the treatment schedule. To do. Similarly, if the patient identified as having such cells is an asymptomatic patient who has been treated with an infection or has previously been treated with an infection (eg, hepatitis B), the present invention Not only allows identification of these patients prior to reversal of symptoms, but also allows for a change in treatment schedule if such MDR cells are detected. Furthermore, diagnostic applications of the present invention allow the diagnosis and imaging of neoplastic, MDR neoplastic or damaged (eg, pathogen infected) cells by using cell surface vimentin markers.

  Diagnostic applications of the present invention include vimentin binding agents, such as probes conjugated to anti-vimentin antibodies, and other detection agents. As used herein, “detectable label” means that the binding agent of the invention is operably linked to a detectable functional group. “Operatively linked” means that the functional group is attached to the binder by a covalent or non-covalent (eg, ionic) bond. Methods for creating covalent bonds are known (eg, Wong, SS, “Chemistry of Protein Conjugation and Cross-Linking”), CRC Press 1991; Burkhart et al., “Amino-crosslinking. Agents, Aminoplast Chemistry and Applications (see “General Chemistry and Application of Amino Crosslinking Agents or Aminoplasts”), John Wiley & Sons Inc., New York, NY, 1999).

  According to the present invention, the detectably labeled binding agent of the present invention comprises a binding agent conjugated to a detectable functional group. Another detectably labeled binding agent of the present invention is a fusion protein, where one is a binding agent and the other is a detectable label. Yet another non-limiting example of a detectable label binding agent includes a binding agent, a first fusion protein comprising a first functional group with high affinity for the second functional group, and a second functional group and detection. A second fusion protein containing a possible label. For example, a binding agent that specifically binds to vimentin protein may be operably linked to a streptavidin functional group. A second fusion protein containing a biotin functional group operatively linked to a fluorescein functional group may be added to the binder-streptavidin fusion protein. In that case, the second fusion protein binds to the binder-streptavidin fusion protein, resulting in a detectably labeled binding agent (ie, a binder operably linked to the detectable label). .

The detectable label of the present invention is a functional group that can be traced, and is not intended to be limiting, but a fluorescent dye (eg, fluorescein (FITC), phycoerythrin, rhodamine), chemical dye, radioactive compound, chemiluminescence It includes compounds, magnetic compounds, paramagnetic compounds, parent magnetic compounds, or enzymes that produce colored products, chemiluminescent products, or magnetic products. In certain embodiments, the detectable label is detectable to the medical imaging device or system. For example, if the medical imaging system is an X-ray device, the detectable label detected by the X-ray device is a radioactive label (eg, ³² P). Note that the binding agent need not be conjugated directly to the detectable functional group. For example, a binding agent (eg, a mouse anti-human vimentin antibody) that is itself specifically bound by a second detectable binding agent (eg, a FITC-labeled goat anti-mouse secondary antibody) has a detectable function. Operably linked to a group (ie, a FITC functional group).

  In certain embodiments, measuring the expression level of vimentin protein on the surface of the test damaged cell comprises contacting an intact test damaged cell with a detectable binding agent that specifically binds the vimentin protein. For example, if a detectable binding agent is detectably labeled by being operably linked to a fluorescent dye, cells that are stained with the fluorescent dye (ie, specifically bound by the binding agent) Can be identified by fluorescence activated cell sorter analysis (see Examples) or routine fluorescence microscopy of clinical specimens prepared on slides.

  Medical imaging devices and systems are known, as are labels detectable by such systems. As previously mentioned, one non-limiting example of such a system and label is an x-ray device capable of detecting a radiolabeled binding agent. Other non-limiting examples of medical imaging systems include: (a) X-ray computed tomography (CT), positron emission tomography (PET), and new mergers and improvements based on these technologies [( PET + CT, spiral CT, single photon emission CT (SPECT), high resolution PET (micro PET), and immunoscintigraphy (using radiolabeled antibodies (Czernin, J. and ME Phelps (2002), Annual Reviews of Medicine 53: 89-112, 2002; Goldenberg, DM (1997), Cancer 80 (12): 2431-2435; Langer, SG et al. (2001) World Journal of Surgery 25: 1428-1437; Middleton, ML. and Shell EG. (2002) Postgrad Med. 111 (5): 89-90, 93-6 ,; (b) Magnetic Resonance Imaging (MRI) (Helbich, TH (2002) Journal of Radiology, 34: 208-219 Langer, SG et al. (2001) World Journal of Surgery 25: 1428-1437; Nabi, HA and Zubeldia, JM (2002) Oncology Journal of Nuclear Medicine Technology, 30 (1): 3-9; (US) (Harvey, CJ ., et al. (2002) Advances in Ultrasound Clinical Radiology, 57: 157-177; Langer, SG et al. (2001) World Journal of Surgery 25: 1428-1437); (c) Optical fiber endoscope (Shelbase DE (2002), Curr. Opin. Pediatr. 14: 327-33); (d) Gun machine scintillation detector (detects gamma emitters, eg 192-Ir) and beta scintillation detector (beta emitters) For example, 90-Sr / Y) (Hanefeld C, Amirie, S. et al. (2002) Circulation 105: 2493-6).

  Labeled antibodies that specifically bind to vimentin polypeptides, and derivatives and analogs thereof, are used for diagnostic purposes to detect, diagnose, or monitor diseases and / or disorders associated with abnormal expression of cell surface vimentin. Is possible. The present invention is a method for detecting abnormal expression of cell surface vimentin, comprising: (a) using one or more antibodies specific for vimentin, and using an individual cell or cell surface membrane fraction of interest. Assaying the expression of the polypeptide, and (b) increasing the quantified cell surface vimentin expression level when compared to the standard expression level, comprising comparing the gene expression level to the standard gene expression level Alternatively, a decrease provides a method that indicates abnormal expression. For example, where multidrug resistance is to be detected in neoplastic cells, the “standard expression levels” to be compared are non-multidrug resistant neoplastic cells of the same or similar origin or cell type. Similarly, if the neoplastic nature of the test cell is to be detected, the “standard expression level” to be compared is a non-neoplastic cell of the same or similar origin or cell type. Further, as “damage” in a test cell (eg, pathogen infection) is to be detected, the “standard expression level” to be compared is the same or similar origin or cell type of uninjured cells (eg, Infected cells).

  With regard to cancer, the presence of a relatively high amount of cell surface vimentin in a biopsy tissue or test cell obtained from an individual may indicate a propensity for the disease to develop or may be an actual clinical condition. There is a possibility to provide a means to detect the disease before the emergence of. When this type of relatively clear diagnosis is obtained, health professionals can take preventive or drastic actions earlier, thereby preventing the development or further progression of cancer.

The antibodies of the invention can be used to quantify protein levels in biological samples using classical immunohistological methods known to those skilled in the art (see, eg, Jalkanen, M., et al. (1985) J. Cell Biol. 101: 976-985; Jalkanen, M., et al., (1987) J. Cell Biol. 105: 3087-3096). Other antibody methods useful for detecting vimentin protein expression include immunoassays, such as enzyme immunoassay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the prior art, enzymes such as glucose oxidase; radioisotopes, such as iodine ⁽¹²⁵ I, ¹²¹ I), carbon ⁽¹⁴ C), sulfur ⁽³⁵ S), tritium ⁽³ H), indium ( ¹¹² I), and technetium ( ⁹⁹ Tc); luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine and biotin.

  One aspect of the invention is the detection and diagnosis of diseases or disorders associated with abnormal expression of cell surface vimentin in animals, eg, mammals, eg, humans. In one embodiment, diagnosis comprises a) administering to a subject an effective amount of a labeled anti-vimentin antibody or other vimentin binding agent that specifically binds to the cell surface vimentin of the animal (eg, B) after administration, the labeled molecule can be concentrated (and unbound) in the subject, preferably at the site where the polypeptide is expressed. Waiting for a sufficient time) so that the labeled molecule can be eliminated and fall to the background level; c) determining the background level; and d) detecting the labeled molecule in the subject, the labeled molecule above the background level Detection of is intended to indicate that the subject has a particular disease or disorder associated with abnormal expression of the polypeptide of interest. Background levels can be quantified in a variety of ways, including comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.

Vimentin-specific, labeled with a suitable detectable imaging functional group, such as a radioisotope (eg ¹³¹ I, ¹¹¹ I, ^99m Tc), a radiopaque substance, or a substance detectable by nuclear magnetic resonance An antibody or antibody portion is introduced into a mammal to be examined for the presence or absence of a disorder (eg, parenterally, subcutaneously, or intraperitoneally). In general, suitable radioisotopes for imaging and detection include radioisotopes that emit alpha, beta, or gamma radiation. Gamma radiation is particularly easy to image using current technology. Examples are gallium, indium, technetium, yttrium, ytterbium, rhenium, platinum, thallium, and astatine, e.g. ⁶⁷ Ga, ¹¹¹ In, ^99m Tc, ⁹⁰ Y, ⁸⁶ Y, ¹⁶⁹ Yb, ¹⁸⁸ Re, ^{195 m} Pt, ²⁰¹ Ti , And ²¹¹ At. It is understood in the prior art that the size of the subject and the imaging system used will determine the amount of imaging functional group needed to produce a diagnostic image. In the case of a radioisotope functional group, for human subjects, the amount of radioactivity injected is typically in the range of about 5 to 20 millicuries of 99mTc. Next, the labeled antibody or antibody fragment preferably accumulates at the cell site expressing the cell surface vimentin protein. Reagents and methods for tumor imaging in vivo (ie, in situ) are known in the prior art, eg, SW Burchiel et al. (1982) “Immunopharmacokinetics of radiolabeled antibodies and fragments thereof ( “Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments”) ”(“ Tumor Imaging. The Radiochemical Detection of Cancer ”), Chapter 13, SW Burchiel and BA Rhodes, Masson Publishing Inc.). For example, antibody labels or markers for in vivo imaging of endokine alpha protein include those detectable by X-ray radiography, NMR, or ESR. For X-ray radiography, suitable labels include radioisotopes such as barium or cesium that emit detectable radiation but are not clearly harmful to the subject. Suitable markers for NMR and ESR include those that can be incorporated into antibodies by labeling the nutrients of the relevant hybridoma and have a detectable characteristic spin, such as deuterium.

  Depending on a number of variables that can be optimized using practices, including the type of label used and the mode of administration, post-dose labeling molecules are sufficient to favor concentration at the site of the subject, And the time interval sufficient for the unbound labeled molecules to be eliminated and fall to the background level is 6 to 48 hours, alternatively 6 to 24 hours, alternatively 6 to 12 hours. In another embodiment, the time interval after administration is 5 to 20 days, alternatively 5 to 10 days.

  In certain embodiments, disease or disorder monitoring is performed by repeating the diagnosis of the disease or disorder, for example, 1 month after the initial diagnosis, 6 months after the initial diagnosis, 1 year after the initial diagnosis, etc.

  Importantly, labeled anti-vimentin antibodies, or other vimentin binding agents, are detected in the patient using known methods for in vivo scanning. These methods depend on the label used. A skilled technician can determine the appropriate method for detecting a particular label. Methods and apparatus that may be used in the diagnostic methods of the present invention include computed tomography (CT), whole body scans such as positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound imaging Law.

For example, in certain embodiments, anti-vimentin antibodies are labeled with a radioisotope and detected in a patient by a radiation-responsive surgical device (Thurston et al., US Pat. No. 5,441,050). In another embodiment, the anti-vimentin antibody is labeled with a fluorescent compound and detected in the patient by a fluorescence reactive scanning device. In another embodiment, the anti-vimentin antibody is labeled with a positron emitting metal and detected in the patient by positron emission tomography. In yet another embodiment, the anti-vimentin antibody is labeled with a paramagnetic label and detected in the patient by magnetic resonance imaging (MRI).

4.5 Vimentin Targeted Therapy The present invention shows that vimentin protein surface markers are present only at negligible levels on the surface of normal cells in the body, but appear on the surface of neoplastic cells, especially in multidrug resistant neoplastic cells. Use the fact that. In contrast, other markers, in particular MDR markers such as P-glycoprotein and multidrug resistance protein (MRP), are present on the surface of a wide variety of normal cells and tissues, liver, kidney, stem cells, and It is present at various levels, including high levels on blood brain barrier epithelial cells. Accordingly, the present invention provides a highly specific method of targeting targeted therapeutic agents against neoplastic cells, particularly multi-drug resistant neoplastic cells, with a binding agent that binds to cell surface vimentin.

Therapeutic agents targeted towards vimentin by the methods of the present invention include antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (eg, Chloretamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin ), Anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin AMC)), and the antibody cell division agents (e.g., vincristine and vinblastine), but can be mentioned, but not limited to.
Anti-vimentin antibodies In one method, an anti-vimentin antibody specific for cell surface-expressed vimentin expressed on injured (eg, pathogen infection), neoplastic, and MDR neoplastic cells is administered systemically to cancer patients. The The adhesion of the antibody to tumor cells results in tumor cell death by activation of the complement system (complement-mediated cytotoxicity) or by activation of T cells (antibody-dependent cellular cytotoxicity). Other antibody-induced anti-tumor effects include inducing apoptosis, enhancing the cytotoxic effect of a second agent (eg, an anti-cancer chemotherapeutic agent), and inducing an anti-idiotype network response. In certain embodiments, humanized anti-vimentin antibodies are utilized. Humanized antibodies avoid the anticipated problem that human patients develop anti-animal (eg, anti-mouse or anti-rat) antibodies. Humanized antibodies consist of human antibodies that contain complementarity determining regions from non-human sources.

  Antibody therapy has been used successfully in some cases. For example, rituximab is a genetically engineered monoclonal that is used to treat non-Hodgkin lymphoma (NHL)-a relatively common malignancy that affects young and older populations. Anti-CD20 antibody. The CD20 antigen normally present on the surface of B cell lymphomas is an ideal target antigen. This is because it is not present on plasma cells, B cell progenitor cells, stem cells, or dendritic (antigen presenting) cells. Rituximab antibody (or Rituxan) is neither released nor taken up by NHL cells nor undergoes post-antigen binding modification. Rituximab was approved by the FDA in 1997 for the treatment of relapsed or refractory CD20 positive B-cell NHL and for low- or follicular lymphoma (Abou-Jawde et al. (2003) Clin. Therap. 25: 2121-37; and Kim (2003) Am. J. Surg. 186: 264-68). This drug functions by mediating antibody-dependent cytotoxicity, inhibits cell growth, sensitizes chemically resistant cells to toxins and chemotherapy, and induces apoptosis in a dose-dependent manner (White et al. al. (2001) Annu. Rev. Med. 52: 125-45; Press (1999) Semin. Oncol. 26 (Suppl. 14): 58-65).

  Another example of an anti-tumor antigen antibody therapy is alemtuzumab. Alemtuzumab is a humanized anti-CD52 monoclonal antibody approved by the FDA in 2001 for the treatment of patients with B-cell chronic lymphocytic leukemia (CLL), the most common form of adult malignancy (Abou- Jawde et al. (2003) Clin. Therap. 25: 2121-37). Although the function of CD52 is not fully specified, alemtuzumab has been shown to cause tumor responses in the presence of a wide range of diseases (Ferrajoli et al. (2001) Expert Opin. Biol. Ther. 1: 1059-1065).

  Yet another example of an anti-tumor antigen antibody therapy is trastuzumab (also called herceptin). This is a humanized, anti-HER2 recombinant monoclonal antibody approved by the FDA in 1998 for the treatment of metastatic breast cancer (Abou-Jawde et al. (2003) Clin. Therap. 25: 2121-37 And Kim (2003) Am. J. Surg. 186: 264-68). HER2 is a member of the epidermal growth factor receptor (EGFR) family that is expressed by many breast cancer tumors, and thus this antibody therapy is effective against solid tumors. Several randomized, controlled-setting experiments have been performed showing improved efficacy and quality of life when treated with trastuzumab in HER2-positive breast cancer patients (Vogel et al. (2002) J Clin. Oncol. 20: 719-26).

Finally, cetuximab is effective in treating several HER1 / erb-B1-expressing solid tumors, including colorectal, rectal, pancreas, non-small cell lung cancer (NSCLC), and head and neck cancer An anti-HER1 chimeric monoclonal antibody (see, for example, O'dwyer et al. (2002) Semin. Oncol. 29 (Suppl. 14): 10-17). Cetuximab competes for EGFR binding and removes receptors from the cell membrane by stimulating intracellular transduction, thereby disrupting cellular processes that lead to proliferation and metastasis (Abou-Jawde et al. (2003) Clin Therap. 25: 2121-37).
Vimentin Targeted Antibody and Ligand Conjugate Complexes In addition to the “naked” antibody method described above, antibodies are conjugated to biological or chemical toxins, or radioisotopes, or otherwise “operable” Can be linked to. The anti-vimentin antibody, or antibody fragment thereof, may be conjugated to a therapeutic functional group such as a cytotoxin, such as a cytostatic or cytocidal agent, therapeutic agent or radioactive metal ion, or otherwise May be operatively coupled in the manner described above. “Operatively linked” means that the therapeutic functional group is attached to the binding agent by covalent or non-covalent (eg, hydrophobic, or ionic) bonds. Methods for creating covalent bonds are known (eg, Wong, SS, “Chemistry of Protein Conjugation and Cross-Linking”), CRC Press 1991; Burkhart et al., “Amino-crosslinking. Agents, Aminoplast Chemistry and Applications (see “General Chemistry and Application of Amino Crosslinking Agents or Aminoplasts”), John Wiley & Sons Inc., New York, NY, 1999). After systemic administration, treatment is directed to cancer cells (or MDR cancer cells or damaged (eg, pathogen-infected) cells) by antibodies.

  The invention further includes a vimentin targeting agent composed of a vimentin targeting element and a toxic agent, such as a biological toxin, a chemical toxin, or a radioisotope. A cytotoxin, or cytotoxic agent, includes all agents that are toxic to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etopside, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mitromycin, actinomycin D 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranol, and puromycin and analogs and homologues thereof. Biological toxins are conjugated to antibodies and to other tumor marker agents or fused genetically in frame. Such biological toxins include ricin, diphtheria toxin, and Pseudomonas exotoxin. After binding to cell surface vimentin, the toxin generally crosses the cell membrane and is then processed before killing the target cell. Toxic effects are typically due to inhibition of protein synthesis by active biological toxins.

  For example, in one embodiment, the anti-vimentin antibody is conjugated to a cobra venom factor. According to the present invention, vimentin-specific antibodies conjugated to cobra venom factor are used to treat cancers, particularly cancers including human multidrug resistant cancers. Methods for conjugate conjugation of antibodies to cobra venom factor are taught in US Pat. No. 5,773,243. In certain embodiments, the binding agent is an immunotoxin (eg, antibody toxin conjugate or antibody drug conjugate).

  Non-limiting examples of immunotoxins include antibody-anthracycline conjugates (Braslawsky GR et al., European Patent EP0398305), antibody cytokine conjugates (Gilles SD, PCT publication WO9953958), and monoclonal antibody-PE conjugates. Body (Roffler SR et al., Cancer Res. 51: 4001-4007, 1991).

Techniques for conjugating other therapeutic functional groups to antibodies are well known. For example, Arnon et al. In “Monoclonal Antibodies For Cancer Targeting In Cancer Therapy”, “Monoclonal Antibodies And Cancer Therapy”. , Reisfeld et al. (Ed.), Pp. 243-56, (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, “Regulatory Drug Delivery” Controlled Drug Delivery ”), (2nd edition), Robinson et al. (Eds.), Pages 623-53 (Marcel Dekker, Inc., 1987); Thorpe,“ Antibody Carriers of Cytotoxic Agents in Cancer Treatment— ”Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review ”),“ Monoclonal Antibodies '84: Biological And Clinical Applications ”), Pincera et al. (Eds.), 475 -506 (1985); "In cancer treatment “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, “Monoclonal antibodies for cancer detection and therapy (” Monoclonal Antibodies For Cancer Detection And Therapy ”), Baldwin et al. (Eds.), Pp. 303-16 (Academic Press 1985), and Thorpe et al.“ Preparation and Cytotoxic Properties of Antibody-Toxin Complexes ” Of Antibody-Toxin Conjugates ")", Immunol. Rev., 62: 119-58 (1982). Each of which is incorporated herein by reference. Alternatively, the antibody can be conjugated to a second antibody and Segal can form an antibody heterozygote as described in US Pat. No. 4,676,980. This patent is incorporated herein by reference.
Drug Adhesion Although several methods have been described in the literature for performing drug and therapeutic agent attachment / release, strategies used for soluble polymer-drug conjugates have recently been reviewed (Soyez, at al. (1966) Adv. Drug Del. Rev. 21: 81-106). Many chemistry of these conjugate conjugation methods are described in textbooks (eg, Ref. Wong (1991) CRC Press, Boca Raton, Florida), attachment sites for antibodies.

  The most used site is the ε-amino group site of the lysine residue. This is because N-succinimidyl-3- (2-pyridylthio) propionate (SPDP) (Carlson, et al., ( 1978) even with heterobifunctional drugs such as Biochem J. 173: 723-737) because it is chemically convenient for use. Since antibodies have varying numbers of lysine residues, and these residues are dispersed throughout the antibody, there is no evidence that there is a relatively reactive subset. Therefore, the probability that the active site lysi residue is modified is the same as that of other residues, and if modified, the antibody activity is lost. The more lysine residues that are modified, the greater the probability of loss of binding. Binding to the lysine ε-amino group also has other effects. First, neutralizing the positive charge of a protein can lead to structural effects or can also affect antibody solubility (Hudecz, et al., (1990) Bioconjug. Chem. 1: 197- 204). However, this disadvantage can be overcome by using a reagent such as 2-iminothiolane that provides thiol functionality but does not change charge (Jue, et al., (1978) Biochemistry 17: 5399-5406 ). Finally, the number of lysine residues that are modified is statistical, so a range of numbers of modified residues will be provided within a population of antibody molecules, and within a single sample A large amount of drug is deposited, resulting in loss of binding activity (Firestone, et al., (1996) BR96-Dox. J. Control. 39: 251-259).

  The second site for modification is a sugar residue attached to the hinge region of the antibody. This is a useful region for attachment because it has a unique chemical reactivity away from the antibody binding site. This site has been used by several research groups to oxidize sugars to provide aldehyde groups (O'Shanessy, et al., (1984) Immunol. Lett. 8: 273- 277; O'Shanessy, et al., (1987) J. Immunol. Methods 99: 153-161; and Rodwell, et al., (1986) Proc. Natl. Acad. Sci. USA 83: 2632-2636). This aldehyde group can be used in several conjugation processes. Aldehyde groups can also be generated on immunoglobulins by enzymatic reactions involving neuraminidase and glucose oxidase (Rodwell, et al., (1986) Proc. Natl. Acad. Sci. USA 83: 2632 -2636 and Stan, et al., (1999) Cancer Res. 59: 115-121). Antibodies can also be modified by genetic engineering to generate new oligosaccharide sites. The oligosaccharide moiety is reported to occupy a more favorable position for carrier-drug molecule attachment (Qu, et al., (1998) J. Immunol. Methods 213: 131-144).

  A third major possibility for attachment is through disulfide bonds inside the antibody. Disulfide links play a major role in antibody structure by providing intrachain and interchain links. Intrachain links that stabilize the domain structure of the antibody are selectively disrupted by dithiothreitol without affecting interchain disulfide bonds that group the antibody chains. This process has a higher binding activity and produces a more soluble complex with a defined number of drug molecules per antibody molecule (Willner et al., (1993) Bioconjug. Chem. 4: 521-527). Surprisingly, this process had no detectable effect on antibody stability and immunoreactivity. Depending on the antibody subclass, the number of available intrachain disulfide sites is limited. The introduction of branched hydrazone linkers to further exploit this binding site by doubling the number of chemically attached drug molecules has not been reported (King, et al., (1999) Bioconjug. Chem. 10: 279-288).

  Antibody fragments have also been used in some drug target experiments. From the perspective of conjugate conjugation, Fab ′ fragments are particularly advantageous in that they possess a single free sulfhydryl group that can be readily used to attach drugs or other macromolecules (Hashida , et al., (1984) J. Appl. Biochem. 6: 56-63).

  One of the simplest attachment methods is the use of peptide bond forming reagents such as carbodiimides or active esters. These are conventionally used to deposit carboxy-containing drugs such as methotrexate (MTX). Early studies on polylysine conjugates showed that biodegradable MTX-poly (-L-lysine) was somewhat cytotoxic, but MTX-poly (-D-lysine) was non-toxic. This suggests that free drug is released by disrupting the polymer (Ryser, et al., (1980) Cancer 45: 1207-1211). In this case, we would like to expect biomolecules like albumin and immunoglobulins to break up and release the free drug. Using this principle, MTX-immunoglobulin (Kanellos, et al., (1985) J. Natl. Cancer Inst. 75: 319-332) and MTX-HSA-immunoglobulin (Garnett, et al., (1983) Int. J. Cancer 31: 661-670) both complexes have been reported. These reactions did not produce a distinct single product, and a mixture of unstable esters and stable amide bonds was formed (Endo, et al., (1988) Cancer Res. 48: 3330-3335 and Hudecz, et al., (1992) Biomed. Chromatogr. 6: 128-132). This ester-binding substance can form up to 24% of the conjugate conjugate drug and is gradually released from the complex by hydrolysis during storage at 4 ° C. for 20 days (Hudecz, et al., (1992 ) Biomed. Chromatogr. 6: 128-132). The complex cytotoxicity is suppressed by ammonium chloride and the lysosomal protease inhibitors leupeptin and E64 (Garnett, et al., (1985) Anti-Cancer Drug Design 1: 3-12). It was suggested to be released by denaturation in the lysosomal compartment. However, a close examination of the release of MTX from the HSA-MTX complex (Fitzpatrik et al., (1995) Anti-Cancer Drug Design 10: 11-24) revealed that low molecular weight drugs released from rat liver tritosomes The amount was found to be very low (5.6% at 55 hours). In addition, only about 10% of the released material was free MTX and the rest were amino acids that were not easily enzymatically cleaved from the drug and were significantly less cytotoxic than unmodified methotrexate (Rosowsky , et al., (1984) J. Med. Chem. 27: 888-893). Complexes designed to release these amino acid derivatives of MTX were also less cytotoxic than complexes that released free MTX. The release of MTX and MTX derivatives from antibody-MTX conjugates is much lower, estimated to be <0.05% over the same period, due to the low substitution ratio to methotrexate (Rokowsky, et al., (1984 ) J. Med. Chem. 27: 888-893) and weak denaturation of the antibody Fab region by tritosomes (Schneider, et al., (1981) J. Cell Biol., 88: 380-387). Has been. Thus, the linker that specifically releases the drug from the complex is the most important component of the target drug complex.

  Aldehyde / Schiff base binding can also be used to attach therapeutic agents to antibodies, or other localization factors. A sugar residue having a vicinal hydroxyl group can be converted to an aldehyde group by oxidation with periodic acid (O'Shanessy et al., (1984) Immunol. Lett. 8: 273-277). This inserts a more useful aldehyde group into a sugar residue in a polysaccharide, such as dextran, or a sugar functional group in a drug, such as a nucleoside sugar group in fluorouridine, or an amino sugar in daunomycin. Is possible. Aldehydes react easily with hydrazides to form hydrazones or react with amines to form Schiff bases. Although this Schiff base itself is relatively unstable (Cordes, et al., (1963) J. Am. Chem. Soc. 85: 2843-2848), it is possible to reform the starting material, eg Usually, it is reduced by a reagent such as sodium borohydride or sodium cyanoborohydride to stabilize the bond. Dialdehydes such as glutaraldehyde are also used to crosslink between drugs and macromolecules, or between macromolecules, where the bond between products is usually irreversible (Wong, ( 1991) CRC Press Boca Raton, FL 101-102). The use of glutaraldehyde as a cross-linking agent has the disadvantage that undesirable cross-linking products and aggregates are readily formed because it is a homo-bifunctional reagent.

  Sulfhydryl binding can also be used to bind therapeutic agents to antibodies or other localization factors. Disulfide bonds are found to bind the chain of plant toxins and are essential for the activity of the toxin (Masuho, et al., (1982) J. Biochem. 91: 1583-1591). That is, it allows the enzyme A chain to dissociate from the binding chain and enter the protoplasm. This need for binding suggested that this would be a possible way to release drugs from antibodies or polymer molecules in the intracellular compartment. Conjugate conjugation of methotrexate to poly (-D-lysine) via disulfide bonds has been shown to be cytotoxic (Shen, et al., (1985) J. Biol. Chem. 260: 10905-10908) . Fragmentation of these complexes was first shown to occur at the cell surface, but not in the endosomal or lysosomal compartments (Freener, et al., (1990) J. Biol. Chem. 265: 18780-18785). It was suggested that the Golgi apparatus is the most likely site where this fragmentation occurs. Although severable intracellularly, this binding has been shown to be unstable in the circulation, thus creating an inhibited disulfide bond that alleviated this problem (Thorpe, et al., (1987) Cancer Res. 47: 5924-5931; and Worrell, et al., (1986) Anti-Cancer Drug Design 1: 179-188). For conjugates that require stable, chemically convenient linkages due to sulfhydryl groups, thioether linkages are more stable and maleimide coupling agents can be added (Hashida, et al., (1984) J. Appl. Biochem. 6: 56-63 and Lau et al., (1995) Bioorg. Med. Chem. 3: 1299-1304) or by using an iodoacetate coupling agent (Rector, et al., (1978 ) J. Immunol. Methods 24: 321-336).

  Acid fragile bonds can also be used to bind therapeutic agents to antibodies or other localization factors. Chemically labile bonds can be used to release the drug in the presence of more acidic conditions. These conditions are reported in tumor environments that are reported to be 0.5-1 pH units more acidic than healthy tissue and blood (Lavie, et al., (1991) Cancer Immunol. Immunther. 33: 223-230 and Ashby ( 1966) Lancet ii: 312-315), or may occur when passing through endosomal / lysosomal compartments where pH of 6-6.8 and 4.5-5.5 are observed, respectively. A major drawback of using acid brittle bonds is that this is a rate-dependent phenomenon, but the breaking rate is proportional to pH, so a 10-fold difference in rate is expected for each decrease in pH unit. This means that hydrolysis is always a compromise between a high speed at low pH of the intracellular compartment and a low speed for serum stability.

  The first acid-sensitive linkage described was the cis-aconityl linkage described by Shen and Ryser (Shen, et al., (1981) Biochem. Biophys. Res. Commun. 102: 1048-1054). First, daunomycin was reacted with anhydrous cis-aconitine, which was then coupled to polylysine using water soluble carbodiimide. This binding was reported to have a half-life of less than 3 hours at pH 4 and greater than 96 hours at pH 6. However, only about 50% of the drug was released from the Affigel matrix. This is thought to be due to improper binding of the gel substrate to the remaining cis-carboxyl group, which causes acid-sensitive release from this linker. Optimal conditions for the use of this reagent are described in detail by (Hudecz, et al., (1990) Bioconjug. Chem. 1: 197-204). An improved version of this release mechanism is also described by (Blaettler et al., (1985) Biochemistry 24: 1517-1524). This is a heterobifunctional factor for binding toxin molecules. In this method, the conjugated junction by the third carboxyl of cis-aconitate is replaced by a specific maleimide group. This eliminates the possibility of acid release inactivation of the cis-carbonyl group.

  A range of acid-sensitive homo- and hetero-dual functional factors originally prepared to perform acid-sensitive release of toxin immune complexes have been described by Srinivasachar as orthoesters, acetals and ketals (Srinivasachur , et al., (1989) Biochemistry 28: 2501-2509). These have the potential to be useful for building chemoimmunoconjugates. These reagents vary in their rate of hydrolysis at the pH found in the intracellular compartment. Hydrazone binding can also be used to bind therapeutic agents to antibodies or other localization factors.

  Hydrazide derivatives are also acid-fragile and are conventionally used to generate vindesine and adriamycin complexes (Laguzza, et al., (1989) J. Med. Chem. 32: 548-555 and Greenfield, et al , (1990) Cancer Res. 50: 6600-6607). In the former (Laguzza, et al., (1989) J. Med. Chem. 32: 548-555), vindesine is first reacted with hydrazine, and then this hydrazine derivative is reacted with the oxidized sugar residue of the antibody. It was. For the adriamycin conjugate (Greenfield, et al., (1990) Cancer Res. 50: 6600-6607), SPDP hydrazine derivatives were prepared and reacted with thiolated antibodies. In any of these complexes, the low molecular weight drug was released under acidic conditions. In the former case, up to about 30% vinca hydrazide was released for 7 days at 37 ° C pH 5.3, and in the latter case, unmodified adriamycin was rapidly released from the complex at pH 4.0-5.5. . Studies on various hydrazone derivatives of adriamycin have been reported (Kaneko, et al., (1991) Bioconjug. Chem. 2: 133-141). Accordingly, it was shown that the acid instability of various linkers was acyl hydrazide> semicarbazide> carboxylic acid dihydrazide> thiosemicarbazide> hydrazine carboxylate = allyl hydrazide. All of these compounds released adriamycin as the only product. With the exception of allyl hydrazide, all of these compounds were stable at pH 7.4. Acylhydrazine releases 85% of the theoretical amount of drug in 3 hours at pH 5.0 and 37 ° C, and its cytotoxicity is similar to free adriamycin when conjugated to anti-transferrin receptor antibody. It was. Maleimidocaproyl hydrazone derivatives are also synthesized to produce thioether-linked complexes, which are more stable in serum and can be easily coupled to the intrachain reduced disulfide groups of antibodies (Firestone, et al. al., (1996) BR96-Dox, J. Control. 39: 251-259). Longer chain alihydrazide linkers for anthracycline-conjugated junctions have been described by (Lau, et al., (1995) Bioorg. Med. Chem. 3: 1299-1304).

  Enzymatically denatureable linkages can also be used to attach therapeutic agents to antibodies or other localization factors. The key criteria for attaching and releasing drugs to macromolecules are linkers that are stable in serum and can be cleaved by specific enzymes in cells. This type of linker containing various amino acids has been described. Some of these are used in drug conjugates conjugated to antibodies, while others are used only in polymer drug conjugates. Daunomycin (Dau) splittable amino acid drug precursor was first manufactured by Levin and Stella (Levin, et al., (1979) FEBS Lett. 98: 119-122). However, these were labeled as low molecular weight drug precursors. The first systematic study on amino acid sequence and length in lysosomal digestion was reported by (Masquelier, et al., (1980) J. Med. Chem. 23: 1166-1170). These studies revealed that Ala-Leu-Dau derivatives can be reverted to free drugs in 2 hours by lysosomal hydrolases. This activity was attributed to a lysosomal dipeptidyl aminopeptidase. This dipeptide derivative was much less active than Dau in vitro but showed greater activity in vivo (Baurain, et al., (1980) J. Med. Chem. 23: 1171-1174). Subsequent work reported by this group (Trouet, et al., (1982) Proc. Natl. Acad. Sci. USA 79: 626-629) showed that daunorubicin was separated by a spacer arm consisting of 1 to 4 amino acids. Resulting in a conjugate linked to succinylated serum albumin. It has been found that a minimum of three or peptide spacers are essential for the successful release of the drug. In the case of an albumin complex containing an Ala-Leu-Ala-Leu-Dau bond, release of 75% of the free drug was achieved in 8 hours, and this complex was stable in the presence of serum (at 24 hours Only 2.5% drug release). In the absence of a peptide spacer, no drug was released from Dau conjugated to succinylated serum albumin by lysosomal enzymes.

  Another tetrapeptide spacer was obtained from a long collaboration between Duncan and Kopecek. In this work, the release of p-nitroaniline as a model drug from poly [N- (2-hydroxypropyl) methacrylamide] copolymers was studied (described in Duncan (Duncan (1986) CRC Crit. Rev. Bicompat. 2: 127-145)). These studies provided a great understanding of the specificity of lysosomal enzymes and the development of Gly-Phe-Leu-Gly-Dau linkers. In the latter case, 80% of the bound p-nitroaniline was released during the 50 hour incubation period. Next, daunomycin was coupled to the polymer delivery system as an antibody-carrier-drug complex (Duncan, et al., (1987) Br. J. Cancer 55: 165-174).

  A tetrapeptide spacer was incorporated into the monoclonal antibody-methotrexate complex by (Umemoto, et al., (1989) Int. J. Cancer 43: 677-684). This is an MTX-Leu-Ala-Leu-Ala-hydrazide linker based on the tetrapeptide described by Trouet. However, in Trouet's study, Dau was attached to the C-terminus of the peptide, whereas in this complex, MTX was attached to the N-terminus of the peptide. Furthermore, since hydrazide is also incorporated into the binding band, there is a possibility that some acid-sensitive release property is imparted to the drug linker portion of the complex. No studies have been reported on the action of lysosomal enzymes on this linker, what products are released, and the rate of product release. However, these linkers resulted in a substantial increase in the efficiency of the complex compared to direct-bound MTX, and the release was mediated by lysosomes by inhibitors such as leupeptin.

  The development of further tetrapeptide spacers as HAS carrier molecules has been described by (Fitzpatrick, et al., (1995) Anti-Cancer Drug Design 10: 1-9). Appropriate spacers have been developed using a lysosomal enzyme denaturation system. In this system, the attachment of the terminal residue of the peptide chain to the ε-aminolysine residue was used as a conjugation model for the protein. Using this system, various amino acids attached to the carboxyl group of MTX can release the free drug, the release rate of the free drug depends on the length of the spacer, and the tetrapeptide spacer is about It was shown to release 90% free drug. By conjugating MTX tetrapeptide to HAS, the release rate of free drug was further reduced to about 30% in 48 hours. These experiments have shown that tetrapeptide spacers are not only overcoming the limitations of the polymer molecule's configuration, but are also related to the efficiency of binding the split to the active part of the enzyme.

  An efficient and general method for linking anthracyclines to peptides by oxime linkages is described by (Ingallinella, et al., (2001) Bioorg. Med. Chem. Lett. 11: 1343-1346). No immune complex using this binding has been reported.

  In general, the simplest way to generate immune complexes is to couple the drug directly to the antibody. This may involve forming a direct bond between the functional group of the drug and one of the functional groups on the antibody, or alternatively inserting a linker or spacer group between the two parts of the complex. Including. The linker group may simply be used to allow conjugation chemistry, but may have a second function of allowing certain types of drug release. If the release is mediated by enzymes that are localized intracellularly or extracellularly, this group is called a spacer group, the purpose of which is to give enough space or to limit the configuration. Relaxing will allow the enzyme to get close enough to the relevant bond.

  Methotrexate is one of the first cytotoxic agents linked to antibodies. These early stages using immune sera (Marthe, et al., (1958) CR Acad. Sci. 246: 1626-1628 and Burstein, et al., (1977) J. Med. Chem. 20: 950-952) In this study, the binding was either by diazotization or by a mixed anhydride process. Both of these processes resulted in a therapeutically effective complex in the mouse model.

  Complexes produced thereafter have been documented in many reviews (eg, Magerstaedt (1991) CRC Press BoCa Raton FL 77-215; Dubowchik, et al., (1999) Pharmacol. Ther. 83: 67- 123; and Pietersz, et al., (1994) Adv. Immunol. 56: 301-387). Early studies on vinblastine-antibody conjugates used various methods to conjugate the drug to the antibody, and one report showed that the conjugate was more cytotoxic than the free drug. (Johnson, et al., (1981) Br. J. Cancer 44: 372). Clinical studies on vinblastine complexes have been reported. The first of these studies involved a complex with rodent monoclonal antibody KS1 / 4 by a half-succinate derivative of DAVLB rather than optimized DAVLBHY (Schneck, et al., (1990) Clin. Pharamacol. Exp. 47: 36-41).

  The two main anthracyclines used in antibody conjugates are daunomycin (agrees with daunorubicin) and adriamycin (agrees with doxorubicin). This differs only at the end C14 of the side chain, which is a methyl group in the former and a less hydrophobic methoxy group in the latter. Daunomycin (Dau) has been reported to be more cytotoxic than daunorubicin (Dox). Idarubicin and epirubicin are slightly more cytotoxic derivatives with improved toxicity profiles compared to Dau or Dox (Arcamone, (1985) Cain Memorial Aware Lecture, Cancer Res. 45: 5995- 5999). Morpholinodoxorubicin and cyanomorpholinodoxorubicin have been reported to be very highly cytotoxic derivatives (Newman, et al., (1985) Science 228: 1544-1546).

  An immunocomplex formulation of anthracyclines (Dau and Dox) against immunoglobulins was evaluated (Hurwitz, et al., (1975) Cancer Res. 35: 1175-1181). (1) Periodic acid oxidation of the sugar function group of the drug, conjugation to the lysyl group of the antibody, subsequent reduction by Schiff base reduction, and (2) glutaraldehyde bond between the sugar amino group and lysyl group of the antibody is there. The drug activity was best preserved with glutaraldehyde activity, but both periodate oxidation complex and glutaraldehyde-linked complex showed good activity against target cells. Periodate oxidation complexes have been evaluated more closely (Levy, et al., (1975) Cancer Res. 35: 1182-1186), retaining approximately 50% of the activity of the free drug and a short time for immune complexes. Specificity was shown in cytotoxicity assays including exposure. The antitumor effect of this complex against PC5 B cell leukemia was superior to the free drug.

  Idarubicin (Ida) immune complex was prepared by 14-bromo-idarubicin, anti-ly2.1 antibody and 1-5 MR (Pietersz, et al., (1988) Cancer Res. 48: 926-931 ).

The conjugate showed selective cytotoxicity but was less active than the free drug against E3 target cells (IC ₅₀ = 430 and 120 nM, respectively). Antitumor activity against E3 thymic tumor xenografts was assessed by decreasing tumor growth rate and was greater than that obtained with Ida alone. Subsequent studies with IDA immunoconjugates prepared from anti-CD19 antibodies resulted in 240 nM activity in the immunocomplex compared to 12 nM free Ida (Rowland, et al., (1993) Cancer Immunol. Immunother. 37: 195-202).

  Anthracyclines are a preferred choice as a targeted transport agent. Since the morpholino group makes it impossible to use the sugar amino group normally used for conjugate conjugation, a side chain C13 linker was used.

An immune complex with 5-fluoro-2'-deoxyuridine (FUDR) reacts an active ester derivative of succinylated FUDR with an anti-ly1.2 monoclonal antibody to conjugate the drug to antibody 7-9MR It was constructed by forming a body (Krauer, et al., (1992) Cancer Res. 52: 132-137). Both antigen-positive E3 cell lines showed similar cytotoxicity for both succinylated FUDR and immune complex (IC ₅₀ = 5 and 3 nM, respectively), but about 10 times more than free drug (0.4 nM) It was low. In vivo, immune complexes showed greater suppression of E3 thymic tumor growth than equivalent amounts of free drug.

  Taxol immune complexes have been reported by Guillemard and Saragovi (Guillemard, et al., (2001) Cancer Res. 61: 694-699). Taxol was first modified with anhydrous glutar to give the drug a cleavable ester bond and then conjugated directly to the antibody by carbodiimide. An immune complex against anti-mouse and anti-rat IgG was produced as antibody 1 against the drug MR. Cytotoxicity tests appeared to indicate that the conjugate has higher cytotoxicity than the free drug. In vivo, immune complexes significantly reduced tumor growth, albeit slightly, against neuroblastoma xenografts.

  Antibody concentration is a critical factor in determining the rate of drug intake. Thus, if more drug molecules can be conjugated per antibody molecule, cytotoxicity should increase. On the other hand, loss of antibody binding activity is a limiting factor on the number of drug molecules, but the use of carrier molecules for targeted drug conjugates provides a solution to this problem. Several types of complexes have been investigated, many using dextran, human serum albumin, or hydroxypropyl methacrylamide (HPMA) as the carrier molecule and using the drugs doxorubicin or methotrexate. The earliest delivery complex is conjugated phenylenediamine mustard to the antibody via a polyglutamic acid delivery agent and exhibits a 45: 1 molar substitution ratio (Rowland, et al., (1975) Nature 255: 487- 488).

  Another solution to the difficulty of delivering enough drug molecules to kill cancer cells is to use more powerful drugs. This is because such drugs require fewer drug molecules to kill the cells. Several such molecules have been discovered and investigated as anti-tumor drug candidates. As such, CC-1065-like alkylating agents such as duocarmycin; enynein including dynemicin; calicheamicin / esperamicin, chromoprotein (Borders, et al., (1994) Marcel Dekker New York) (eg neoplastic Cardinostatin, and calicheamicin); and macrolide antibiotics such as geldanamycin and maytansine.

  Application of tumor targeted cytotoxicity therapy has been successful for many tumor antigens. For example, gemtuzumab ozogamicin, used to treat acute, myeloid tumor-derived leukemia (AML), is composed of an anti-CD-33 antibody attached to calicheamicin, an antitumor chemotherapeutic agent . Since the CD33 antigen is present in bone marrow progenitor cells but not in hematopoietic stem cells, CD33 targeted therapeutics are selective for AML cells but leave important normal cell types. Binding this drug to the CD33 antigen present in AML cells results in the formation of a complex that is taken up into the cell. After intracellular uptake, the antitumor functional group of this drug is released into bone marrow cells, leading to cell death (Naito, et al., (2000) Leukemia 14: 14636-43). This drug has been approved by the FDA for the treatment of relapsed or refractory AML in patients over 60 years old (Abou-Jawde et al., (2003) Clin. Therap. 25: 2121-37). In addition, gemtuzumab ozogamicin has been found to be effective in treating recurrent AMS and may therefore rescue many patients.

Immunotoxins The therapeutic agent, or drug component, should not be considered limited to classical chemical or radiotherapeutic agents. For example, the drug component may be a protein or polypeptide having the desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin, as well as other biologically active proteins such as tumor necrosis factor, alpha-interferon, beta-interferon, Nerve growth factor, platelet growth factor, tissue plasmogen activator, thrombotic factor or anti-angiogenic factor such as angiostatin or endostatin, or biological response modifier such as lymphokine, IL-1, IL-2, IL -4, Il-5, Il-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-15, granulocyte giant phase colony stimulating factor ("GM-CSF"), Granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

  Immunotoxins include ligands such as growth factors, monoclonal antibodies or antibody fragments that are connected to protein toxins. After the ligand subunit binds to the surface of the target cell, the molecule enters and the toxin kills the cell. Bacterial toxins directed against cancer cells include Pseudomonas exotoxin and diphtheria toxin, which are suitable for forming single or double stranded recombinant fusion toxins. Plant toxins include ricin, abrin, pokeweed antiviral protein, saponin, and gelonin, which are generally connected to the ligand by disulfide bond chemistry. Immunotoxins are produced to target hematological malignancies and solid tumors via a variety of growth factor receptors and antigens.

  The purpose of immunotoxin therapy is to fire a cytotoxic agent toward a cell surface molecule, which takes up the cytotoxic agent and results in cell death. Because immunotoxins differ greatly from chemotherapy in mode of action and toxicity profile, immunotoxins provide improvements in systemic treatment of tumors.

  An immunotoxin is simply defined as a protein containing an antibody and a toxin. Toxins discussed herein include catalytic proteins produced by plants or bacteria that kill target cells. The term “immunotoxin” generally refers to a toxin targeted by an intact IgG, Fab fragment, or Fv fragment, although toxins targeted by growth factors or other ligands are also referred to as “chimeric toxins”. In some immunotoxins or chimeric toxins, the binding between the ligand and the toxin is made chemically, but the protein may be referred to as a “chemically coupled conjugate”. Alternatively, if the linkage is a peptide bond produced by genetic engineering, the protein is called a “recombinant toxin” or “fusion toxin”. Finally, if a selected group of immunotoxins contains an Fv sequence fused to the toxin, these proteins that are both immunotoxins and recombinant toxins are often referred to as “recombinant immunotoxins”.

  Protein toxins are preferred because they are extremely active. One or several molecules of protein toxins have been shown to be able to kill cells when injected into the protoplasm (Yamaizumi, et al., (1978) Cell 15: 245-250 and Eiklid, et al., (1980) Exp. Cell Res. 126: 321-326).

  Plant toxins exist in nature as holotoxins and hemitoxins. Holotoxins (also called class II ribosomes in activated proteins) include ricin, abrin, mistletoe lectin and modexin, which contain a binding domain disulfide bonded to the enzyme domain. Hemitoxins such as pokeweed antiviral protein (PAP), saponin, and gelonin contain an enzyme domain but no binding domain.

  To produce an immunotoxin, generally a plant toxin is chemically conjugated to a ligand (see, eg, Kreitman, et al., (1998) Adv. Drug Del. Rev., 31: 53-88).

  Two bacterial toxins commonly used to produce immunotoxins, Pseudomonas exotoxin (PE) produced by Pseudomonas aeruginosa, and Corynebacterium diphtherae The diphtheria toxin (DT) produced is mentioned. Both PE and DT catalyze ADP ribosylation of cytosolic EF-2 (Carroll, et al., (1987) J. Biol. Chem. 262: 8707-8711; Uchida, et al., (1972) Science 175: 901-903; and Uchida, et al., (1973) J. Biol. Chem. 248: 3838-3844).

Mutant and truncated forms of DT and PE can also be used (see Kreitman, et al., (1998) Adv. Drug Del. Rev., 31: 53-88). For example, mutant toxins are designed in which the binding domain of the toxin has been deleted or rendered non-functional by mutation. In the case of DT, this can be done chemically by treating the toxin with trypsin and purifying the A chain (Masuho, et al., (1979) Biochem. Biophys. Res. Commun. 90: 320. -326). Recombinant toxins with full length and mutant binding domains include PE ^4E , which is a substitution of a base residue with glutamate at positions 57, 246, 247 and 249 of PE, and CRM107, which is DT In which leucine at position 390 and serine at position 525 are substituted with phenylalanine (Greenfield, et al., (1987) Science 238: 536-539 and Chaudhary, et al., (1990) J. Biol Chem. 265: 16306-16310).

  A wide variety of trace immunotoxins and recombinant toxins have been produced and tested against malignant target cells (see Kreitman, et al., (1998) Adv. Drug Del. Rev., 31: 53-88).

  Next generation immunotoxins include recombinant toxins. Immunotoxins that contain antibodies chemically conjugated to toxins or antibody fragments such as Fab 'have several disadvantages. First, tumor penetration is reduced due to the large size (100-200 kDa). Second, for conjugation to antibodies, derivatives of toxins such as PE40 and PE38 must be formed by reagents that modify lysine residues, and much of lysine is near the carboxyl terminus. Similarly, antibodies also require derivatization of lysine residues within the antigen binding site. Thus, the resulting immunotoxin is a heterogeneous mixture not only with respect to antibody and toxin attachment sites, but also with respect to the number of toxins and antibody components per immunotoxin molecule. Finally, chemical complexes are difficult to manufacture. This is because the toxin and antibody must be purified and conjugated separately and then the product must be purified again.

  Toxins can target cells without chemically conjugating the ligand and toxin if both are connected as a single polypeptide unit.

  Bacterial toxins PE and DT are optimal for producing these fusion toxins. Because each toxin contains a proteolytic process site in the disulfide loop, which allows the catalytic domain to separate from the rest of the toxin after entry into the cell, allowing efficient transfer to the cytosol. (Chiron, et al., (1994) J. Biol. Chem. 269: 18167-18176; Frying, et al., (1992) Infect. Immun. 60: 497-502; Ogata, et al., (1992) J. Biol. Chem. 267: 25396-25401; and Williams, et al., (1990) J. Biol. Chem. 265: 20673-20677).

  In 1981, Mab B3 / 25 was reported to be conjugated to DT or RTA with rounded stumps and used to inhibit human melanoma growth in nude mice (Trowbridge, et al., (1981) Nature 294 : 171-173). The above and similar immunotoxins have shown anti-cancer activity against various solid tumors including gastrointestinal adenocarcinoma, mesothelioma, cervical cancer, and glioblastoma (Griffin, et al., (1998) J Biol. Response Mod. 7: 559-567; Griffin et al., (1987) Cancer Res. 47: 4266-4270; and Martell, et al., (1993) Cancer Res. 53: 1348-1353) . Mab HB21 was conjugated to full-length PE and delivered intraperitoneally to increase survival of mice bearing human ovarian cancer (FitzGerald, et al., (1986) Proc. Natl. Acad. Sci. USA 83: 6627-6630). HB21 and its Fab ′, (Fab ′) 2 and Fv fragments were also conjugated or fused to cleaved PE or DT, but have been shown to cause antitumor activity in various models (Batra, et al. al., (1989) Proc. Natl. Acad. Sci. USA 86: 8545-8549; Debinski, et al., (1991) Cancer Res. 52: 5379-5385; and Batra, et al., (1991) Mol. Cell Biol. 11: 2200-2205).

  Many of the cell lines targeted by immunotoxins that target various antigens are relatively resistant to chemotherapy. Immunotoxins can also specifically target cells that are resistant to a number of chemotherapeutic agents by targeting the p-glycoprotein, which is responsible for enhancing the excretion of chemotherapeutic agents from the cell. is there. Mab MRK16 conjugated to PE showed very high cytotoxicity against cell lines that were extremely resistant to chemotherapy by expression of p-glycoprotein (FitzGerald, et al., ( 1987) Proc. Natl. Acad. Sci. USA 84: 4288-4292). This immunotoxin also killed multidrug resistant epithelial cancer cells in MDR transgenic mice (Mickisch, et al., (1993) J. Urol. 149: 174-178). This antibody was also conjugated to saponin to form an immunotoxin capable of expelling multidrug resistant cells from the bone marrow (see Dinota, et al., (1990) Cancer Res. 50: 291-294. ).

Targeted radiotherapy For vimentin targeted therapy, radioisotopes may be used as cytotoxic agents. The anti-vimentin antibody of the present invention may be conjugated to one or more therapeutic agents. A radionuclide is mentioned as a suitable medicine in this respect. Suitable radionuclides include ⁹⁰ Y, ¹²³ I, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹¹ At, and ²¹² Bi. Specific carriers for radionuclide drugs to promote attachment to vimentin targeting agents include radiohalogenated small molecules and chelate compounds. For example, US Pat. No. 4,735,792 discloses representative radiohalogenated small molecules and methods for their synthesis. Radionuclide chelates can be formed from chelating compounds including metals or metal oxides and those containing nitrogen and sulfur atoms as donor atoms for binding the radionuclide. For example, US Pat. No. 4,673,562 issued to Davison et al. Discloses representative chelating compounds and methods for their synthesis.

  An ideal radioligand therapeutic agent accumulates selectively in target cells. The effects of radiation therapy are due to the destruction of dividing cells caused by radiation-induced damage to cellular DNA (eg WD Bloomer et al., (1977) “Therapeutic application of iodine-125 labeled iododeoxyuridine in an early ascites tumor model ( “Therapeutic Application of Iodine-125 Labeled Iododeoxyuridine in an Early Ascites Tumor Model”), Current Topics in Radiation Research Quarterly 12: 513-25). In both therapeutic and imaging applications, even if there are unbound circulating radioligands, they are quickly eliminated by the excretory system, thus helping to protect normal organs and tissues. Radioligands are also denatured by biological processes, which promote free radioisotope excretion (Wiseman et al., (1995) “Treatment of neuroendocrine tumors with radiolabeled MIBG and somatostatin analogs (“ Therapy of Neuroendocrine Tumors with Radiolabelled MIBG and Somatostatin Analogues ”)”, Seminars in Nuclear Medicine, Volume XXV, No. 3, pages 272-278).

Radioisotopes most suitable for therapeutic treatment include Auger electron-emitting radioisotopes such as ¹²⁵ I, ¹²³ I, ¹²⁴ I, ¹²⁹ I, ¹³¹ I, ¹¹¹ In, ⁷⁷ Br, and other radiolabeled halogens Is mentioned. The selection of a suitable radioisotope is optimized based on a variety of factors including the type of radiation emitted, the radiant energy, the distance at which the energy is stored, and the physical half-life of the radioisotope. In one example, the radioisotope used is one that has a radioactive half-life that matches or is longer than the biological half-life of the vimentin-targeted therapeutic agent. For example, in certain embodiments, the radioisotope has a half-life of about 1 hour to 60 days, preferably 5 hours to 60 days, more preferably 12 hours to 60 days. ¹²⁵ I has advantages over other emissive elements that generate high energy gamma rays that require hospitalization and isolation (ie, ¹¹¹ In and ¹³¹ I). ¹²⁵ I allows the development of outpatient treatment because of the limited amount of radiation that escapes from the body.

  Radiolabeled therapeutic agents are administered by intravenous bolus infusion (eg, HP Kalofonos, et al., (1989) “of brain gliomas with radiolabeled monoclonal antibodies directed against epidermal growth factor receptor and placental alkaline phosphatase, Antibody-Induced Diagnosis and Therapy ("Antibody Guided Diagnosis and Therapy of Brain Gliomas using Radiolabeleld Monoclonal Antibodies Against Epidermal Growth Factor Receptor and Placental Alkaline Phosphatase") ", The Journal of Nuclear Medicine, Vol. 30, pp. 163-145; L. Virgolini et al., (1994), “Vasoactive Intestinal Peptide-Receptor Imaging for the Localization of Intestinal Adenocarcinomas and Endocrine Tumors”. "The New England Journal of Medicine, 331, 1116-21; GA Wiseman et al., (1995)" Radiolabeled MIBG and Somatos. Treatment of Neuroendocrine Tumors with Tachin Analogues (“Therapy of Neuroendocrine Tumors with Radiolabelled MIBG and Somatostatin Analogues”), Semiars in Nuclear Medicine, Vol. Somatostatin-Receptor Imaging in the Localization of Endocrine Tumors, The New England Journal of Medicine, 323, 126-49; EP Krenning et al., (1992 ) "Somatostatin Receptor Scintigraphy with Indium 111-DTPA-D-Phe-1-octreotide in Humans: Metabolism, Volumetric Measurement, Comparison with Iodine-123-Tyr-3-Octreotide (" Somatostatin Receptor Scintigraphy with Indium-123- Tyr-3-Octretide in Man: Metabolism, Dosimetry, and Comparison with Iodine-123-Tyr-3-Octreotide ”)” The Journal of Nuclear Medicine, 33, 652-58; EP Krenning et al., (1989) "Somatostatin Localization of Endocrine-Related Tumors with Radioiodinated Analogue of Somatostatin (see The Lancet, Vol. 1989, No. 1, pages 242-244).

Targeted gene therapy It is also possible to use gene vectors as cytotoxic agents for vimentin targeted therapy. For example, a gene vector encoding an antibody gene (or a fragment thereof) is introduced into tumor cells. The transgene expression product binds to intracellular proteins, such as those derived from oncogenes, thereby down-regulating oncogene protein expression. Labeled gene therapy uses a dual-functional cross-linker that targets adenovirus and retroviral vectors, thereby adding a short target peptide and a larger polypeptide binding domain to the coat protein of several different viral vectors. It is facilitated by insertion and by using a replication competent vector (see Wand and Liu (2003) Acta Biochimica et Biophysica Sinica 35 (4): 311-6). In addition, non-viral therapeutic agents including DNA complexes and bacterial vehicles have also been developed. Gene therapy for vimentin targeted compositions and methods of the invention may be taken from gene therapy known in the prior art or alternatively, U.S. Patent Nos. 5,871,726, 5,885,806, 5,888,767, 5,981,274, 6,207,426, 6,210,708, 6,232,120. 6,498,033, 6,537,805, 6,555,107 and 6,569,426.

  In one method, a target replicating or non-replicating viral vector may be used to deliver the gene therapy agent. For example, adenoviral gene therapy vectors have been modified to target neoplastic cells (see Rots, et al., (2003) Journal of Controlled Release 87: 159-165). Selective targets for adenoviral vectors limit inflammation and immune responses to viral vectors and reduce therapeutic toxicity. This is because a low dose of virus can be used. Adenovirus infection is usually triggered by binding to target cells by the C-terminal part of the adenovirus fiber protein called know, and to primary cell receptors, Coxsackie B virus and adenovirus receptors (CAR). After this stage, the virus enters the cell by the interaction of the viral penton base protein RGD (arg-gly-asp) sequence with cellular integrins. Selective targeting of adenoviral vectors is feasible. Ligation (eg, conjugate conjugation) of a vimentin-specific antibody to an adenoviral vector targets the resulting construct towards vimentin-expressing neoplastic, MDR neoplastic, and damaged (eg, pathogen-infected) cells. For example, this strategy has been successfully performed by modifying adenovirus to be directed to the EGP-2 antigen present on tumor cells (Heiderman et al., (2001) Cancer Gene Ther. 8: 342-51). . The modification was achieved by conjugating a neutralized anti-fibrotic protein to an antibody against epithelial cell adhesion molecule (EGP-2). The resulting EGP-2 adenovirus was fired towards cancer cells expressing EGP-2, indicating that the infection was independent of CAR. Another strategy is to use bispecific antibodies to bridge cell surface vimentin to therapeutic gene delivery vectors (eg, adenoviral vectors) (eg, Haisma et al., (2000) Cancer Gene 7 : 901-4; Grill et al. (2001) Clin. Cancer Res. 7: 641-50; Krasnykh et al., (1998) J. Virol. 72: 1844-52; and van Beusechem et al., (2000 ) See Gene Ther. 7: 1940-46).

In general, the terms “viral vector” and “virus” are used interchangeably herein to refer to any genuine intracellular regulator that has no protein synthesis or energy production mechanisms. The viral genome is RNA or DNA encased by a covering structure composed of lipid membrane proteins. As used herein, the terms virus (s) and viral vector (s) are used interchangeably. Viruses useful for practicing the invention include recombinant modified coated or uncoated DNA and RNA viruses, preferably baculoviruses, parvoviruses, picornoviruses, herpesviruses, poxviruses, or adenoviruses It was chosen from the genus. The virus may be a naturally occurring virus, or the viral genome may be modified by recombinant DNA methods to include expression of an exogenous transgene, and may not be replicable or conditional. It may be processed so that there is replication or replication capability. Chimeric viral vectors (see, eg, Feng, et al., (1997) Nature Biotechnolog y 15: 866-870) that take advantage of the respective characteristics of the parent vector may also be useful in the practice of the present invention. . A minimal vector system in which the viral backbone contains only the sequences necessary for packaging of the viral vector and optionally a transgene expression cassette may also be produced according to the practice of the invention. In general, it is preferred to use a virus obtained from the species to be treated, but in some cases it is advantageous to use a vector obtained from another species that has the preferred pathogenicity. For example, equine herpesvirus for human gene therapy is described in WO98 / 27216 published on August 5, 1998. This vector is described as being useful for human therapy because equine viruses are not pathogenic to humans. Similarly, sheep adenovirus may be useful for human gene therapy. This is because the virus is said to circumvent antibodies against human adenovirus vectors. Such a vector is described in WO97 / 06826 published on April 10, 1997.

  The term “non-replicatable” refers to a vector that is not replicable in wild-type mammalian cells. In order to produce such vectors in large quantities, the production cell line must be co-transfected with helper virus or modified to compensate for the lost function. For example, 293 cells have been engineered to compensate for adenovirus E1 deletion, allowing passage of E1 deleted replication-incompetent adenovirus vectors in 293 cells. The term “replicating viral vector” refers to a viral vector capable of infection, DNA replication, packaging, and degradation of infected cells. The term “conditional replication competent viral vector” refers to a replication competent vector designed to achieve selective expression in a particular cell type, while avoiding an undesirably broad spectrum of infection. . Such conditional replication operably links tissue-specific, tumor-specific, or cell-type-specific or selectively induced regulatory sequences to early genes (eg, the adenovirus E1 gene). Can be realized.

  In addition to targeted snipability, cell type specificity by viral vectors is improved by using a pathway-responsive promoter that drives a repressor of viral replication. The term “pathway responsive promoter” refers to a DNA sequence that binds to a protein but causes a neighboring gene to transcriptionally react to the binding of the protein in normal cells. Such promoters are generated by incorporating reaction elements that are sequences to which transcription factors bind. Such reactions are generally inductive. However, there are some cases where increasing protein levels lowers transcription. Pathway responsive promoters may occur naturally or may be synthetic. A pathway-responsive promoter is constructed based on the pathway, ie, the functional protein being targeted. For example, naturally occurring p53 pathway responsive promoters may include transcriptional control elements that are activated by the presence of a functional p53, such as the p21 or bax promoter. Alternatively, synthetic pathway responsive promoters may be generated using synthetic promoters that contain a p53 binding site upstream of a minimal promoter (eg, SV40 TATA box region). Synthetic pathway responsive promoters are generally constructed from one or more copies of a sequence that matches a common binding motif. Such a common DNA binding motif can be easily determined. Such consensus sequences are generally arranged in series or as a head-to-tail repeat sequence separated by a few base pairs.

  Examples of pathway responsive promoters useful in the practice of the present invention include synthetic insulin pathway responsive promoters that contain a common insulin binding sequence (Jacob, et al., (1995) J. Biol. Chem. 270: 27773-27779). , Cytokine pathway responsive promoters, glucocorticoid pathway responsive promoters (Lange, et al., (1992) J. Biol. Chem. 267: 15673-80), IL-1 and IL-6 pathway responsive promoters (Won KA and Baumann H. (1990) Mol. Cell Biol. 10: 3965-3978), T3 pathway responsive promoter, thyroid hormone pathway promoter containing common motif, TPA pathway responsive promoter (TRE), TGF-beta pathway responsive promoter (Grotendorst, et al., (1996) Cell Growth and Differentiation 7: 469-480). In addition, natural or synthetic E2F pathway responsive promoters may be used. One example of an E2F pathway responsive promoter is described in Parr et al. (1997) Nature Medicine 3: 1145-1149. This paper describes an E2F-1 promoter containing a 4E2F binding site, which is reportedly active in tumor cells and rapidly repeats the cell cycle. Examples of other pathway responsive promoters are well known in the art and can be identified in the transcriptional regulatory region database of the eukaryotic genome accessible via the Internet.

  In certain embodiments of the invention, the viral vector is an adenovirus. The term “adenovirus” is synonymous with the term “adenovirus vector” and refers to a virus of the genus adenovirus. The term adenovirus genus collectively refers to animal adenoviruses of the genus mast adenovirus, i.e., adenoviruses including humans, cows, sheep, horses, dogs, pigs, rodents, and simian adenovirus subgenera It is not limited to. In particular, human adenoviruses include the A-F subgenus, their individual serotypes, individual serotypes and A-F subgenera, and human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Ad11A and Ad 11P), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91 However, it is not limited to them. The term bovine adenovirus includes but is not limited to bovine adenovirus types 1, 2, 3, 4, 7, and 10. The term canine adenovirus includes, but is not limited to, canine type 1 (CLL, Glaxo, R126, Utrecht, Toronto 26-61 strain) and 2. The term equine adenovirus includes, but is not limited to, equine types 1 and 2. The term porcine adenovirus includes but is not limited to porcine types 3 and 4. The term viral vector includes non-replicatable, replicative, and conditional replicated viral vectors.

  Particularly useful are vectors derived from human adenovirus types 2 and 5. These vectors may incorporate certain modifications to enhance their therapeutic capabilities. For example, these vectors may contain deletions of the E1a and E1b genes. Certain other regions may be emphasized or deleted to achieve specific characteristics. For example, up-regulation of the E3 region to reduce the immunogenicity associated with human adenoviral vectors administered to human subjects has been described. The E4 region is thought to be important for transgene expression by the CMV promoter, whereas the E4orf 6 protein has been described to lead to denaturation of p53 in target cells in the presence of the large E1b protein. (Steegenga, et al. (1998) Oncogene 16: 345-347).

  The therapeutic gene delivered is generally a cytotoxic gene, a tumor suppressor gene, a toxin gene, an apoptosis precursor gene, a drug activation precursor gene, or a cytokine gene. The term “cytotoxic transgene” is a nucleotide sequence, the expression of which in a target cell induces apoptosis of the cell. The term cytotoxic transgene includes, but is not limited to, a tumor suppressor gene, a toxin gene, a static cell gene, a drug activated precursor gene, or an apoptotic gene. The vectors of the present invention may be used such that one or more therapeutic transgenes are generated in series by using IRES elements or through independently regulated promoters.

  The term “tumor suppressor gene” is a nucleotide sequence whose expression in a target cell is capable of suppressing a neoplastic phenotype and / or inducing apoptosis. Examples of tumor suppressor genes useful in the practice of the present invention include p53 gene, APC gene, DPC-4 gene, BRCA-1 gene, BRCA-2 gene, WT-1 gene, retinoblastoma type gene (Lee, et al., (1987) Nature 329: 642), MMAC-1 gene, adenocarcinoma colon polyposis protein (US Pat. No. 5,783,666), colon cancer deletion gene (DCC), MMSC-2 gene, NF-1 gene, chromosome Nasopharyngeal epithelial tumor suppressor gene mapped to 3p21.3 (Cheng, et al. (1998) Proc. Nat. Acad. Sci. 95: 3042-3047), MTS1 gene, CDK4 gene, NF-1 gene, NF2 gene And the VHL gene.

  The term “toxin gene” is a nucleotide sequence whose expression in a target cell causes a toxic effect. Examples of such toxin genes include nucleotide sequences that encode Pseudomonas exotoxin, ricin toxin, diphtheria toxin and the like.

  The term “apoptotic precursor gene” is a nucleotide sequence whose expression in a target cell results in a programmed cell death in the cell. Examples of apoptotic precursor sequences include p53, adenovirus E3-11.6K, adenovirus E4orf4 gene, p53 pathway gene, and gene encoding caspase.

  The term “drug-activated precursor gene” is a nucleotide sequence, the expression of which results in the production of a protein capable of converting a non-therapeutic compound into a therapeutic compound, whereby the cell Becomes susceptible to killing by foreign factors or induces toxic conditions in the cells. One example of a drug activation precursor gene is the cytosine deaminase gene. Cytosine deaminase converts 5-fluorocytosine to 5-fluorouracil, a potent antitumor agent. Tumor cell lysis results in the death of many surrounding tumor cells because it results in a local burst of cytosine deaminase that allows the conversion of 5FC to 5FU at the local point of the tumor. This will kill a large number of tumor cells without the need to infect these cells with adenovirus (so-called “rolling action”). In addition, in the case of the thymidine kinase (TK) gene (see US Pat. Nos. 5,631,236 and 5,601,818), cells expressing the TK gene product become sensitive to selective killing by ganciclovir administration. Such genes may be used.

  The term “cytokine gene” is a nucleotide sequence whose expression in a target cell produces a cytokine. Examples of such cytokines include GM-CSF, interleukins, in particular IL-1, IL-2, IL-4, IL-12, IL-10, IL-19, IL-20, alpha, beta, And gamma subtypes of interferons, particularly interferon alpha-2b, and fusions such as interferon alpha-2-alpha-1.

  Modifications and / or deletions to the aforementioned genes to change to encode functional subfragments of the wild-type protein can be easily adapted for use in the practice of the present invention. For example, reference to the p53 gene includes not only the wild type protein but also the modified p53 gene. Examples of such modified p53 proteins include modifications to p53 to increase nuclear maintenance, deletion of delta 13-19 amino acids to remove calpain common cleavage sites, modification of oligomerization domains (Bracco, et al. al., PCT publication WO 97/0492, or US Pat. No. 5,573,925).

  The invention further includes the use of gene targeted non-viral vectors. A “non-viral vector” as used in this aspect of the invention is an autonomously replicating extrachromosomal circular DNA molecule that is independent of the normal genome and affects the expression of the DNA sequence of the target cell. DNA molecules that are not essential for cell survival under dynamic conditions. Plasmids replicate autonomously in bacteria and facilitate bacterial production. In order to be able to select or screen for the presence of a recombinant vector, additional genes such as genes encoding drug resistance may be included. Such additional genes can include, for example, genes encoding neomycin resistance, multidrug resistance, thymidine kinase, beta-galactosidase, dihydrofolate reductase (DHFR), and chloramphenicol acetyltransferase.

  In order to target therapeutic genes to neoplastic, MDR neoplastic, and damaged (eg, pathogen-infected) cells, in some cases, additional elements that facilitate cellular targeting are incorporated into non-viral gene delivery systems. It is advantageous to incorporate. For example, a lipid-coated expression plasmid may incorporate a vimentin antibody or ligand that facilitates targeted shooting. Simple liposome formulations may be administered, but liposomes filled or decorated with the desired composition of the present invention can be delivered systemically or directed to the desired tissue Then, in that tissue, the liposomes carry the selected therapeutic / immunogenic peptide composition. Vimentin antibodies and ligands used in this application include antibodies, monoclonal antibodies, humanized antibodies, single chain antibodies, chimeric antibodies, or functional fragments thereof (Fv, Fab, Fab '). Alternatively, non-viral vectors can be linked to the target component via a polylysine component, as described in Wu et al., US Pat. Nos. 5,166,320 and 5,635,383.

Liposome formulation Another strategy that may be used for vimentin targeted delivery of therapeutic agents is the use of immunoliposomes. Immunoliposomes take up antibodies against tumor-associated antigens into the liposomes. Liposomes carry therapeutic drugs or enzymes that activate drug precursors that remain inactive if not activated (see, eg, Lasic et al., (1995) Science 267: 1275-76). Several preclinical reports have reported that targeted targeting has been successful and enhanced anti-cancer efficacy with immunoliposome drugs (Maruyama et al., (1990) J. Pharm. Sci. 74: 978- 84; Maruyama et al., (1995) Biochim. Biophys. Acta 1234: 74-80; Otsubo et al., (1998) Antimicrob. Agents Chemother. 42: 40-44; Lopes de Menezes et al., (1998) Cancer Res. 58: 3320-30).

  Alternatively, non-antibody vimentin binding agents, such as modified LDL, can also be used as tumor-specific ligands in targeted targeting of liposome formulations of therapeutic agents. For example, folate-binding liposomes can be used to fire a therapeutic agent toward a tumor that overexpresses folate receptors. Folate-binding liposomes are suitably delivered to cancer cells that overexpress the folate receptor, both in vitro and in vivo (Lee and Low (1994) J. Biol. Chem. 269: 3198-204; Lee and Low (1995) Biochim. Biophys. Acta 1233: 134-144; Rui et al., (1998) J. Am. Chem. Soc. 120: 11213-18; and Gabizon et al., (1999) Bioconj. Chem 10: 289-98). In fact, several preclinical reports describe successful targeted targeting of liposomal drugs conjugated with such ligands (Ichinose et al., (1998) Anticancer Res. 18: 401-4 ; Yamamoto et al., (2000) Oncol. Rep. 7: 107-11; Rui et al., (1998) J. Am. Chem. Soc. 120: 11213-18; and Gabizon et al., (1999) Bioconj. Chem. 10: 289-98). Transferrin has been used as a target ligand to direct liposomal drugs to various cancer cells in vivo (Ishida and Maruyama (1998) Nippon Rinsho 56: 657-62; Kirpotin et al. (1997) Biochem. 36:66 -75). PEG-immunoliposomes conjugated with anti-transferrin antibody at the distal end of PEG are favorably associated with C6 glioma cells in vitro and treated with tumor-specific long-circulating liposomes containing doxorubicin followed by inoculation of glioma with doxorubicin Increased significantly (Eavarone et al., (2000) J. Biomed. Mater. Res. 51: 10-14).

  Methods for forming liposomal micelles / drug formulations are known in the prior art. For example, therapeutic drug micelles can be formed by mixing a therapeutic drug and a phosphatidylglycerol lipid derivative (PGL derivative). Briefly, the therapeutic agent and the PGL derivative are mixed in the range of 1: 1 to 1: 2.1 to form a therapeutic agent mixture. Alternatively, the ratio of therapeutic agent to PGL derivative is 1: 1.2, or 1: 1.4, or 1: 1.5, or 1: 1.6, or 1: 1.8, or 1: 1.9, or 1: 2.0, or 1: 2.1. Is in range. This mixture is then mixed with at least 20% organic solvent, such as ethanol, to form micelles containing the therapeutic agent. Methods for encapsulating antibodies or tumor targeting ligands in micelle formulations to produce immunoliposomes are known in the prior art and are described below. For example, immunoliposomes are prepared and used in U.S. Pat. Has been.

As used herein, the term “phosphatidylglycerol lipid derivative (PGL derivative)” is any lipid derivative that has the ability to form micelles and has a net negatively charged head group. Such includes, but is not limited to, dipalmitoyl phosphatidyl glycerol (DPPG), dimyristoyl phosphatidyl glycerol, and dicapryl phosphatidyl glycerol. In one aspect, phosphatidyl derivatives having a carbon chain of 10 to 28 carbon atoms and having an unsaturated aliphatic side chain are within the scope of the present invention. When a therapeutic agent and a negatively charged phosphatidylglycerol lipid are complexed with varying molecular ratios, the net is positively charged (1: 1), neutral (1: 2), or slightly negative Charged (1: 2.1) particles are obtained, which allows for targeted sniping against various tissues of the body after administration. On the other hand, complexing a therapeutic agent with a negatively charged PGL has been shown in many cases to enhance the solubility of the therapeutic agent, which is why it is necessary for effective anti-neoplastic therapies. Capacity is reduced. Furthermore, the complex formation between the therapeutic agent and the negatively charged PGL greatly increases the capsule formation efficiency, thereby minimizing drug loss during the manufacturing process. These complexes are stable, do not form a precipitate, and retain therapeutic efficacy for at least 4 months after storage at 4 ° C. In order to exert maximum therapeutic effect by avoiding rapid elimination from the blood circulation by the reticuloendothelial system (RES), the immunoliposome drug formulation may include components such as polyethylene glycol (PEG) (Klibanov et al., (1990) FEBS Lett. 268: 235-7; Maruyama et al., (1992) Biochim. Biophys. Acta 1128: 44-49; Allen et al., (1991) Biochim. Biophys. Acta 1066: 29-36). Conjugation of PEG to immunoliposomes has been shown not only to prolong liposome circulation in the blood but also to enhance the therapeutic efficacy of liposome formulations (Daemen et al., (1997) J. Control. Rel. 44: 1-9; Storm et al. (1998) Clin. Cancer Res. 4: 111-115; Vaage et al., (1997) Br. J. Cancer 75: 482-6; Gabizon et al., (1994) Cancer Res. 54: 987-92). Long-circulating immunoliposomes fall into two types. Those with antibodies bound to lipid head processes (Maruyama et al., (1990) J. Pharm. Sci. 74: 978-84) and those with antibodies bound to the distal end of PEG (Maruyama et al. al., (1997) Adv. Drug Del. Rev. 24: 235-42). In some cases, it may be advantageous to place a tumor-specific antibody at the distal end of the PEG polymer so that efficient target binding can be achieved by avoiding interference due to PEG chain configuration. This type of immune liposome formulations, the lungs.... (Maruyama et al , (1995) Biochim Biophys Act a 1234:... 74-80), brain (Huwyler et al, (1996) P roc Natl Acad Sci. USA 93: 14164-69) and tumors (Allen et al., (1995) Biochem. Soc. Transact. 23: 1073-79) have been used successfully with in vivo targets.

  Effective delivery of drugs by immunoliposome formulations is generally enhanced by active uptake by endocytosis of bound immunoliposomes. Human scFv antibodies can be selected from a phage display library to optimize uptake into tumor cells and ensure optimal targeting and delivery of the immunoliposomes in which they are taken up ( Poul et al., (2000) J. Mol. Biol. 301: 1149-61; Schier et al., (1996) J. Mol. Biol. 263: 551-67).

  Another strategy associated with antibody-mediated tumor targeting is antibody directed enzyme drug precursor (ADEPT). This is a two-step therapy designed to form high concentrations of anticancer drugs near the tumor cell membrane (Springer et al., (1996) Adv. Drug Deliv. Rev. 22: 351-64). Using this strategy, an enzyme-antibody complex, which preferably binds to any tumor-associated antigen, is first administered and then a non-toxic drug precursor is injected. This is activated by the action of the target enzyme. An improved ADEPT has been developed and tested that uses immunoliposomes as target carriers for drug precursor-activating enzymes instead of enzyme-antibody conjugates (Storm et al., (1997) Adv. Deliv. Rev. 24: 225 -31; Vingerhoeds et al., (1993) FEBS Lett. 336: 485-90).

Therapeutic Methods The present invention includes, but is not limited to, cancers, i.e., neoplasias, tumors, metastases, or diseases or disorders characterized by unregulated cell growth, and in particular, its multi-drug resistant forms. -Treatment or prevention of cancer, which provides treatment and prevention performed by administration of a therapeutically or prophylactically effective amount of an anti-vimentin antibody or a nucleic acid molecule encoding said antibody. Examples of types of cancer and proliferative disorders that are treated by the vimentin-targeted treatment of the present invention include leukemias (eg, myeloblastic, promyeloblastic, myelomonocytic, monocytic leukemia, erythroleukemia, Chronic myelogenous (granulocyte) leukemia and chronic lymphocyte leukemia), lymphoma (eg, Hodgkin's disease and non-Hodgkin's disease), fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma, endothelium Sarcoma, Ewing tumor, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell cancer, basal cell cancer, adenocarcinoma, renal cell cancer, hepatoma, Wilms tumor, cervical cancer, uterine cancer, testicular tumor, lung cancer Small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, oligodendroglioma, melanoma, neuroblastoma, retinoblastoma, dysplasia, and Hyperplasia and the like. In certain embodiments, the therapeutic compound of the invention is a prostate cancer (eg, prostatitis, benign prostatic hyperplasia, benign prostatic hyperplasia (BPH), prostate paraganglioma, prostate cancer, prostate intraepithelial neoplasia, prostate and rectum). It is administered to men with fistula and atypical prostate matrix lesions. Treatment and / or prevention of cancer includes, but is not limited to, alleviating symptoms associated with cancer, suppressing cancer progression, and enhancing immune response. In one embodiment, commercially available or naturally occurring anti-vimentin antibodies, functionally active fragments or derivatives thereof are used in the present invention.

  Vimentin therapeutic agents may be administered alone or in combination with other types of cancer treatments (eg, radiation therapy, chemotherapy, hormone therapy, immunotherapy, and anti-tumor agents). Examples of anti-tumor agents include cisplatin, ifosfamide, paclitaxel, taxene, topoisomerase I inhibitors (eg CPT-11, topotecan, 9-AC, and GG-211), gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil ( 5-FU), leucovorin, vinorelbine, temodarl, and taxol, but are not limited to these. In one embodiment, one or more anti-vimentin antibodies are administered to an animal, preferably a mammal, most preferably a human, following surgical resection of the cancer. In another embodiment, one or more anti-vimentin antibodies are administered to an animal, preferably a mammal, most preferably a human, with chemotherapy or radiation therapy. In another embodiment, the one or more anti-vimentin antibodies are administered to the animal, preferably to a mammal, most preferably to a human, prior to administering plasma to the animal for cancer prevention or treatment (e.g., 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), after administration (eg, 1 minute) , 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week later) .

  The anti-vimentin antibodies and other vimentin-targeted therapeutic agents described herein can be used in animals, preferably mammals, most preferably humans, IgG antibodies, IgM antibodies, for cancer prevention or treatment, And / or prior to administration of one or more complement components to the animal (eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours 24 hours, 2 days, or 1 week before), after administration (eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours) Administered at time, 2 days, or 1 week later) or simultaneously with administration. In another embodiment, one or more anti-vimentin antibodies and other vimentin-targeted therapeutic agents are used in animals, preferably in mammals, most preferably in humans, for the prevention or treatment of cancer. (Eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours) 24 hours, 2 days, or 1 week before), after administration (eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours) Administered at time, 2 days, or 1 week later) or simultaneously with administration. In yet another embodiment, the one or more anti-vimentin antibodies and other vimentin-targeted therapeutic agents are administered to an animal, preferably a mammal, most preferably a human, for the prevention or treatment of cancer. Prior to administration of an antibody that has been used for treatment to an animal (eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours 2 days or 1 week before), after administration (eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 Administered daily or weekly) or concurrently with administration. Examples of such antibodies include Herceptin, Retxan, Ovalex, Panorex, BEC2, IMC-C225, Vitaxin, Campus I / H, Smart M195, Lymphoside, Smart ID10, and Oncolim It is not limited.

  The invention further relates to a method for the treatment or prophylaxis of viral and other pathogen infections in animals, preferably mammals, most preferably humans, comprising an anti-vimentin antibody, or a nucleic acid molecule encoding said antibody, or Other methods are provided comprising administering a therapeutically or prophylactically effective amount of a vimentin-targeted therapeutic described herein. Examples of viral infections that can be treated or prevented according to the present invention include retroviruses (eg, human T-cell tropic virus (HTLV) types I and II, human immunodeficiency virus (HIV)), herpes virus ( For example, herpes simplex virus (HSV) types I and II, Epstein-Barr virus and cytomegalovirus), arena virus (eg Lassa fever virus), paramyxovirus (eg morbillivirus, virus, human respiratory syncytial Viruses and lung viruses), adenoviruses, bunya viruses (eg, hantavirus), cornaviruses, filoviruses (eg ebola virus), flaviviruses (eg hepatitis C virus (HCV)), yellow fever virus, and , Japanese encephalitis virus), hepadnavirus (eg, hepatitis B virus) Lus (HBV)), orthomyovirus (eg, Sendai virus, and influenza viruses A, B and C), papovavirus (eg, papilloma virus), picoma virus (eg, rhinovirus, enterovirus, and hepatitis A virus) ), Poxviruses, reoviruses (eg, rotavirus), togaviruses (eg, rubella virus), and rhabdoviruses (eg, rabies virus), but are not limited to these. Treatment and / or prevention of viral infection includes, but is not limited to, alleviation of symptoms associated with the infection, suppression or prevention of viral replication, and enhancement of immune response.

  The vimentin targeted therapeutics described herein may be administered alone or in combination with antiviral agents or other anti-pathogenic agents. Examples of antiviral agents include cytokines (eg, IFN-alpha, IFN-beta, and IFN-gamma); reverse transcriptase inhibitors (eg, AZT, 3TC, D4T, ddC, ddI, d4T, 3TC, adefovir, Efavirenz, delavirdine, nevirapine, abacavir, and other dideoxynucleosides or dideoxyfluoronucleosides); inhibitors of viral mRNA capping, such as ribavirin; protease inhibitors, such as HIV protease inhibitors (eg, amprenavir, indinavir, nelfinavir, Ritonavir and saquinavir); amphotericin B; catanospermine as an inhibitor of glycoprotein processing; neuraminidase inhibitors such as influenza virus neuraminidase inhibitors (eg zanamivir and oseltamivir) Topoisomerase I inhibitors (eg, camptothecin and analogs thereof); including but not limited to amantadine and rimanatazine; For example, one or more anti-vimentin antibody-drug conjugates may be administered to an animal, preferably a mammal, most preferably a human, before plasma is administered to the animal for the prevention or treatment of viral infection (eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before) after administration (eg, 1 Minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week later) The

  In another example, one or more vimentin-targeted therapeutic agents are used in animals, preferably in mammals, most preferably in humans, for the prevention or treatment of viral infections, IgG antibodies, IgM antibodies, and / or Before administering one or more complement components to an animal (eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days or 1 week before), after administration (eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days Or one week later) or at the same time as administration. In another preferred embodiment, the anti-vimentin antibody is an immunospecific antibody against one or more viral antigens for the prevention or treatment of viral infection in animals, preferably in mammals, most preferably in humans. (Eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week) Before), after administration (for example, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week later) Or simultaneously with administration. Examples of antibodies that are immunospecific for viral antigens include, but are not limited to, Synagis, RTM, PRO542, ostavir, and protovir.

  The present invention further comprises a method of treating or preventing microbial infection in an animal, preferably a mammal, most preferably a human, comprising administering a therapeutically or prophylactically effective amount of an anti-vimentin targeted therapeutic agent. I will provide a. Examples of microbial infections that can be treated or prevented according to the present invention include, but are not limited to, yeast infections, fungal infections, protozoal infections, and bacterial infections. Bacteria that cause microbial infections include Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoea, Neisseria meningitie and Neisseria meningitidis diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumae, Klebsiella pneumae ), Klebsiella rhinoscleromotis, Staphylocos aureus (Staphyloco) ccus aureus), Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (febr), Campylobacter i・ Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudorsber pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimuri um), Treponema pallidum, Troponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Borrelia burgdorferi, Borrelia burgdorferi Radiepae (Leptospira icterohemorrhagiae), Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii, Francisella tularenella bull, Serra , Brucella suis, Brucella meritensis, Mycoplasma multiple species, Rikettsia prowazeki, Rickettsia Tsu Hydrophilidae (Rickettsia tsutsugamushi), chlamydia (Chlamydia) more, and, although Helicobacter pylori (Helicobacter pylori) are exemplified, but not limited to these. Treatment and / or prevention of microbial infection includes, but is not limited to, alleviation of symptoms associated with said infection, suppression or prevention of replication, and enhancement of immune response.

  Vimentin-targeted therapeutic agents may be administered alone or in combination with other types of antimicrobial agents. Examples of antimicrobial agents include antibiotics such as penicillin, amoxicillin, ampicillin, carbenicillin, ticarcillin, piperacillin, cephalosporin, vancomycin, tetracycline, erythromycin, amphotericin B, nystatin, metroidazole, ketoconazole, and pentamidine. However, it is not limited to these. In one embodiment, the vimentin-targeted therapeutic agent is administered to an animal, preferably to a mammal, most preferably to a human, prior to administering plasma to the animal for prevention or treatment of microbial infection (e.g., 1 minute). 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before) after administration (eg, 1 minute, 15 Minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week later) or at the same time as administration.

  In one example, one or more vimentin-targeted therapeutic agents are used in animals, preferably mammals, most preferably humans, IgG antibodies, IgM antibodies, and / or 1 for the prevention or treatment of microbial infections. Prior to administering more than one species of complement component to the animal (eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 Day or 1 week before), after administration (eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, Or after 1 week) or at the same time as administration. In another example, one or more vimentin targeted therapeutic agents are immunized against one or more microbial antigens for the prevention or treatment of microbial infections in animals, preferably mammals, most preferably humans. Prior to administration of specific antibodies to animals (eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 week before), after administration (eg, 1 minute, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, or 1 Administered weekly) or simultaneously with administration. Examples of antibodies immunospecific for microbial antigens include, but are not limited to, antibodies that are immunospecific for LPS and capsule polysaccharide 5/8. In certain embodiments, animals having an increased risk of contracting a viral or bacterial infection are administered a composition of the invention. Examples of such animals include, but are not limited to, human burn patients, infants, immunocompromised or immunocompromised humans, and the elderly.

4.6 Kits The present invention further provides kits used for therapies, including neoplastic and multidrug resistant neoplasms diagnostic and prognostic methods. The diagnostic kit is useful, for example, in detecting a cell surface vimentin-expressing neoplasm in a patient sample or in a patient's body and monitoring the emergence of multidrug resistance. For example, monitoring of cell surface vimentin and other MDR-related markers described herein provides valuable information regarding the effectiveness of treatment and avoiding the development of multidrug resistance during the course of patient chemotherapy. can get. For example, the kit includes a labeled compound or agent capable of detecting cell surface vimentin protein in a biological sample, a means for measuring the amount of cell surface vimentin in the sample; and the sample vimentin amount as a standard (eg, Means for comparison with normal non-neoplastic cells or non-MDR neoplastic cells) can be included. The compound or drug can be packaged in a suitable container. The kit may further include instructions for using the kit to detect MDR-related markers, including cell surface vimentin protein. Such a kit can include, for example, one or more antibodies capable of specifically binding to at least a portion of a cell surface vimentin protein.

4.7 Vimentin vaccines Immunological compositions comprising vaccines and other pharmaceutical compositions comprising vimentin protein, or portions thereof, are included within the scope of the present invention. One or more types of vimentin protein, or an active or antigenic fragment thereof, or a fusion protein thereof can be used alone as a vaccine using methods and materials known to those skilled in the art. Or in combination with other antigens and can be packed. The immune response can be used therapeutically or prophylactically and can provide antibody immunity or cellular immunity similar to that produced by T lymphocytes.

  In order to emphasize immunogenicity, the protein may be conjugated to a carrier molecule. Suitable immunogenic carriers include proteins, polypeptides or peptides such as albumin, hemocyanin, thyroglobulin and its derivatives, in particular bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), polysaccharides, carbohydrates , Polymers, and solid bodies. Other protein-derived substances or non-protein-derived substances are known to those skilled in the art. The immunogenic carrier usually has a molecular weight of at least 1,000 daltons, preferably greater than 10,000 daltons. The carrier molecule often contains reactive groups to facilitate covalent conjugation to the hapten. The carboxyl group or amino group of amino acids, or the sugar group of glycoproteins are often used in this way. Carriers lacking such groups often react with suitable chemicals to produce such groups. Preferably, the immune response is elicited when the immunogen is injected into animals such as mice, rabbits, rats, goats, sheep, guinea pigs, chickens, and other animals, most preferably mice and rabbits. . Alternatively, a protein or polypeptide, or multiple antigen peptide containing multiple copies of an antigen or as an immunologically equivalent polypeptide, is sufficiently antigenic to improve immunogenicity without the use of a carrier. May have.

  Vimentin protein or a portion thereof, eg, a common or variable sequence amino acid motif, or a combination of proteins may be administered with an amount of adjuvant sufficient to enhance the immune response against the conjugate. One adjuvant that is widely used in humans is alum (aluminum phosphate or aluminum hydroxide). Saponin and its purified component QuilA, complete Freund's adjuvant, and other adjuvants used in research and veterinary applications are also available. Chemically defined formulations such as muramyl dipeptide, monophosphoryl lipid A, phospholipid conjugates such as Goodman-Snitkoff et al. (1991) J. Immunol. 147: 410-415 (through citation Encapsulated in proteoliposome capsules, such as Miller et al. (1992) J. Exp. Med. 176: 1739-1744 (incorporated herein by reference). ) And proteins can also be utilized in lipid particles such as Novasome® (Micro Vascular Systems, Inc., Nashua, NH) lipid particle capsules.

  The invention includes fragments or partial sequences of the intact vimentin polypeptide shown in FIG. 12A (SEQ ID NO: 1). This vimentin polypeptide partial sequence, or in the case of DNA vaccines, is the corresponding nucleic acid sequence encoding them, but these are preferably selected so as to be highly immunogenic. Antigenic principles for anti-vimentin vaccine production purposes include vimentin used as an immunogen for the production of anti-vimentin polyclonal and monoclonal antibodies used in the vimentin-based diagnostic and therapeutic methods described herein. The same applies to the use of polypeptide sequences.

  Computer-aided algorithms for predicting the antigenicity of polypeptide subsequences are widely available. For example, the “antigenic” appearance of a predicted antigenic region is represented by the Kolaskar method (Kolaskar and Tongaonkar (1990) FEBS Letters 276: 172-174 “a semi-empirical method for predicting antigenic determinants on protein antigens ( “A semi-empirical method for prediction of antigenic determinants on protein antigens”)). In the first experiment, Kolaskar and Tongaonkar experimentally tested 169 antigens. 156 less than 20 amino acids per determinant were selected (total 2066 residues). f (Ag) was calculated as the frequency of occurrence of each residue in the antigenic determinant [f (Ag) = Epitope_occurrence / 2066]. Hydrophobicity, accessibility, and elasticity values were obtained from Parker et al. (Parker, et al., (1986) Biochemistry 25: 5425-5432). For any protein, the average for each 7-mer was calculated and a number assigned to the central residue of that 7-mer. If any of these 7mers exceeds the protein average, the residue is considered to be on the surface. Using this result, f (s) was obtained as the surface appearance frequency of amino acids. This prediction algorithm includes the following steps. That is, calculate the average propensity for each overlapping 7mer and assign the result to the central residue (i + 3) of the 7mer; calculate the average for all proteins; if the average for all proteins is greater than 1.0, calculate 1.0 All residues above may be antigenic; if the average for all proteins is below 1.0, then residues that exceed the average for all proteins (note: the original paper shows a broken expression here) All may be antigenic; seek 6-mers where all residues are selected in step 3.

  Another method for determining the antigenicity of a polypeptide partial sequence is the Hopp and Woods algorithm (Hopp and Woods (1981) Proc. Natl. Acad. Sci. 86: 152-6). There are websites available to the general public for Hopp and Woods algorithmic analysis of user input polypeptide sequences and convenient graphical output of the analysis results (see for example). Using this algorithm to analyze the human vimentin full-length sequence shown in Figure 12A reveals several preferred sequences with high Hopp and Woods antigenic readings that are long enough for immunogenicity It was made. These are vimentin amino acid residues: 45-60 (ie RPSTSRSLYASSPGGV); 295-315 (ie FADLSEAANRNNDALRQAKQE); and 330-345 (ie VDALKGTNESLERQMR).

  Furthermore, the present invention provides a composition comprising the vimentin protein or polypeptide fragment of the present invention together with a suitable adjuvant. The composition can be present in a pharmaceutically acceptable carrier as described herein. As used herein, an “adjuvant” or “appropriate adjuvant” is a substance that can be mixed with a vimentin protein or polypeptide to enhance a subject's immune response without having a toxic effect on the subject. Is described. Suitable adjuvants include, for example, immunostimulatory cytokines; 5 percent (weight / volume) squalene (DASF, Parsippany, NJ), 2.5 percent pluronic, L121 polymer (Aldrich Chemical, Milwaukee) and 0.2 percent polysorbate (Tween 80 , Sigma) in a phosphate buffer saline solution include, but are not limited to, SYNTEX adjuvant formulation 1 (SAF-1). Other suitable adjuvants are well known in the art, but include QS-21, Freund's adjuvant (complete and incomplete), alum, aluminum phosphate, aluminum hydroxide, N-acetyl-muramyl-L-threonyl- D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP11637, called nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L- Alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP 19835A, referred to as MTP-PE) and RIBI, which are bacteria 3 components extracted from the above, monophosphoryl lipid A, trehalose dimycolate, and cell wall skeleton (MPL + TDM + CWS) dissolved in 2% squalene / Tween 80. Adjuvants such as immunostimulatory cytokines may be administered prior to administration of vimentin protein or vimentin-encoding nucleic acid sequence, simultaneously with administration of vimentin protein or vimentin-encoding nucleic acid sequence, or up to 5 days after administration of vimentin protein or vimentin-encoding nucleic acid sequence. It can be administered to a subject. QS-21 can be administered within several hours of administration of the fusion protein, like Alum, Freund's complete adjuvant, SAF, and the like.

  The present invention may also utilize a plurality of immunostimulatory cytokines that are co-administered prior to, simultaneously with, and subsequent to administration of a mixture of a plurality of adjuvants, eg, vimentin protein or nucleic acid encoding vimentin. For example, a mixture of multiple adjuvants, such as multiple immunostimulatory cytokines, may include two or more immunostimulatory cytokines of the invention, such as GM / CSF, interleukin-2, interleukin-12, interferon-gamma, interferon It may consist of leukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoietic factor flt3L, CD40L, B7.1 costimulatory molecule, and B7.2 costimulatory molecule. The effect of an adjuvant or adjuvant mixture is determined by measuring the immune response directed against the vimentin polypeptide with and without the adjuvant or adjuvant mixture using standard procedures described herein. Is possible.

  Furthermore, the present invention provides compositions comprising a vimentin protein or a nucleic acid encoding vimentin and an adjuvant, such as an immunostimulatory cytokine, or a nucleic acid encoding an adjuvant, such as an immunostimulatory cytokine. The composition can be present in a pharmaceutically acceptable carrier as described herein. Immunostimulatory cytokines used in the present invention are GM / CSF, interleukin-2, interleukin-12, interferon-gamma, interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoietic factor flt3L, CD40L, B7.1 costimulatory molecule, and B7.2 costimulatory molecule may also be used.

  As used herein, the term “vaccine” refers to a DNA vaccine, which is a nucleic acid molecule encoding vimentin or an antigen portion thereof, eg, a DNA vaccine in which a common or variable sequence amino acid motif is administered to a patient as a pharmaceutical composition. Including. For genetic immunization, suitable delivery methods known to those skilled in the art include direct injection of plasmid DNA into muscle (Wolff et al. (1992) Hum. Mol. Genet. 1: 363), specific proteins Transport of DNA complexed with the carrier (Wu et al., (1989) J. Biol. Chem. 264: 16985), coprecipitation of DNA and calcium phosphate (Benvenisty and Reshef (1986) Proc. Natl. Acad. Sci 83: 9551), encapsulation of DNA in liposome capsules (Kaneda et al. (1989) Science 243: 375), particle firing (Tang et al., (1992) Nature 356: 152; and Eisenbraun et al. (1993) ) DNA Cell Biol. 12: 791) and in vivo infection with cloned retroviral vectors (Seeger et al. (1984) Proc. Natl. Acad. Sci. 81: 5849).

  In another embodiment, the present invention relates to a polynucleotide, which is a flanking nucleic acid that can be expressed to produce vimentin or an immunostimulatory gene product upon introduction of said polynucleotide into eukaryotic tissue in vivo. A polynucleotide comprising a sequence. The encoded gene product preferably operates as an immunostimulant or as an antigen capable of generating an immune response. Thus, the nucleic acid sequence in this embodiment encodes an immunogenic epitope and optionally a member of a cytokine or T cell costimulatory element, eg, a B7 family protein.

Advantages of immunizing with a gene rather than a gene product include: First, it is relatively simple that it is possible to present natural or nearly natural antigens to the immune system. Recombinantly expressed mammalian proteins, even in bacteria, yeast, or even mammalian cells, require extensive processing to ensure proper antigenicity. A second advantage of DNA immunization is the possibility that the immunogen enters the MHC class I pathway and triggers a cytotoxic T cell response. Immunization of mice with DNA encoding the influenza A nucleoprotein elicited a CD8 ⁺ response to NP, which protected mice from challenge by heterologous strains of influenza (Montgomery, DL et al. (1997) Cell Mol. Biol. 43 (3): 285-92; and Ulmer, J. et al. (1997) Vaccine 15 (8): 792-794). Cellular immunity is important in terms of controlling infection. DNA immunization can elicit both humoral and cellular immune responses, thus providing a relatively easy way to investigate the potential use of these vaccines for many vimentin genes and gene fragments The biggest advantage would be.

The present invention also includes known preparations or uses of tumor antigen vaccines used for the treatment or prevention of cancer. For example, US Pat. No. 6,562,347 is a use of a fusion polypeptide comprising a chemokine and a tumor antigen that, when administered as a protein or nucleic acid vaccine, elicits an effective immune response to treat or prevent cancer. Teaches the usage of polypeptides. Chemokines are a group of normally small secreted proteins (7-15kDa) that are triggered by inflammatory stimuli and are involved in directing the migration, leakage, and activation of blood migrating leukocytes that mediate inflammatory responses (Wallack (1993) Annals New York Academy of Sciences 178). Chemokines communicate their function through interactions with specific cell surface receptor proteins (23). As defined by the cysteine signature motif, at least four chemokine subfamilies called CC, CXC, C, and CX ₃ C have been identified. In this nomenclature, C is cysteine and X is any amino acid residue. From structural studies, at least CXC and CC chemokines share a very close three-dimensional structure (monomer), but have a different four-dimensional structure (dimer). In many cases, the configurational difference is localized at the loop portion or at the N-terminus. In the present invention, for example, a human vimentin polypeptide sequence (as shown in FIG. 12A), or a polypeptide fragment thereof, and a chemokine sequence are fused and used in an immune vaccine. The chemokine portion of the fusion may be a human monocyte chemotropic protein-3, a human macrophage derived chemokine, or a human SDF-1 chemokine. The vimentin portion of the fusion is preferably expected to have strong antigenicity on routine screening.

4.8 Pharmaceutical Formulation and Treatment The present invention provides both prevention and treatment for patients with neoplastic disease. A subject at risk of suffering from such a disease is characterized, for example, by the diagnostic or prognostic assays described herein. Prophylactic agents and administration can be performed prior to the appearance of symptoms characteristic of the neoplasm such that the development of the neoplasm is blocked or otherwise slowed. In general, prophylaxis or therapy involves treatment of a compound comprising a vimentin targeting moiety capable of binding to and binding to a neoplastic cell, particularly a cell surface vimentin present on a multidrug resistant neoplastic cell. Administration of an effective amount to a subject.

  Examples of vimentin target compounds include monoclonal anti-vimentin antibodies and fragments thereof. Examples of suitable therapeutic ingredients include conventional chemotherapeutic agents such as actinomycin, adriamycin, altretamine, asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine , Dactinomycin, daunorubicin, docetaxel, doxorubicin, epoetin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, lomustine, mechlorethamine, melphalantomitremito TRON, paclitaxel, pentostatin, procarba Emissions, taxol, teniposide, topotecan, vinblastine, vincristine, and include vinorelbine. Other examples of suitable therapeutic ingredients include Pseudomonas exotoxin, diphtheria toxin, plant ricin toxin, plant abrin toxin, plant saponin toxin, plant gelonin toxin, and pokeweed antiviral protein. Such immunotoxins are shot by vimentin-targeting components of therapeutic compounds toward vimentin-expressing neoplastic cells or multidrug resistant neoplastic cells, and when bound to cell surface vimentin, they are captured and taken up into the cell, Kill the neoplastic cell or prevent its growth.

4.8.1 Effective doses Toxicity and therapeutic efficacy of this compound is determined by standard pharmaceutical processes in cell cultures or experimental animals, eg LD ₅₀ (dose that is lethal to 50% of the population) and ED ₅₀ (50% of the population). The dose can be quantified by a process for quantification. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD ₅₀ / ED ₅₀ . Compounds that exhibit large therapeutic indices are preferred. Compounds that exhibit toxic side effects may be used, but if such compounds are directed to the affected tissue, a delivery system should be designed to reduce the side effects by minimizing the expected damage to uninfected cells. Must be taken into account.

Data obtained from cell culture assays and animal studies is used to formulate a dosage range for use in humans. Dose of the compound, most, or preferably within a range of circulating concentrations that include the ED ₅₀ with no completely nontoxic. The dose may vary within this range depending on the dosage form used and the route of administration utilized. All compounds used in the methods of the invention are first estimated in a therapeutically effective amount by cell culture assays. Animal models formulate doses that achieve a circulating plasma concentration range that includes the IC ₅₀ (ie, the concentration of the test compound that achieves half-maximal suppression of symptoms) as determined in cell culture. More accurately determine useful doses in humans, plasma levels can be measured, for example, by high performance liquid chromatography.

4.8.2 Formulation and Use The pharmaceutical compositions used in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and physiologically acceptable salts and solvents can be obtained, for example, by infusion, inhalation, or swallowing (from the mouth or from the nose) or by oral, buccal, parenteral or rectal administration. It may be formulated to be administered.

  In this therapy, the compounds of the present invention can be formulated for a variety of dosage loads, i.e., systemic and topical, or loads including localized administration. In general, techniques and formulations can be found in Remmington's “Pharmaceutical Sciences”, Meade Publishing Co., Easton, Pennsylvania. For systemic administration, infusion including intramuscular, intravenous, intraperitoneal, and subcutaneous is preferred. For injection, the compounds of the invention are formulated as solutions, preferably formulated in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds may be formulated as a solid form and redissolved or suspended immediately before use. Also included are lyophilized forms.

  For oral administration, the pharmaceutical composition may be prepared by conventional means with the following, for example, in the form of tablets or capsules. Pharmaceutically acceptable excipients such as binders (eg pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (eg lactose, microcrystalline cellulose, Or calcium hydrogen phosphate); lubricant (eg, magnesium stearate, talc, or silica); disintegrant (eg, potato starch or sodium starch glycolate); or wetting agent (eg, sodium lauryl sulfate) It is. The tablets may be coated by methods known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, but the preparations may be dried before being used for constitution with water or other suitable vehicle. It may be presented as a product. Such liquid preparations may be prepared by conventional means with the following pharmaceutically acceptable additives. Additives include, for example, suspensions (eg, sorbitol syrup, cellulose derivatives, or hydrogenated edible fat); emulsifiers (eg, lecithin, or acacia); non-aqueous vehicles (eg, almond oil, oily esters, ethyl alcohol) Or fractionated vegetable oils); and preservatives (eg, methyl or propyl-p-hydroxybenzoate, or sorbic acid). The formulations may also suitably contain buffer salts, flavoring agents, coloring agents and sweetening agents.

  Preparations for oral administration are suitably formulated to achieve controlled release of the active compound. For buccal administration, the formulation may take the form of tablets or lozenges formulated in conventional manner. For inhalation administration, the compounds used according to the invention can be obtained from a pressurized pack or nebulizer from a suitable driver, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable By using a simple gas, it is suitably transported in the form of an aerosol spray. In the case of a pressurized aerosol, the dosage unit is determined by providing a valve to deliver the specified dose. For example, gelatin capsules and cartridges for use in aspirators or inhalers are formulated to contain a powder mixture of the compound and a suitable base powder, such as lactose or starch.

  The compounds may be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Injectable formulations may be presented as unit dosage forms, for example in ampoules or in multi-dose containers, with the addition of preservatives. The composition may take the form of a suspension, solution, emulsion in an oily or aqueous vehicle, and may contain formulations such as suspending, stabilizing and / or dispersing agents. Good. Alternatively, the active ingredient may be present in powder form for constitution with a suitable vehicle, such as sterile, pyrogen-free water, prior to use.

  The compound may also be formulated as a rectal composition, eg, a suppository, or a retention enema, eg, a conventional suppository base, eg, a composition comprising cocoa butter or other glycerides. Good.

  In addition to the aforementioned formulations, the compounds may be further formulated as a retention formulation. Such long lasting formulations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with a suitable polymer, or a hydrophobic material (eg, as a suspension in an acceptable oil), or as an ion exchange resin, or a sparingly soluble derivative, eg, It may be formulated as a sparingly soluble salt. Other suitable transport systems include microspheres. This allows the drug to be delivered locally non-invasively over a long period of time. This technique utilizes precapillary-sized microspheres that are injected through a coronary catheter into any selected part, such as the heart or other organs, without causing inflammation or ischemia. The administered therapeutic agent is slowly released from the microsphere and taken up by surrounding tissue cells (eg, epithelial cells).

  Systemic administration can also be performed by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the prior art, and examples include bile salts and fusidic acid derivatives for transmucosal administration. In addition, surfactants may be used to facilitate penetration. Transmucosal administration is performed using a nasal spray or suppository. For topical administration, the oligomers of the invention are formulated into ointments, coatings, gels, or creams as is generally known in the art. To accelerate healing, it is also possible to use a washing solution to treat trauma or inflammation locally.

  In the clinical setting, the vimentin targeted therapeutic agent treatment system and gene delivery system can be introduced into a patient in any manner selected from several methods familiar to the prior art. For example, a formulation of a vimentin-targeted therapeutic can be introduced systemically, for example, by intravenous injection.

  Formulations of the vimentin-targeted therapeutics of the present invention, consisting essentially of a compound dissolved in an acceptable diluent, or can include a sustained release substrate in which the gene delivery vehicle or compound is embedded.

  The composition may be presented in a pack or dosing device containing one or more unit dosage forms containing the active ingredient, if desired. The pack may include, for example, a metal or plastic pack such as a water bubble pack. The pack or dosing device may be accompanied by instructions for administration.

Examples The present invention is more specifically described by the following examples. However, this example should not be considered limiting. The contents of all references, patents, and published patent applications cited throughout this application are hereby incorporated by reference. The nucleotide and amino acid sequences referred to herein and deposited in public databases are also included herein by reference. Those skilled in the art will recognize and be able to ascertain numerous equivalents to the specific materials and processes described herein with only routine experimentation. Such equivalents are intended to be encompassed in the scope of the following claims.

5.1 Overexpression of 53 kDa protein in MDR solid tumor / cancer cells Determine which protein, if any, is differentially expressed in multidrug resistant tumor cell lines when compared to their drug-sensitive alleles An experiment was conducted for this purpose. The seven different cell lines used in this example are shown in the table below.

Suspension cells were grown in RPMI or α-MEM media containing 10% to 15% fetal calf serum (Hyclone, Inc. purchased from Logan, Utah). Cells were grown without antibiotics at 37 ° C. in a humidified atmosphere of 5% CO 2 and 95% air, and the culture was passaged at 1 × 10 ⁶ cells / ml. Multi-drug resistant cells (HL60 / AR, NB4 / VLB, NB4 / DOX, CEM / VLB, CEM / DOX, HSB2 / VLB, HSB2 / DOX, Molt4 / VLB, Molt4 / DOX) (Aurelium Biopharma Inc., Montreal (Quebec) ), Canada) were continuously grown with appropriate concentrations of cytotoxic drugs. Similarly, adherent cells were grown in α-MEM medium (MCF-7) or DMEM (MDA) containing 10% fetal bovine serum. Multidrug resistant cells (MCF-7 / AR, MDA / AR and MDA / MITO) were continuously grown with appropriate concentrations of cytotoxic drugs. All cell lines were examined using a commercially available (eg, Stratagene Inc., San Diego, Calif.) PCR Mycoplasma Detection Kit based on the manufacturer's instructions to confirm no mycoplasma contamination. All multi-drug resistant cell lines were frequently tested for multi-drug resistance using a set of different drugs representing different classes of drugs. MDR cells expressed other MDR markers on their cell surface.

  Various extracts were prepared from each cell type. Cells were concentrated and lysed according to standard methods to obtain whole cell extracts from the cells (eg Ausubel et al. “Current Protocols in Molecular Biology”), John Wiley & Sons Inc., New York, New York, 1993). Separately, the cells were first biotinylated on the surface and then lysed to obtain a biotinylated whole cell extract (as shown in the examples below). To perform this experiment, intact drug susceptibility (CEM, HL60 and MCF-7 / AR) and multidrug resistant cells (CEM / VLB, HL60 / AR, and MCF-7 / AR) were removed from the membrane. Biotinylated with a permeabilized biotinylation agent, Sulfo-NHS-LC-LC-biotin (Pierce # 21338). For this, the cells were washed three times with 50 ml PBS pH 8 and biotinylated. Next, Sulfo-NHS-LC-LC-biotin, a membrane-impermeable reagent, was prepared at 0.1-0.5 mg / ml and added to the cells. Incubation with Sulfo-NHS-LC-LC-biotin was continued for 1 hour at 4 ° C. with rotation. The reaction was stopped by washing the cells once with 50 ml PBS pH 8 containing 10 mM glycine and several times with 50 ml PBS without glycine. The cells are then treated with 200 μl of buffer A (1% SDS and 0.05%) containing a protease inhibitor (protease inhibitor: 1 μg / ml pepstatin; 1 μg / ml leupeptin; 1 μg / ml benzamidine; 0.2 mM PMSF). M Tris / HCl, pH 7.4) and incubated on ice for 5 minutes. Next, this cell lysate was sonicated by placing it on ice for 3 × 10 seconds according to Vibracell ultrasonic transmitter amplitude 40 setting # 25 and for 1 minute between ultrasonic irradiations. The sonicated cell lysate was mixed with 800 μl buffer B (1.25% Triton-X100, 0.05 M Tris / HCl, pH 7.4, 190 mM NaCl) containing protease inhibitors and incubated on ice for 5 minutes. The cell lysate was then centrifuged at 14,000 rpm for 5 minutes in an Eppendorf microcentrifuge. The supernatant was removed and the protein concentration was quantified using a DC protein assay kit obtained from BIORAD according to the manufacturer's instructions (BioRad Laboratories, Hercules, CA) (also Lowry et al., J. Biol. Chem). 193: 265-275, 1951).

  By using this Sulfo-LC-LC-biotinylating agent, modification of the ε-amino group of the lysine side chain of the protein exposed on the cell surface was ensured. Conversely, it was expected that intracellular proteins were not biotinylated. This is because this sulfobiotin cannot cross the cell membrane of intact cells.

In addition, cell membrane specimens were prepared for each type of surface biotinylated or non-biotinylated cells. To do this, suspend 3x10 ⁹ cells (of each cell type) in 12.5 ml of hypotonic buffer 1 (10 mM NaCl, 1.5 mM MgCl ₂ , 10 mM Tris-HCl, pH 7.4) and incubate on ice for 10 minutes. did. The cells were then homogenized with a Dounce glass homogenizer type B (15 ml). The extent of cell lysis was confirmed by microscopic examination of the cells. Approximately 40 strokes were required to destroy 85% of the cells. Immediately after homogenization, add half (6.25 ml) of 2.5X buffer II (buffer 1X; 210 mM mannitol; 70 mM sucrose; 5 mM Tris-HCl pH7.5, 1 mM EDTA pH7.5) to the cell homogenate and mix did. The homogenate was spun for 5 minutes at 1300xg (3300 rpm) in a Sorvall centrifuge using an SS34 rotor (brake off). The pellet containing the nuclear fraction was separated from the supernatant containing the cell membrane and organelles. The supernatant after nuclear separation was rotated at 17000xg (11900 rpm) for 15 minutes in a Sorvall centrifuge using an SS34 rotor (brake off). The mitochondrial-enriched pellet was separated from the membrane-enriched supernatant fraction (post-mitochondrial fraction). The latter supernatant was centrifuged at 100,000 × g for 2 hours at 4 ° C. in a Sorvall ultracentrifuge using a PA ultra clear tube procured from AH-629 rotor and Beckman # cat344058. The cytosol-enriched supernatant was carefully removed and the membrane-enriched membrane pellet was resuspended in 300 μl of buffer 1 and mixed well using a 27 gauge needle. The cell membrane was further concentrated by digesting the final membrane pellet with a discontinuous sucrose gradient (16%, 31%, 45%, 60% w / v sucrose / buffer 1). Briefly, after a centrifugation step of 100,000xg, an equal amount of 32% w / v sucrose dissolved in buffer 1 (finally 16% w / v) was added to the resuspended pellet of concentrated membrane material (finally 16% w / v) About 350 μl) was added. A sucrose gradient was prepared with 6.9 ml 60% at the bottom of the tube followed by 9.9 ml 45% sucrose, 13.9 ml 31% sucrose, and 6.9 ml 16% sucrose. Slowly pour 16% sucrose containing unpurified cell membrane onto the top of the gradient and pour this sample at 4 ° C in a Sorvall ultracentrifuge using a PA ultra-clear tube sourced from AH-629 rotor and Beckman # cat344058. And centrifuged for 18 hours. The intervening layer between 16% and 31% sucrose containing the highly concentrated cell membrane was collected and washed once by centrifugation with buffer 1. After centrifugation at 100,000 × g in an ultracentrifuge, this highly concentrated cell membrane fraction was resuspended in an appropriate volume (about 50 μl) of buffer 1 and stored at −80 ° C.

Proteins from surface biotinylated whole cell extracts and cell membrane specimens for each cell type (HL60, HL60 / AR, NB4, NB4 / DOX, NB4 / VLB, CEM, CEM / VLB, Molt4, Molt4 / AR, Molt4 / VLB , HSB2, HSB2 / VLB, HSB2 / DOX, MCF-7, MCF-7 / AR, MDA, MDA / AR, MDA / MITO), two-dimensional (2-D) gel electrophoresis And visualized by silver staining or immunoblot with anti-VIM antibody or streptavidin-HPR complex (streptavidin binds to biotin). This allowed the sample to be decomposed according to the difference in equipotential points in the first dimension and according to the molecular weight in the second dimension. In the first dimension, isoelectric focusing was performed using a 13 cm fixed pH gradient strip (Amersham Pharmacia Biotech, Piscataway, NJ). Briefly, 13 cm strips were rehydrated in a 250 μl rehydration buffer containing a protein sample (0.5 mg-2 mg protein) in a ceramic strip holder. Next, an electrode pad was placed on each electrode, and proteins were separated with an IPGphor apparatus according to the following program.

13cm strip (pH4-7) -500V For 500Vh
-1000V For 1000Vh
-8000V For 16000Vh

The strips were then rinsed with water, equilibrated with 1% DTT dissolved in equilibration buffer for 15 minutes, and then equilibrated with 4% iodoacetamide dissolved in equilibration buffer for 15 minutes.

  In the second dimension, the isoelectric strip is subjected to 8% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli (Lemmli UK, Nature 227: 680-685, 1970). This was done using a gel. Molecular weight markers were loaded on 2x3 mm filter paper and placed at one end of the strip. Next, the strip and molecular weight marker filter paper were sealed in a polyacrylamide gel containing 0.5% agarose solution as a transfer buffer. Protein was slowly transferred from the strip to the gel for 1 hour at 30 V at room temperature. Separations were carried out at 4-8 ° C. for 17-18 hours at 70 V or 75 V in 8% or 10% gels, respectively.

  The gel was then dyed with silver and photographed. (At this point, the gel-degraded protein can also be transferred to a Hybond C nitrocellulose membrane and then immunoblotted with antibody or streptavidin-HRP as described below in the Examples below. Be careful).

  A protein of about 53 kDa (pI of about 5) was identified in a silver-stained 2-D gel of whole cell extracts of MCF-7 and MCF-7 / AR cells. This 53kDa protein was extremely overexpressed in whole-cell extracts of multidrug resistant MCF-7 / AR cells compared to drug-sensitive MCF-7 cells (Figures 1 (A) and (B)) .

5.2 Identification of 53 kDa protein in MCF-7 / AR breast cancer cells as vimentin To clarify the identity of the 53 kDa protein in breast MCF-7 and MCF-7 / AR cells, MCF-7 or MCF-7 / AR The whole cell extract was loaded onto a 2-D gel (2 x 750 μl, pI 4 to 7, 10% gel), silver stained and the 53 kDa spot was excised. The spots were then processed using gel staining / decolorization, trypsin digestion, peptide extraction, and peptide purification optimization steps. Briefly, gels were dyed with SilverQuest silver stain (Invitrogen, Carlsbad, CA) according to the manufacturer's specified procedure. The protein spots of interest were cut out with a razor blade washed with acid, cut into small pieces on a clean (washed with acid) glass plate, and transferred to a 200 μl PCR tube (MeOH treated). Mix this gel piece with 50 μl destain A and 50 μl destain B (provided with the SilverQuest kit) (or 100 μl freshly prepared destain, from premix) Incubated for 15 minutes at room temperature without agitation. The washing solution was removed using the capillary tip. Water was added to the gel pieces, mixed and incubated at room temperature for 10 minutes. The subsequent process was repeated three times. The gel pieces were then dehydrated in 100 μl 100% methanol for 5 minutes at room temperature and then rehydrated with 30% methanol / water. The gel pieces were then washed twice with water for 10 minutes, twice with 25 mM Ambic (ammonium bicarbonate) / 30% acetonitrile (5 minutes), and then the gel drying step. Trypsin digestion of decolorized and washed gel pieces by adding approximately 1 volume trypsin solution (130 ng enzyme dissolved in 25 mM ammonium bicarbonate, 5 mM CaCl ₂ ) to 1 volume gel piece Was left on ice for 45 minutes. The digestion was allowed to proceed for 15-16 hours at 37 ° C. The digested peptide was extracted with acetonitrile for 15 minutes at room temperature with shaking. The gel pieces / solvent were sonicated for 5 minutes and re-extracted with 25 mM Ambic: 50% acetonitrile without sonication. The digested peptide was further extracted with freshly prepared 5% formic acid: 50% acetonitrile: 45% water for 15 minutes while shaking at room temperature. Mixing was completed with 1 volume of acetonitrile and the gel pieces / solvent were sonicated for 5 minutes and similarly re-extracted without ultrasound. The collected materials were combined and dried. Extracted peptides were resuspended in 5% methanol containing 0.2% trifluoroacetic acid (or 0.5% formic acid) and then loaded onto an equilibrated C18 bed (by Ziptip, Millipore). The loaded Ziptip was washed with 5% acetonitrile containing 0.2% TFA (or 0.5% formic acid) and then eluted with 10 μl of 60% acetonitrile. The eluted peptide solution was dried and analyzed by MALDI mass spectrometry (Mann M. et al. Ann. Rev. Biochem. 70: 437-473, 2001).

  The mass spectrum of the 53kDa spot (after purification and protease digestion) consists of more than 20 trypsinized peptides, 16 of which are derived from the 53kDa protein and the rest of the peptides are trypsin autolysis products (data diagram). Not shown). The 53 kDa peptide was further analyzed using a shareware software program for searching a sequence database called ProFound (registered trademark) (http://www.proteomics.com/prowl-cgi/Profound.exe). PROFOUND was used to search public databases for protein sequences (eg, non-redundant collection of sequences from the National Center for Biotechnology Information (NCBI)). The NCBI database contains the protein sequences of the PDB, SWISS-PROT, and PIR databases as well as the translated protein sequences of all collections of DNA stored in GenBank.

  Using the ProFound® program, the 53 kDa protein was identified as vimentin (Chan et al., Biochemistry 28: 1033-1039, 1989; Hale and Mansfield, Nuclear Acids Res. 17 (23): 10112, 1989). This identification was made with a probability Z score of 2.43 (entering the 99.9th percentile) based on analysis of 16 trypsinized peptide sequences covering 43% of the complete amino acid sequence of vimentin (Figure 12). Two values were taken into account when evaluating the PROFOUND analysis results. That is, the Z probability score and percent occupancy (which is the percentage of the peptide sequence relative to the complete amino acid sequence of the identified protein). Z = 1.65-2.43 is an acceptable range of scores. Z = 1.65 means that the result falls in the 95th percentile, and Z = 2.43 means that the result falls in the 99.9th percentile. Thus, a Z score of 2.43 indicated that this 53 kDa spot peptide sequence matched the vimentin with a very high probability.

  The sequence of the 16 peptides diffused throughout the vimentin molecule and together represented 43% of the complete vimentin protein amino acid sequence. Thus, the identified protein is a full-length protein, not a fragment or a fusion protein.

  To confirm that vimentin is overexpressed in MCF-7 / AR cells, the whole extract was digested with 2D-PAGE, transferred to nitrocellulose, and anti-vimentin (MS-129-P, NeoMarker) Blotted.

  As shown in FIGS. 2 (A) and (B), vimentin is at least 11 in the whole cell extract of multidrug resistant MCF-7 / AR cells compared to the whole cell extract of drug sensitive MCF-7 cells. It was expressed twice as much.

5.3 Cell surface expression of vimentin in MDR lymphoblastic leukemia cells To determine whether vimentin is inside or outside the cell membrane, intact lymphoblastic leukemia cells (CEM and CEM / VLB) were Similarly, whole cell extracts were prepared from both drug sensitivity (CEM) and multidrug resistance (CEM / VLB) by treatment with a membrane-impermeable biotinylation reagent that reacts with lysine. For CEM and CEM / VLB, two equivalent sets of 2-D gels were prepared, silver stained, transferred to nitrocellulose and blotted with streptavidin-HRP.

  3A and B show 2-D streptavidin-HRP blots of CEM and CEM / VLB whole surface biotinylated cell extracts, respectively. The displayed area corresponds to the expected site of vimentin in a 2-D gel (MW = 53 kDa, pI = 5.1). The 50 kDa protein was expressed at least a 2-fold excess on the surface of multidrug resistant CEM / VLB cells compared to drug sensitive CEM cells (compare FIGS. 3A and B).

  To confirm the identity of this protein is vimentin, a 50 kDa spot was positioned on a silver-stained gel using the coordinates obtained from the corresponding spot, cut out and processed as described in Example 2. A sample for MALDI analysis was prepared. The mass spectrum of this 50 kDa spot (after trypsin digestion and peptide purification) consists of more than 20 trypsin-treated peptides, of which 15 peptides are derived from the 53 kDa protein and the remaining peptides are trypsin autolysis products ( Data not shown). The 53 kDa peptide was further analyzed using a shareware software program for searching a sequence database called ProFound (registered trademark) (http://www.proteomics.com/prowl-cgi/Profound.exe).

  The 50 kDa protein was identified as vimentin (Chan et al., Biochemistry 28: 1033-1039, 1989; Hale and Mansfield, Nuclear Acids Res. 17 (23): 10112, 1989). This identification was made with a probability Z score of 2.43 (entering the 99.9th percentile) based on analysis of 14 trypsinized peptide sequences covering 52% of the complete amino acid sequence of vimentin. A Z score of 2.43 indicated that this approximately 50 kDa spot peptide sequence matched the vimentin with a very high probability.

  Full-length vimentin protein was expressed on the surface of CEM / VLB cells and biotinylated in one place. That is, a lysine residue located relatively close to the C-terminus of the molecule. The sequence of the 15 peptides diffused throughout the vimentin molecule and together represented 52% of the complete vimentin protein amino acid sequence. Thus, the identified protein is again a full-length protein, not a fragment or a fusion protein. This biotinylated region was identified in two ways. First, the bound biotin component changes the mass of the peptide containing it as compared to a peptide that is not biotinylated. Second, biotinylated peptides bind to streptavidin beads (immobilized streptavidin beads, commercially available from Pierce # 20347, Dallas, TX), but not non-biotinylated peptides.

5.4 Cell surface expression of vimentin in MDR promyelocytic leukemia cells To determine whether vimentin is inside or outside the cell membrane, intact premyelocytic leukemia cells (HL60 and HL60 / AR) were Similarly, whole cell extracts were prepared from both drug sensitivity (HL60) and multidrug resistance (HL60 / AR) by reaction with a membrane-impermeable biotinylated reagent that reacts with the amino acid lysine.

  For this, the cells were washed three times with 50 ml PBS pH 8 and biotinylated. The membrane-impermeable biotinylation reagent Sulfo-NHS-LC-LC-biotin (Pierce Chemicals, Dallas, TX) was then prepared at 0.1-0.5 mg / ml and added to the cells. Incubation with Sulfo-NHS-LC-LC-biotin was continued for 1 hour at 4 ° C. with rotation. The reaction was stopped by washing the cells once with 50 ml PBS pH 8 containing 10 mM glycine and several times with 50 ml PBS without glycine. The cells are then treated with 200 μl of buffer A (1% SDS and 0.05%) containing a protease inhibitor (protease inhibitor: 1 μg / ml pepstatin; 1 μg / ml leupeptin; 1 μg / ml benzamidine; 0.2 mM PMSF). M Tris / HCl, pH 7.4) and incubated on ice for 5 minutes. Next, this cell lysate was sonicated by placing it on ice for 3 × 10 seconds according to Vibracell ultrasonic transmitter amplitude 40 setting # 25 and for 1 minute between ultrasonic irradiations. The sonicated cell lysate was mixed with 800 μl buffer B (1.25% Triton-X100, 0.05 M Tris / HCl, pH 7.4, 190 mM NaCl) containing protease inhibitors and incubated on ice for 5 minutes. The cell lysate was then centrifuged at 14,000 rpm for 5 minutes in an Eppendorf microcentrifuge. The supernatant was removed and the protein concentration was quantified using a DC protein assay kit obtained from BIORAD according to the manufacturer's instructions (BioRad Laboratories, Hercules, CA) (also Lowry et al., J. Biol. Chem). 193: 265-275, 1951).

  In addition, surface biotinylated whole cell extracts of HL60 and HL60 / AR cells can be strept using immobilized streptavidin (commercially available from Pierce, catalog number # 20347, or Amersham Pharmacia Biotech RPN1231 (Piscataway, NJ)). Avidin purified biotinylated protein was prepared. For this purpose, a 50 μl sample containing 500 μg to 2 mg of protein was diluted to a final 450 μl with buffer C containing protease inhibitors (1: 4 v / v mixture of buffer A and B). The sample was then centrifuged for 1 minute at 14,000 rpm in an Eppendorf microcentrifuge. The supernatant was transferred to a new Eppendorf tube and mixed with 100 μl of streptavidin-linked Sepharose beads. The proteolysate combined with the linked Sepharose beads was incubated overnight at 4 ° C. while rotating. The mixture was centrifuged at 14,000 rpm for 30 seconds in an Eppendorf microcentrifuge. The supernatant was removed and the protein-loaded beads were washed 3 times with buffer C, then washed with 500 mM NaCl dissolved in buffer C and again with buffer C. The protein was eluted from the streptavidin-linked beads after boiling for 10 minutes with SDS sample buffer. The elution was repeated and the eluates were pooled.

  Equivalent proteins from HL60 and HL60 / AR (promyelocytic leukemia) cell surface biotinylated whole cell extracts and streptavidin purified cell surface biotinylated extracts were analyzed by SDS-PAGE according to the method of Laemmli (described above). Degraded and subjected to Western blot analysis, anti-vimentin antibody (mouse monoclonal antibody MS-129-P, NeoMaker) or horseradish peroxidase (HRP) -linked streptavidin (which specifically binds to biotinylated protein, Amersham RPN1231 ) Probe search. For this purpose, gels containing separated proteins were prepared according to the method of Towbin (Towbin, HT, Proc. Natl. Acad. Sci. USA 76: 4350-4354, 1979), Hybond C nitrocellulose membrane (Amersham, Piscataway, NJ). ). The nitrocellulose membrane was then probed with antibodies or HPR-streptavidin. Antibody binding was detected by HRP-conjugated goat anti-mouse secondary antibody (BioRad). Both secondary antibody and HRP-linked streptavidin were detected using an ECL chemiluminescence detection kit commercially available from Pierce. Relative protein levels were detected by exposure to XAR film (Kodak, Rochester, NY) in the dark.

  Anti-vimentin antibodies bound to vimentin protein were expressed at significantly higher levels in surface biotinylated whole cell extracts of multidrug resistant HL60 / AR cells compared to HL60 cells (see FIG. 4A). Vimentin was also overexpressed in streptavidin purified cell surface biotinylated protein of HL60 / AR cells compared to HL60 cells (see FIG. 4B).

  To confirm that cell surface vimentin is truly biotinylated, surface biotinylated whole cell extracts from HL60 and HL / 60AR cells were immunoprecipitated with anti-vimentin antibodies and the immunoprecipitates were The sample was resolved by SDS-PAGE and Western blotted. For this, samples were prepared as described above using protein A sepharose beads instead of streptavidin beads. To elute the protein from the Protein A Sepharose beads, the loaded beads were washed with buffer D (0.03% SDS, 0.05 M Tris-HCl, pH 7.4, 0.1% Triton X containing protease inhibitors as described above. -100, 5 mg / ml BSA fraction V, 150 mM NaCl) and washed once with buffer E (150 mM NaCl, 0.05 M Tris-HCl, pH 7.4). Protein elution from the beads was repeated one or more times, and the eluate was pooled. Proteins were resolved by SDS-PAGE and Western blot with anti-VIM monoclonal antibody or HRP-labeled streptavidin as before.

  The blot was probed with anti-vimentin antibody and streptavidin-HRP. As expected, vimentin was detected in immunoprecipitates in both cell types, but significantly larger amounts of cell surface biotinylated vimentin were found in anti-vimentin immunoprecipitates in HL60 / AR cells compared to HL60 cells. (See FIGS. 4C and 4D). Vimentin has a very sensitive protease reaction site at D90, which may be responsible for the low molecular weight of the band observed when vimentin is immunoprecipitated with anti-vimentin.

  Together, these results suggest that translocation of vimentin across the cell membrane to the cell surface is associated with multidrug resistance in HL60 / AR cells, as well as additional novel synthesis of vimentin.

5.5 Cell surface expression of vimentin in MDR breast cancer To determine whether vimentin is inside or outside the cell membrane, intact breast cancer cells (MCF-7 and MCF-7 / AR) are treated with the amino acid lysine. Whole cell extracts were prepared from both drug-sensitive (MCF-7) and multi-drug resistant (MCF-7 / AR) treated with a permeabilized biotinylation reagent. In addition, anti-vimentin immunoreactive precipitates, including streptavidin-purified biotinylated proteins, were prepared from surface biotinylated whole cell extracts of MCF-7 and MCF-7 / AR cells, and digested with SDS-PAGE. Transferred to nitrocellulose as in Example 4.

  Anti-vimentin antibodies bound to vimentin protein were expressed at significantly higher levels in surface biotinylated whole cell extracts of multidrug resistant MCF-7 / AR cells compared to MCF-7 cells (see FIG. 5A). ). Vimentin was also overexpressed in the streptavidin purified cell surface biotinylated protein of MCF-7 / AR cells compared to MCF-7 cells (see FIG. 5B). In addition, immunoprecipitation blots were probed with anti-vimentin antibody and streptavidin-HRP. As expected, significantly larger amounts of cell surface biotinylated vimentin were present in the anti-vimentin immunoprecipitates of MCF-7 / AR cells compared to MCF-7 cells (see FIGS. 5C and 5D).

  Together, these results suggest that translocation of vimentin across the cell membrane to the cell surface is associated with multidrug resistance in MCF-7 / AR cells, as well as additional novel synthesis of vimentin.

5.6 Cell surface expression of vimentin in MDR promyelocytic leukemia cells
HL60 and HL60 / AR cells were analyzed by cell surface staining and fluorescence activated cell sorter (FACS) analysis to determine the difference in cell surface expression of vimentin in the two cell lines. To this end, indirect immunofluorescence analysis was performed using 1 μg rabbit polyclonal antibody as the primary antibody (CBL46, Cymbus Biotech, Hampshire, UK), followed by goat anti-rabbit IgG FITC-conjugated secondary antibody (Cat # F9887 , Sigma).

  The following indirect staining method was used to prepare cells for FACS analysis.

Cells were washed 3 times with 50 ml PBS, pH 7.4 and 0.1% NaN ₃ and counted.

1 × 10 ⁶ cells per sample were placed in 12 × 75 mm tubes or in deep 96 well plates in 100 μl PBS and 0.1% NaN ₃ . A primary antibody (rabbit polyclonal anti-vimentin antibody) was added and incubated at 37 ° C. for 20 minutes. 3 ml (or 1.25 ml for plates) of PBS pH 7.4 and 0.1% NaN ₃ was added and the mixture was rotated for 5 minutes (1000 rpm / 200xg). The supernatant was discarded and the pellet was resuspended in 100 μl PBS and 0.1% NaN ₃ .

Secondary Ab (FITC-conjugate) was added. This mixture was diluted 1/2 with PBS pH 7.4 and 0.1% NaN ₃ and rotated at 4 ° C. at maximum speed for 30 minutes. Separated from the pellet, 1/10 was used for staining. Incubate at 37 ° C for 20 minutes. 3 ml (or 1.25 ml for plates) PBS pH 7.4 and 0.1% NaN ₃ were added and the mixture was rotated for 5 minutes (1000 rpm / 200 × g). The supernatant was discarded and the addition of PBS and NaN ₃ was repeated. The mixture was again rotated for 5 minutes (1000 rpm / 200xg).

1 μg-2 μg / μl of EMA was added. The mixture was incubated on ice for 10 minutes in white light. 3 ml (or 1.25 ml for plates) of PBS pH 7.4 and 0.1% NaN ₃ was added and the mixture was rotated for 5 minutes (1000 rpm / 200xg). The supernatant was discarded and the pellet was resuspended in 100 μl PBS and 0.1% NaN ₃ . 3 ml (or 1.25 ml for plates) of PBS pH 7.4 and 0.1% NaN ₃ was added and the mixture was rotated for 5 minutes (1000 rpm / 200xg). The supernatant was discarded and the addition of PBS and NaN ₃ was repeated. Resuspended in 500 μl PBS pH 7.2 / 2% paraformaldehyde and stored at 4 ° C. in the dark.

For each sample, 10,000 cells were analyzed with a fluorescence activated cell sorter (Beckman Coulter XL MCL). The fluorescence emission corresponding to the specifically stained cells was determined by subtracting the emission measured on the cells at 530 nm by a pre-excluded rabbit IgG negative control. In some cases, rabbit IgG controls were also removed beforehand for HL60 or HL60 / AR cells. This is to reduce non-specific staining in each cell line. This was done by incubating 1 μg rabbit IgG with 1-2 × 10 ⁶ cells in 50-100 μl PBS for 10 minutes at 37 ° C. The mixture was then spun and the supernatant was used as a negative control or an additional 1 or 2 incubations were performed. The supernatant was incubated with HL60 and HL60 / AR, respectively, and used as a rabbit IgG control for HL60 and HL60 / AR.

  The results showed that vimentin was overexpressed at a 5-fold higher level on the surface of HL60 / AR cells than the vimentin level expressed on the surface of HL60 cells (see FIG. 6).

  As expected, the difference between the vimentin expression level on the HL60 / AR cell surface and the vimentin expression level on the HL60 cell surface was better observed when using the pre-excluded rabbit IgG control. Furthermore, the difference between the vimentin expression level on the surface of HL60 / AR cells and the vimentin expression level on the surface of HL60 cells was better observed with low levels of primary antibodies. Therefore, indirect immunofluorescence analysis was performed using 0.1 μg of polyclonal anti-vimentin as the primary antibody (CBL46, Cymbus Biotech, Hampshire, UK) instead of 1 μg of polyclonal anti-vimentin. When 0.1 μg instead of μg was used as a negative control, the fold difference in vimentin expression levels in HL60 / AR versus HL60 cells was 26 (previously excluded) and 9 (non-excluded), respectively.

  In addition, monoclonal anti-VIM was increased to 10 μg-20 μg-40 μg as the primary antibody (commercially available from NeoMarker, catalog number MS-129-P), followed by anti-mouse IgG-FITC conjugate secondary antibody (Chemicon Indirect immunofluorescence analysis was performed using 1:10 dilution of catalog number # AP181F, commercially available from The fluorescence emission corresponding to the specifically stained cells was determined by subtracting the emission measured in the cells at 530 nm by the mouse IgG1 isomer control. 40 μg of anti-VIM was saturating for HL60 / AR.

  Similarly, experiments were performed with antibodies ranging from 2.5 μg to 40 μg to determine the concentration of anti-vimentin that saturates HL60 cells. The obtained fluorescence intensity did not increase in a concentration-dependent manner and was in the range of 0.2 and 0.9 RFI. This suggests that the cells are already saturated at 2.5 μg. Therefore, 40 μg of anti-vimentin was used to quantify the number of vimentin molecules present on the surface of HL60 and HL60 / AR cells.

  At a saturating concentration (40 μg of monoclonal anti-vimentin), vimentin was expressed at the surface of HL60 / AR cells at a level 37 times higher than the level of vimentin expressed on the surface of HL60 cells. The number of vimentin molecules expressed on the surface of HL60 / HL60 / AR cells was quantified using a flow cytometry quantification kit (Serotec # FCSC814: 498-44298 molecule, Raleigh, NC). The QSC system consists of approximately lymphocyte-sized microbeads that bind to goat anti-mouse antibodies. A set of 5 populations of microbeads carrying various known amounts of goat anti-mouse antibody is provided in the kit. The microbeads are incubated with a saturating amount of the mouse monoclonal antibody of interest. Plot the fluorescence intensity obtained from the microbeads to generate a standard curve of fluorescence intensity against the number of antibody molecules bound to the beads. The signal obtained from the cells (in saturating amount) is then correlated to the number of antibody molecules bound per cell using a standard curve. This value corresponds to the number of antigens present on the cell surface. Under saturation conditions of antibodies against beads and cell lines, HL60 and HL60 / AR were found to carry about 186 and 5052 molecules of vimentin, respectively, when using mouse monoclonal anti-vimentin purchased from NeoMarker (Figure 7). reference). This corresponds to a multi-drug resistant H60 / AR cell line with a 27-fold increase in cell surface vimentin expression compared to the corresponding non-MDR HL60 cell line.

5.7 Characterization of cell surface vimentin expression in MDR lymphoblastic leukemia cells
Molt4 and Molt4 / DOX cells were analyzed by cell surface staining and fluorescence activated cell sorter (FACS) analysis to determine the difference in cell surface expression of vimentin in the two cell lines. To this end, indirect immunofluorescence analysis was performed using 20 μg mouse monoclonal anti-vimentin antibody as the primary antibody (MS-129-P, NeoMarker) and then goat anti-mouse IgG FITC-conjugated secondary antibody (Chemicon, catalog). # AP181F).

  Cells were prepared according to the procedure described above in Example 6. For each sample, 10,000 cells were analyzed on a fluorescence activated cell sorter (Beckman Coulter XL MCL, Fullerton, CA). The fluorescence emission corresponding to specifically stained cells was determined by subtracting the radiation measured on the cells at 530 nm with 20 μg of mouse IgG1 isomer control.

  The results of FACS analysis showed that vimentin is expressed at the surface of Molt4 / AR cells at a level 1.5 times higher than the level of vimentin expressed on the surface of Molt4 cells (see FIG. 8).

5.8 Characterization of cell surface vimentin expression in MDR breast cancer cells
HS574M, MCF-7, MCF-7 / AR, MDA, MDA / AR and MDA / MITO breast cancer cells were analyzed using cell surface staining and fluorescent activated cells to determine the difference in cell surface expression of vimentin in the two cell lines. Analyzed by sorter (FACS) analysis. To this end, indirect immunofluorescence analysis was performed using 5 and / or 20 μg of murine monoclonal anti-vimentin antibody as the primary antibody (MS-129-P, NeoMarker) and then goat anti-mouse IgG FITC-conjugated secondary antibody. (Chemicon, catalog # AP181F, Temecula, Calif.).

  Cells were prepared according to the procedure described above in Example 6. For each sample, 10,000 cells were analyzed with a fluorescence activated cell sorter (Beckman Coulter XL MCL). The fluorescence emission corresponding to specifically stained cells was determined by subtracting the radiation measured on the cells at 530 nm with 5 μg or 20 μg of mouse IgG1 isomer control.

  The results of FACS analysis show that vimentin is not expressed on the surface of HS574M cells but is expressed in MCF-7 / AR cells at a level 1.5 times higher than the level of vimentin expressed on the surface of MCF-7 cells. (See FIG. 9). Similarly, vimentin was expressed in MDA / AR cells at a level 3 to 5 times higher than the level of vimentin expressed on the surface of MDA cells (see FIG. 9). Vimentin was expressed in MDA / MITO cells at a level 3 times higher than the level of vimentin expressed on the surface of MDA cells (see FIG. 10).

  The number of vimentin molecules expressed on the surface of MCF-7 and MCF-7 / AR cells was determined using a flow cytometry quantification kit (Serotec # FCSC814: 498-44298 molecule) in the same manner as described in Examples 5 and 6. And quantified. When using mouse monoclonal anti-vimentin purchased from NeoMarker under antibody saturation conditions on beads and cell lines (50 μg), MCF-7 and MCF-7 / AR carry about 14556 and 22010 molecules of vimentin, respectively. Turned out to be. This corresponds to a 1.5-fold difference (see FIG. 11).

5.9 Characterization of vimentin expression in normal, neoplastic, MDR neoplastic cells Next, normal cells were compared to hematological cancer cells and MDR hematological cancer cells to determine the difference in cell surface expressed vimentin levels. For this, human blood samples (heparin tubes) were obtained from donors and processed within 1 hour. Briefly, red blood cells were separated from white blood cells and plasma on a Ficoll hypaque gradient (Histopaque Sigma 1077-1, St. Louis, MO).

Specifically, half of 15 ml of blood was diluted with phosphate buffer saline pH 7.4 and placed in an equal Ficoll gradient. Cells were separated by centrifugation at 400 xg for 30 minutes at room temperature. The upper layer (plasma) was removed from the plasma / Ficoll boundary layer to 0.5 cm. Mononuclear cells (boundary layer) were placed in a 50 ml Falcon tube. Ficoll was removed and red blood cells (at the bottom of the tube) were collected. All cell types were washed twice with PBS and counted by spinning at 250 xg for 10 minutes at 4 ° C. The cells were then resuspended in PBS to 1 × 10 ⁷ cells / ml and 100 μl (1 × 10 ⁶ cells) were used for flow cytometry (FACS) analysis.

  Cells were stained with primary and secondary antibodies for FACS analysis as described above in Example 6. For each sample, 10,000 cells were analyzed with a fluorescence activated cell sorter (Beckman Coulter XL MCL). The fluorescence emission corresponding to the specifically stained cells was determined by subtracting the radiation measured on the cells at 530 nm with an isomer matched control.

  Each FACS experiment was run with several controls. For cells only to quantify autofluorescence emission; cell plus EMA to identify dead cells under analysis; cell plus secondary antibody (Ab) only to identify non-specific interactions with secondary antibodies; Cell plus isomer-matched antibody (Abs) or appropriate host primary Abs control; cells (mononuclear cells) plus CD45-PC5 mouse monoclonal, phycoerythrin-cyanine 5 complex to define and introduce various leukocyte populations (Immunotech PN IM2653); and erythrocyte plus glycophorin A-FITC mouse monoclonal, Cymbus CBL 409F as a positive control for erythrocytes.

  Red blood cells or white blood cells obtained from normal, healthy donors were stained with polyclonal anti-vimentin antibodies (CBL46, Cymbus Biotech) of 0.25 μg-0.5 μg or 1 μg (3, 6 and 21 donors, respectively) In contrast, normal RBC and WBC were found to express no vimentin on their cell surface, unlike HL60 and HL60 / AR stained with 1 μg of polyclonal anti-vimentin antibody (results not shown) .

  Thus, in addition to showing an increase in MDR neoplastic cells over the same type of non-MDR neoplastic cells, cell surface vimentin expression is also increased in neoplastic cells relative to non-neoplastic cells of the same or similar origin. Thus, vimentin levels on the cell surface can be used in the detection and / or diagnosis of neoplasms as well as MDR neoplasms.

5.10 Vimentin vaccine against MDR blood cancer cells and MDR breast cancer cells
To determine whether full-length vimentin protein expressed on the surface of MDR blood cancer cells is useful as a vaccine to immunize animals against MDR blood cancer cells, purified vimentin protein is mixed with an adjuvant (eg, Freund). Were administered to several groups of mice suffering from blood cancer caused by the presence of blood cancer cells. One such non-limiting blood cancer is acute lymphocyte leukemia.

  To this end, mice were injected with blood cancer cells compatible with the mouse MHC type (or injected into SCID mice). Some of these mice were injected with tumor cells before being immunized with full-length vimentin protein. Others were immunized with purified full-length mouse vimentin protein and then injected with blood cancer cells. Note that purified vimentin protein may be administered with an adjuvant. Appropriate controls were provided for each group of mice (ie, one control group was injected only with purified mouse vimentin protein, and another group was injected with only blood cancer cells).

  Tumors formed in mice were weighed and measured (eg, the number of tumor cells was counted, tumors were removed and weighed, or tumors were measured with calipers). Mice vaccinated with vimentin prior to tumor cell injection were found to have smaller tumors after treatment than those not vaccinated with vimentin prior to tumor cell injection.

  Subsequent experiments evaluated the efficacy of vimentin as an antigen against MDR breast cancer cells (MCF / AR). Briefly, 6-week-old female mice receive 30-250 μg full-length vimentin or control antigen, with or without adjuvant, SC and IP, or hind paw pads 1, 7, 14, 21, Injections on days 28 and 35. Blood samples were collected from the retroorbital venous plexus at various intervals and used for anti-vimentin antibody assays. On day 59, mice were challenged with 1.5 × 104 live MDR mammary carcinoma cells (MCF / AR) administered S.C. on the right flank. Mice were examined twice a week, and the tumor appearance rate was determined from the number of mice bearing tumors. Tumor size was measured with calipers. Survival was measured up to 80 days after adenocarcinoma challenge.

5.11 Vimentin-Targeted Treatment for MDR Hematopoietic Cancer Cells To determine whether it is useful to target-fire therapeutic agents toward cell surface vimentin to treat existing cancer conditions, Tumors were formed by administration to MHC matched mice. The mice were then dosed with vincristine (or another chemotherapeutic agent) at a dose that killed many of the tumor cells inside the mouse, but not necessarily all cells. Mice known to have developed multidrug resistant tumor cells were administered a composition comprising vincristine and a binding agent that specifically binds to mouse vimentin protein. This binder was operably linked to ricin toxin.

  Mice that received the composition had a better prognosis (ie, tumors were smaller and tumor cells were fewer) than mice that received only the binder or only vincristine.

Subsequent experiments evaluated the effect of vimentin-targeted therapy in the treatment of MDR breast cancer cells (MCF / AR). Briefly, athymic nude mice were used for MCF-7 / ADR xenotransplantation. Male mice aged 5-7 weeks and weighing 18-22 g were used. Subcutaneous (sc) injections of 0.5 × 10 ⁶ cells per inoculation were made under the mouse shoulder. After subcutaneous implantation of cells, when the size of the subcutaneous tumor was approximately 5.5 mm, mice were randomly assigned to a treatment group consisting of 4 animals including controls. The group consists of vincristine or doxorubicin alone (4 mg / kg) intraperitoneally (ip) every 2 days; antivimentin alone (100 μg-1 mg / kg); vincristine or doxorubicin plus antivimentin mAb (100 μg- 1 mg / kg) ip. Animal weights were measured every 4 days. Each animal was tagged with an ear tag and individually tracked throughout the experiment. Tumor growth starting on day 1 of dosing was measured and graft volume was monitored every 4 days. Mice were anesthetized and sacrificed when the average tumor weight in the control group exceeded 1 g. Tumor tissue was excised from the mouse and its weight was measured.

5.12 Qualitative analysis of cell surface vimentin expression in MDR breast cancer cells
MCF-7 and MCF-7 / AR cells were analyzed by immunostaining to determine if there was a difference in cell surface expression of vimentin in the two cell lines. To this end, immunofluorescence analysis was performed on cells that were permeabilized or not permeabilized using 0.5 μg of anti-vimentin as the primary antibody (clone V9, NeoMarker MS-129-P). Ethidium monoazide (EMA) was used to stain the nuclei of permeabilized or damaged cells.

  13A and 13B are flowcharts depicting the steps taken to stain cells. Control cells were MCF-7 and MCF-7 / AR stained with 0.5 μg mouse IgG1, and no staining was observed in permeation enhanced or non-permeation enhanced cells. As shown in FIG. 14, vimentin was clearly expressed on the surface of untreated multidrug resistant MCF-7 / AR, whereas MCF-7 showed no staining. When cells were permeabilized, MCF-7 showed negligible staining, whereas MCF-7 / AR showed a rich network of vimentin filaments (Figure 14).

5.13 Quantitative analysis of cell surface vimentin expression in MDR promyeloblastic leukemia cells
For HL60 and HL60 / AR cells, 125 iodine-labeled anti-vimentin was analyzed for vimentin surface exposure by binding directly to the cell surface. To this end, anti-vimentin (NeoMarker, MS-129-PABX), mouse IgG1 (an isomer that matches the negative control, Sigma, M-9035), and anti-CD33 (positive control, Serotec, # MCA1271) Pre-coated iodinated tubes (Pierce, # 28601) were inoculated according to the manufacturer's instructions (average activity obtained: 7.5 μCi / μg).

The cells were washed twice with RPMI 1640 and resuspended as 10 ⁶ cells / 100 μL in the same solvent. Survival was assessed by trypan blue staining and was less than 5%. Cells (1 × 10 ⁶ ) were aliquoted into borosilicate tubes and incubated for 1 hour on ice in the presence of 1 μg of radiolabeled anti-vimentin, IgG1, or anti-CD33. After incubation, the cells were washed 3 times with 1 ml RPMI 1640 and the cell pellet was counted in a gamma counter.

  The results of the intact cell radioimmunoassay are shown in the bar graph of FIG. Numbers represent cell pellet counts per minute minus IgG1 background. Vimentin was expressed on the surface of resistant HL60 / AR cells at three times the level of vimentin expressed on the surface of drug-sensitive HL60 cells.

5.14 Cytotoxic effect of radioiodine labeled anti-vimentin on HL60 and HL60 / AR cells Cells were washed twice with RPMI 1640 and resuspended as 10 ⁶ cells / 100 μL in the same solvent. Survival was assessed by trypan blue staining and was less than 5%. Cells (1 × 10 ⁶ ) are aliquoted into borosilicate tubes and 37 μl in the presence of 5 μg of sterile filter radiolabeled anti-vimentin, IgG1, or anti-CD33 (iodination step is described in Example 5.13). Incubated for 4 hours at 0 ° C (0.5% CO ₂ ). After incubation, cells are washed once with 1 ml RPMI 1640, 10% FBS, 1 mM HEPES, resuspended in 1 ml of the same solvent, and plated in flat-bottom tissue culture 96-well plates at 5,000 per well. It was. The cytotoxicity of the radiolabeled antibody was assessed after 72 hours by assay with MTT. Anti-CD33 was used as a positive control for antibody surface binding, and 120 nM doxorbucin (Doxo) was used as a positive control for cytotoxicity.

  The results of the cytotoxicity assay are shown in the bar graph of FIG. Numbers are expressed as percent survival. Percent survival values in anti-vimentin and anti-CD33 antibody-125I conjugates were normalized to the values obtained with radiolabeled IgG1 (100% survival with negligible background binding). The values obtained with the anticancer drug doxorubicin were normalized to the values obtained in cells that were not drug-treated.

  The results obtained showed that both radiolabeled anti-vimentin had a cytotoxic effect on HL60 and HL60 / AR that both express vimentin on the surface. The slight increase in cytotoxicity observed for HL60 / AR (10%) may be due to the fact that HL60 / AR drug resistant cells express more vimentin on their surface than HL60 drug sensitive cells. Absent.

5.15 Molecular quantification of cell surface vimentin in drug-sensitive and drug-resistant breast and ovarian cancer cells Breast cancer MCF-7, MCF-7 / AR, MDA, MDA / mito, and ovarian cancer SKOV3, SKOV / T320 (resistant to taxol) 2008 And for 2008 / T320 (taxol resistant) cells, 125 iodine-labeled anti-vimentin was analyzed for vimentin surface exposure by binding directly to the cell surface. For this purpose, anti-vimentin (NeoMarker, MS-129-PABX) and mouse IgG1 (isomer matching the negative control, Sigma, M-9035) and anti-CD33 (positive control, Serotec, # MCA1271) IODOGEN® pre-coated iodinated tubes (Pierce, # 28601) were iodinated using the manufacturer's instructions (average activity obtained: 7.5 μCi / μg).

Cells were seeded in 96 Stripwell® (Costar, # 9102) plates at 20,000 per well. After overnight growth in complete medium (37 ° C, 0.5% CO ₂ ), the wells are gently washed with 100 μl medium containing 1% FBS, and 1% FBS and 0.1% sodium azide and Incubated overnight at 37 ° C. in 100 μl culture medium containing 100 ng of radiolabeled anti-vimentin (or IgG1) (37 ° C., 0.5% CO ₂ ). Prior to incubation, mortality was checked by trypan blue staining, which was usually less than 1%. After the incubation, the culture solution was discarded, and the wells were washed twice with 350 μl of the culture solution containing 1% FBS, and individually counted with a gamma counter.

  Binding experiments were performed on MDA / mito cells using increasing amounts of radiolabeled anti-bimethidine in an intact cell radioimmunoassay. Scatchard analysis was used to calculate the apparent dissociation constant (Kd) and the number of antibody molecules per cell. The average number of bound anti-vimentin molecules per cell was obtained from the average of 5 independent Scatchard results and is listed in the table of FIG. 17A. FIG. 17B shows the Scatchard plot of the first experiment described in the table of FIG. 17A.

The average number of vimentin epitopes present on the MDA / mito cell surface was 2.49 × 10 ⁶ per cell and the average Kd was 5.7 nM. Kd was determined for MDA cells in a separate experiment and was 9.3 nM (see table in FIG. 17C). This was equivalent to the Kd measured for MDA / mito cells.

The number of vimentin epitopes present on MCF-7, MICF-7 / AR, MDA, SKOV3, SKOV / T320, 2008 and 2008 / T320, the fold difference in surface exposure between these cells and MDA / mito Calculated from This fold was determined by examining the same number (approximately 20,000 cells) of MDA / mito cells and the aforementioned cells using the aforementioned intact cell radioimmunoassay with 100 ng radiolabeled anti-vimentin. The latter number of surface vimentin epitopes represents the average of multiple independent experiments (n = 2 to 5). These numbers are listed in the table of FIG. 17C, although MCF-7 / AR, MDA, MDA / mito, SKOV3 and SKOV / T320 express a large number of vimentins on their surface (maximum MDA / mito 2.5x10 ⁶ ), while the numbers of MCF-7, 2008 and 2008 / T320 are relatively low ("2.5.E + 06" represents an index with a base of 10, ie 2.5x10 ⁶ ) . The fold difference in vimentin surface exposure between sensitive and resistant cells is given as R / S, and for MCF-7 / AR, MDA / mito, SKOV / T320 and 2008 / T320, 41.2, 4, 1.6 and 2.

5.16 Induction of vimentin cell surface exposure in breast and ovarian cancer cells According to the findings reported in Example 5.15 above, various breast and ovarian cancer cells selected with doxorubicin, mitoxantrone, or taxol were compared to their sensitive counterparts. Thus, it has been shown to increase vimentin surface exposure, but given this point, it is of interest to determine whether these agents induce vimentin surface exposure in MDA and SKOV3 cell lines. For this, 20,000 cells were plated on 96 Stripwell® (Costar, # 9102) plates. After 5 hours, the culture solution was removed, replaced with a culture solution containing various drugs (as shown in FIG. 18), and incubated at 37 ° C. for 12 to 16 hours (0.5% CO ₂ ). After incubation, the wells are gently washed with 100 μl culture medium containing 1% FBS, 1% FBS and 0.1% sodium azide and 100 ng radiolabeled anti-vimentin (or IgG1) Incubated for 1 hour at 37 ° C. in 0.5 μl (0.5% CO ₂ ). Prior to incubation, mortality was checked by trypan blue staining, which was usually less than 1%. After the incubation, the culture solution was discarded, and the wells were washed twice with 350 μl of the culture solution containing 1% FBS, and individually counted with a gamma counter. FIG. 18 represents the surface exposure of vimentin when MDA cells were incubated with 1 or 10 μM taxol, 1 or 10 μM doxorubicin, or 0.1 or 1 μM mitoxantrone. Values were corrected for nonspecific binding by IgG1. Although slight induction of vimentin surface exposure was observed for all drugs, mitoxantrone showed the most prominent effect. A similar but less intense effect was observed when SKOV3 cells were treated with 1 μM taxol. These findings are important when considering immunotherapy combined with drugs.

5.17 Uptake of ¹²⁵ I-labeled anti-vimentin by MCF-7 / AR, MDA, MDA / AR, and MDA / mito cells Breast cancer cells suspended in liquid (see Figure 19A) or cells attached to 96-well plates In FIG. 19B, cell surface vimentin uptake was measured. FIG. 19A was obtained as follows. 10 Subculture ⁶ cells in dissociation buffer (Gibco, # 13150-016), then transfer to borosilicate tubes, wash with PBS, 200 μl alpha-MEM, 1 μg radiolabeled anti-vimentin Resuspended in 3% BSA containing or as a positive control in 1 μg anti-mucin 1 or as a background control in 1 μg IgG1. Mucin 1 (CD227) is a highly glycosylated protein that is widely present in many human tissues, is often produced at high levels in tumor cells, and exhibits an unusual glycosylation pattern. Mouse antibodies are used in clinical trials for the purpose of treating such cancers and are commercially available (eg, Fitzgerald Industries, Inc. Concord, Mass.). After 1 hour incubation at 4 ° C., the cells were washed twice and incubated for an additional 4 hours at 4 ° C. or 37 ° C. According to trypan blue staining of the cells before incubation with radiolabeled IgG, the cell viability was 100%. After incubation, the cells were washed and radiolabel was quantified by counting the samples with a gamma counter. The cells were then stripped (striped) with 50 mM L-Gly, pH 2.8, 150 mM NaCl for 10 minutes at RT, washed and counted for residual activity with a gamma counter. The graph shown in Figure 19A shows the percentage of surface-related radiolabels in MCF-7 / AR (removed with L-Gly) and captured (remaining after stripping) at 4 ° C and 37 ° C. Represents. The percent uptake was derived from the difference in uptake measured at 4 ° C. and 37 ° C., 38.2% for vimentin and 12.5% for mucin 1.

These results indicate that anti-vimentin antibodies attached to radionuclide therapeutic agents (or diagnostic probes) bind to surface vimentin present on the surface of multidrug resistant breast epithelial neoplastic cells and are actively taken up in a temperature-dependent manner. It was done. This temperature dependence suggests that the antibody- ^125I complex is actively taken up by endocytosis. Whatever the mechanism of ingestion, the result is that the anti-vimentin antibody recognizes MDR neoplastic cells and puts a therapeutic agent (or diagnostic agent) in that cell for the treatment (or diagnostic detection) of that cell Indicates that it can be transported.

  For FIG. 19B, cells were prepared as in the intact cell radioimmunoassay (20,000 cells per well). Briefly, after overnight culture, wells were washed and preincubated for 1 hour at 4 ° C. with 100 ng radiolabeled anti-vimentin or 100 μl culture medium plus 1% FBS containing IgG1 as background control. Prior to the previous incubation, mortality was checked by trypan blue staining and was less than 1%. After the previous incubation, the culture medium was discarded and the wells were washed with 350 μL culture medium containing 1% FBS and further incubated at 4 ° C. or 47 ° C. for 4 hours. After incubation, the wells were washed as before and the cells were stripped and washed with 50 mM L-Gly, pH 2.8, 150 mM NaCl for 10 min at RT. Next, the peeled material and well activity were counted with a gamma counter. The graph shown in Figure 19B shows the percentage of surface-related cpm (stripped with L-Gly) in MCF-7 / AR, MDA, MDA / AR, MDA / mito at 4 ° C and 37 ° C. (Remaining after stripping). The percent uptake of vimentin is derived from the difference in uptake measured at 4 ° C and 37 ° C, with MCF-7 / AR, MDA, MDA / AR and MDA / mito at 11.3, 25.3, 24.8 and 8.9%, respectively. there were.

This result further supports the utility of anti-vimentin antibodies in targeting and incorporating therapeutic and diagnostic agents linked to both neoplastic and MDR neoplastic cells.
5. Equivalents Those skilled in the art will recognize, and be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically described herein. I will. Such equivalents are intended to be encompassed in the scope of the claims recited in the introductory text.

FIG. 1A is a photographic record of a silver stained 2-D gel developing a whole cell extract of MCF-7 cells, indicating the presence of vimentin in a 53 kDa spot. FIG. 1B is a photographic record of a silver stained 2-D gel developing a whole cell extract of multidrug resistant MCF-7 / AR cells, showing the presence of vimentin at the 53 kDa spot. FIG. 2A is a recording of an anti-vimentin immunoblot of a whole cell extract of MCF-7 cells developed on a 2-D gel and transferred to nitrocellulose. FIG. 2B is a recording of an anti-vimentin immunoblot of a whole cell extract of MCF-7AR cells developed on a 2-D gel and transferred to nitrocellulose. FIG. 3A is a streptavidin-HRP blot record of a surface biotinylated whole cell extract of CEM cells developed on a 2-D gel and transferred to nitrocellulose. FIG. 3B is a recording of a streptavidin-HRP blot of surface biotinylated whole cell extracts of multidrug resistant CEM / VLB cells developed on 2-D gels and transferred to nitrocellulose. FIG. 4A is a photographic record of Western blot analysis with anti-vimentin antibody of 10% SDS-polyacrylamide gel developed biotinylated whole cell extracts prepared from HL60 and HL60 / AR cells. Figure 4B is a photograph of a Western blot analysis with anti-vimentin antibody of a 10% SDS-polyacrylamide gel-developed biotinylated whole cell extract prepared from a streptavidin purified extract prepared from a surface biotinylated whole cell extract. It is a shooting record. FIG. 4C is a photographic recording of a 10% SDS-polyacrylamide gel-developed biotinylated whole cell extract prepared from a surface biotinylated whole cell extract anti-vimentin immunoprecipitate and Western blot analysis with anti-vimentin antibody. is there. FIG. 4D is a photographic recording of a Western blot analysis with a streptavidin-HRP probe of a 10% SDS-polyacrylamide gel-developed biotinylated whole cell extract prepared from an anti-vimentin immunoprecipitate of a biotinylated whole cell extract. It is. FIG. 5A is a photographic record of Western blot analysis with anti-vimentin antibody of 10% SDS-polyacrylamide gel developed biotinylated whole cell extracts prepared from MCF-7 and MCF-7 / AR cells. FIG. 5B is a photograph of a Western blot analysis with anti-vimentin antibody of a 10% SDS-polyacrylamide gel-developed biotinylated whole cell extract prepared from a purified streptavidin extract prepared from a surface biotinylated whole cell extract. It is a shooting record. Figure 5C is a photographic recording of a 10% SDS-polyacrylamide gel-developed biotinylated whole cell extract prepared from a surface biotinylated whole cell extract anti-vimentin immunoprecipitate and Western blot analysis with anti-vimentin antibody. is there. FIG. 5D is a photograph of a Western blot analysis with a HRP-streptavidin probe of a 10% SDS-polyacrylamide gel-developed biotinylated whole cell extract prepared from an anti-vimentin immunoprecipitate of a surface biotinylated whole cell extract It is a record. FIG. 6 is a graphical representation of FACS analysis results of vimentin surface expression in HL60 (white bar) and HL60 / AR (gray bar) cell lines with polyclonal anti-vimentin antibodies. FIG. 7 is a graphical representation of FACS analysis results of vimentin surface expression in HL60 (white bar) and HL60 / AR (gray bar) cell lines with saturating amounts of monoclonal anti-vimentin antibodies. FIG. 8 is a graphical representation of the results of FACS analysis of vimentin surface expression in Molt4 (white bar) and Molt4 / DOX (gray bar) cell lines with monoclonal anti-vimentin antibodies. FIG. 9 is a graphical representation of FACS analysis results of vimentin surface expression in a breast cancer drug sensitivity and drug resistance model cell line by monoclonal anti-vimentin antibody. FIG. 10 is a graphical representation of the results of FACS analysis of vimentin surface expression in MDA drug sensitive and MDA / MITO drug resistant cell lines with monoclonal anti-vimentin antibodies. FIG. 11 is a graphical representation of the results of quantitative FACS analysis of vimentin surface expression in MCF-7 and MCF-7 / AR cell lines. FIG. 12A is a schematic diagram of the polypeptide sequence of human vimentin corresponding to GenBank accession number P08670 (SEQ ID NO: 1). FIG. 12B is a schematic diagram of the nucleotide sequence of the nucleic acid sequence encoding human vimentin corresponding to GenBank accession number X56134 (SEQ ID NO: 2). The start and stop codons of the vimentin protein open reading frame are underlined. FIG. 13A is a flowchart showing the procedure of immunohistochemical staining of adherent tumor cells that are not penetration enhanced. FIG. 13B is a flowchart showing the procedure of immunohistochemical staining of adherent tumor cells that are not penetration enhanced. FIG. 14 shows permeation-enhanced and non-permeation-enhanced MCF-7 and MCF-7 / AR cells immunostained with anti-vimentin antibody (clone V9, NeoMarker MS-129-P) using the procedure described in FIGS. 13A and B It is a photography record. Mouse IgG1 was used as a negative isotype matching control and showed no staining (not shown). Only MCF-7 / AR showed vimentin exposed on the surface. FIG. 15 is a graphical representation of untreated cell radioimmunoassay results for vimentin surface exposure in HL60 drug sensitivity and HL60 / AR drug resistant cell lines. CD33 was used as a positive control for surface exposure. FIG. 16 is a graphical representation of cytotoxicity results of radiolabeled anti-vimentin against HL60 and HL60 / AR cells. CD33 was used as a positive control for surface exposure. FIG. 17A is a table showing the results of five independent Scatchard analyzes for vimentin surface exposure in MDA / mito cells. The R ² value represents the quality of the non-linear fit used to determine Kd and Bmax. Analysis was performed with Prizm GraphPad. FIG. 17B is a display of the Scatchard analysis performed in the first experiment of Table 17A. FIG. 17C is a table showing the number of vimentin molecules present on the surface of various breast and ovarian cancer cell lines. R / S indicates the fold overexpression of surface vimentin when compared to sensitive and resistant corresponding cells. FIG. 18 is a graphical representation of the results of an intact cell radioimmunoassay examining the induction of vimentin surface expression by doxorubicin, taxol, and mitoxantrone. FIG. 19A is a graphical representation of vimentin uptake evaluation results in MCF-7 / AR cells kept in suspension during the experiment. FIG. 19B is a graphical representation of the evaluation results of vimentin uptake in adherent MCF-7 / AR, MDA, MDA / AR, and MDA / mito cells.

Claims (108)

A method of detecting multidrug resistance or potential multidrug resistance in a test neoplastic cell, comprising:
a) measuring the level of cell surface expressed vimentin protein in test neoplastic cells of any origin or cell type; and
b) comparing the cell surface expressed vimentin protein level of the test neoplastic cell with the cell surface expressed vimentin level in non-resistant neoplastic cells of the same origin or cell type;
If the level of cell surface expressed vimentin in the test neoplastic cell is higher than the vimentin level of cell surface expression in non-resistant neoplastic cells of the same arbitrary origin or cell type, the test neoplastic cell is multidrug resistant or A method characterized by having the possibility of becoming drug-resistant.   Measuring cell surface expressed vimentin levels in said test neoplastic cells comprises isolating a plasma membrane fraction from the cells and measuring vimentin levels in the plasma membrane fraction. The method of claim 1, wherein   Measuring the cell surface expressed vimentin level in the test neoplastic cell comprises contacting the cell with an anti-vimentin antibody and measuring the level of antibody bound to the cell surface vimentin. The method of claim 1.   4. The method of claim 3, wherein measuring the level of antibody bound to the cell surface vimentin is by immunofluorescence emission.   4. The method of claim 3, wherein measuring the level of antibody bound to the cell surface vimentin is by a radioactive label.   2. The method of claim 1, wherein the test neoplastic cell is selected from the group consisting of promyelocytic leukemia cells, T lymphoblastoid cells, breast epithelial cells, and ovarian cells.   The non-resistant neoplastic cells are derived from a drug sensitive cell line selected from the group consisting of HL60, NB4, CEM, HSB2, Molt4, MCF-7, MDA, SKOV-3, and 2008. 1 way.   The test neoplastic cell is selected from the group consisting of lymphoma cells, melanoma cells, sarcoma cells, leukemia cells, retinoblastoma cells, hepatoma cells, myeloma cells, glioma cells, mesothelioma cells, and epithelial cancer cells. The method of claim 1, characterized by:   The test neoplastic cell is derived from a tissue selected from the group consisting of blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, digestive tract, eye, breast, prostate, and ovary. The method of claim 1, wherein A method for detecting multidrug resistant cells in a patient comprising:
(a) administering to the patient a vimentin binding agent operably linked to a detectable label; and
(b) detecting a label operably linked to the vimentin binding agent, wherein the vimentin binding agent specifically binds to cell surface expressed vimentin present on the multidrug resistant cells of the patient. .   11. The method of claim 10, wherein the vimentin binding agent is an antibody or fragment thereof.   The vimentin binding agent is selected from the group consisting of modified LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein and peripherin, fimbrin, RhoA-binding kinase alpha, and protein phosphatase 2A. 11. The method of claim 10, wherein   11. The method of claim 10, wherein the vimentin binding agent is selected from the group consisting of a natural ligand, a synthetic small molecule, a drug, a nucleic acid, a peptide, a protein, and an antibody.   The detectable label comprises a fluorescent dye, a chemical dye, a radioactive compound, a chemiluminescent compound, a magnetic compound, a paramagnetic compound, a parent magnetic compound, an enzyme that produces a colored product, an enzyme that produces a chemiluminescent product, and a magnetic product. 11. The method of claim 10, wherein the method is selected from the group consisting of enzymes that produce   15. The method of claim 14, wherein the multidrug resistant cell is a neoplastic cell.   The neoplastic cells include breast cancer cells, ovarian cancer cells, myeloma cells, lymphoma cells, melanoma cells, sarcoma cells, leukemia cells, retinoblastoma cells, hepatoma cells, glioma cells, mesothelioma cells, and epithelial cancer cells. 16. The method of claim 15, wherein the method is selected from the group consisting of:   16. The method of claim 15, wherein the neoplastic cells are selected from the group consisting of promyelocytic leukemia cells, T lymphoblastoid cells, breast epithelial cells, and ovarian cells.   11. The method of claim 10, wherein the patient is a human.   19. The method of claim 18, wherein the patient is suffering from a disease or disorder caused by the presence of multidrug resistant cells. A kit for diagnosing or detecting multidrug resistance in a test neoplastic cell,
a) a first probe for detection of vimentin, and
b) A kit comprising a second probe for detecting a multidrug resistance marker selected from the group consisting of nucleophosmin and HSC70. A kit for diagnosing or detecting multidrug resistance in a test neoplastic cell,
a) a first probe for detection of vimentin, and
b) A kit comprising a second probe for detecting a marker selected from the group consisting of MDR1, MDR3, MRP1, MRP5, and LRP.   The kit according to claim 20 or 21, wherein the probe for vimentin detection is an anti-vimentin antibody.   The probe for detecting vimentin is a vimentin ligand selected from the group consisting of LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein and peripherin, fimbrin, RhoA-binding kinase alpha, and protein phosphatase 2A. 22. Kit according to claim 20 or 21, characterized in that   21. The kit of claim 20, wherein the second probe is selected from the group consisting of a nucleophosmin antibody and an HSC70 antibody.   21. The kit of claim 20, wherein the second probe is selected from the group consisting of a nucleophosmin ligand and an HSC70 ligand.   The kit according to claim 20 or 21, wherein the first probe detects vimentin present on the surface of a test neoplastic cell.   The kit according to claim 20 or 21, wherein the second probe detects a marker present on the surface of a test neoplastic cell.   22. The kit of claim 21, wherein the second probe is selected from the group consisting of MDR1 antibody, MDR3 antibody, MRP1 antibody, MRP3 antibody, and LRP antibody.   A probe for detecting cell surface vimentin in a patient, comprising a vimentin binding component and a detectable label for detection in the body.   30. The probe according to claim 29, wherein the vimentin-binding component is an antibody.   30. The probe according to claim 29, wherein the detectable label is technetium.   A cell surface vimentin targeting agent for treating or preventing a multidrug resistant neoplasm comprising a vimentin binding component and a therapeutic component, the vimentin binding component targeting the therapeutic component towards the multidrug resistant neoplasm, A vimentin targeting agent, characterized in that it treats multidrug resistant neoplasms.   33. The agent of claim 32, wherein the vimentin binding component is an anti-vimentin antibody.   The vimentin binding component is selected from the group consisting of LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein, peripherin, fimbrin, RhoA-binding kinase alpha, and protein phosphatase 2A. 33. The medicament of claim 32.   33. The agent of claim 32, wherein the vimentin binding component is selected from the group consisting of a natural ligand, a synthetic small molecule, a drug, a nucleic acid, a peptide, a protein, an antibody, and a vimentin binding fragment thereof.   The therapeutic ingredients are actinomycin, adriamycin, altretamine, asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxin epoxorpoxe , Etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, lomustine, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoteline, mitoxantrone, paclitaxol pentotaxol The topoteka , Vinblastine, vincristine, and characterized in that it is selected from the group consisting of vinorelbine, drug claim 32.   33. The agent of claim 32, wherein the therapeutic component is present as a liposomal formulation.   33. The agent of claim 32, wherein the therapeutic component is a radioisotope. The radioactive ^{isotope, 90 Y, 125 I, 131} I, 211 At, and characterized in that it is selected from the group consisting of ²¹³ Bi, agents of claim 38.   33. The agent of claim 32, wherein the therapeutic component is a toxin capable of killing or inducing killing of targeted multi-drug resistant neoplastic cells.   41. The agent of claim 40, wherein the toxin is selected from the group consisting of Pseudomonas exotoxin, diphtheria toxin, plant ricin toxin, plant abrin toxin, plant saponin toxin, plant gelonin toxin, and pokeweed antiviral protein. .   42. The vimentin binding component binds to a target cell surface and the therapeutic element is taken up to prevent cell growth, compromise cell survival, or kill the cell. Any drug.   A vaccine for treating or preventing a multi-drug resistant neoplasm comprising a vimentin polypeptide, or a partial sequence of the vimentin polypeptide, and at least one pharmaceutically acceptable vaccine component.   44. The vaccine of claim 43, wherein the vimentin polypeptide or polypeptide subsequence is the human vimentin polypeptide sequence of SEQ ID NO: 1.   44. The vaccine of claim 43, wherein the vimentin polypeptide subsequence is at least 8 amino acids long.   46. The vaccine of claim 45, wherein the vimentin polypeptide subsequence comprises a hapten.   44. The vaccine of claim 43, wherein the pharmaceutically acceptable vaccine component is an adjuvant.   The adjuvant is aluminum hydroxide, aluminum phosphate, calcium phosphate, oil suspension, bacterial product, whole inactivated bacteria, endotoxin, cholesterol, fatty acid, aliphatic amine, paraffin-like compound, vegetable oil, monophosphoryl lipid 48. The vaccine of claim 47, wherein the vaccine is selected from the group consisting of A, saponin, and squalene.   42. A method of treating or preventing a multidrug resistant neoplasm in a subject comprising administering a cell surface vimentin targeted therapeutic according to any one of claims 32 to 41.   The neoplasm is selected from the group consisting of breast cancer, ovarian cancer, myeloma, lymphoma, melanoma, sarcoma, leukemia, retinoblastoma, hepatoma, glioma, mesothelioma, and epithelial cancer, 49. The method according to item 49.   50. The method of claim 49, wherein the subject is a human patient.   52. The method of claim 51, wherein the human patient suffers from a disease or disorder caused by the presence of multidrug resistant cells.   The neoplasm is derived from a tissue selected from the group consisting of blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, digestive tract, eye, breast, prostate, and ovary. 50. The method of claim 49.   49. A method of treating or preventing a multidrug resistant neoplasm in a subject comprising administering a vimentin vaccine according to any one of claims 43 to 48.   The neoplasm is selected from the group consisting of breast cancer, ovarian cancer, myeloma, lymphoma, melanoma, sarcoma, leukemia, retinoblastoma, hepatoma, glioma, mesothelioma, and epithelial cancer, 54. The method according to item 54.   55. The method of claim 54, wherein the subject is a human patient.   57. The method of claim 56, wherein the human patient suffers from a disease or disorder caused by the presence of multidrug resistant cells.   The neoplasm is derived from a tissue selected from the group consisting of blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, digestive tract, eye, breast, prostate, and ovary. 55. The method of claim 54. A method for detecting whether a test cell is a neoplasm,
a) measuring the level of cell surface expressed vimentin protein in a test cell of any origin or cell type; and
b) comparing the cell surface expressed vimentin protein level of the test cell with the cell surface expressed vimentin level in non-neoplastic cells of the same origin or cell type;
A method wherein a test cell is neoplastic if the level of cell surface expressed vimentin in the test cell is higher than the cell surface expressed vimentin level of a non-neoplastic cell of the same origin or cell type.   Measuring the vimentin level of cell surface expression in the test cell comprises isolating a plasma membrane fraction from the cell and measuring the vimentin level in the plasma membrane fraction, 60. The method of claim 59.   Measuring the vimentin level of cell surface expression in the test cell comprises contacting the cell with an anti-vimentin antibody and measuring the level of antibody bound to the cell surface vimentin. 60. The method of claim 59.   62. The method of claim 61, wherein measuring the level of antibody bound to the cell surface vimentin is by immunofluorescent radiation.   62. The method of claim 61, wherein measuring the level of antibody bound to the cell surface vimentin is by a radioactive label.   The test cell is derived from a tissue selected from the group consisting of blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, digestive tract, eye, breast, prostate, and ovary. 60. The method of claim 59.   The non-neoplastic cell is derived from a tissue selected from the group consisting of blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, digestive tract, eye, breast, prostate, and ovary. 60. The method of claim 59. A method for detecting neoplastic cells in a patient comprising:
(a) administering to the patient a vimentin binding agent operably linked to a detectable label; and
(b) detecting a label operably linked to a vimentin binding agent, wherein the vimentin binding agent specifically binds to cell surface expressed vimentin present on a patient's neoplastic cells.   68. The method of claim 66, wherein the vimentin binding agent is an antibody or fragment thereof.   The vimentin binding agent is selected from the group consisting of modified LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein and peripherin, fimbrin, RhoA-binding kinase alpha, and protein phosphatase 2A. 68. The method of claim 66.   68. The method of claim 66, wherein the vimentin binding agent is selected from the group consisting of natural ligands, synthetic small molecules, drugs, nucleic acids, peptides, proteins, antibodies, and fragments thereof.   The detectable label may be a fluorescent dye, a chemical dye, a radioactive compound, a chemiluminescent compound, a magnetic compound, a paramagnetic compound, a parent magnetic compound, an enzyme that produces a colored product, an enzyme that produces a chemiluminescent product, or a magnetic product. 68. The method of claim 66, wherein the method is selected from the group consisting of enzymes that produce.   The neoplastic cells include breast cancer cells, ovarian cancer cells, myeloma cells, lymphoma cells, melanoma cells, sarcoma cells, leukemia cells, retinoblastoma cells, hepatoma cells, glioma cells, mesothelioma cells, and epithelial cancer cells. 68. The method of claim 66, wherein the method is selected from the group consisting of:   68. The method of claim 66, wherein the neoplastic cell is selected from the group consisting of promyelocytic leukemia cells, T lymphoblastoid cells, breast epithelial cells, and ovarian cells.   68. The method of claim 66, wherein the patient is a human.   74. The method of claim 73, wherein the patient suffers from a disease or disorder caused by the presence of neoplastic cells. A kit for diagnosing or detecting neoplasia,
a) a first probe for detection of vimentin, and
b) A kit comprising a second probe for detecting a neoplastic marker selected from the group consisting of nucleophosmin and HSC70.   The kit according to claim 75, wherein the probe for detecting vimentin is an anti-vimentin antibody or a binding fragment thereof.   The probe for detecting vimentin is a vimentin ligand selected from the group consisting of LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein and peripherin, fimbrin, RhoA-binding kinase alpha, and protein phosphatase 2A. 76. The kit of claim 75, wherein:   76. The kit of claim 75, wherein the second probe is selected from the group consisting of a nucleophosmin antibody and an HSC70 antibody.   76. The kit of claim 75, wherein the second probe is selected from the group consisting of a nucleophosmin ligand and an HSC70 ligand.   The kit according to claim 75, wherein the first probe detects vimentin present on the surface of the test cell when it is a neoplasm.   The kit according to claim 75, wherein the second probe detects a marker present on a surface of the test cell when the test cell is a neoplasm.   A cell surface vimentin targeting agent for treating cancerous neoplastic cell proliferation, comprising a vimentin binding component and a therapeutic component, wherein the vimentin binding component targets the therapeutic component towards neoplastic cell proliferation and thereby the cancer Vimentin targeting agent characterized by treating   83. The agent of claim 82, wherein the vimentin binding component is an anti-vimentin antibody.   The vimentin binding component is selected from the group consisting of LDL, NLK1 protein, vimentin, desmin, glial fibrillary acidic protein and peripherin, fimbrin, RhoA-binding kinase alpha, and protein phosphatase 2A. 83. The medicament of claim 82.   85. The agent of claim 82, wherein the vimentin binding component is selected from the group consisting of a natural ligand, a synthetic small molecule, a drug, a nucleic acid, a peptide, a protein, an antibody, and a vimentin binding fragment thereof.   The therapeutic ingredients are actinomycin, adriamycin, altretamine, asparaginase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxin epoxorpoxe , Etoposide, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, lomustine, mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoteline, mitoxantrone, paclitaxol pentotaxol The topoteka , Vinblastine, vincristine, and vinorelbine and combinations thereof, characterized in that it is selected from the group consisting of, a medicament according to claim 82.   83. The agent of claim 82, wherein the therapeutic component is present as a liposomal formulation.   83. The agent of claim 82, wherein the therapeutic component is a radioisotope. The radioactive ^{isotope, 90 Y, 111 In, 125} I, 131 I, 211 At, and ²¹³ characterized in that it is selected from the group consisting of Bi, according to claim 88 drugs.   83. The agent of claim 82, wherein the therapeutic component is a toxin capable of killing or inducing killing of labeled neoplastic cells.   95. The agent of claim 90, wherein the toxin is selected from the group consisting of Pseudomonas exotoxin, diphtheria toxin, plant ricin toxin, plant abrin toxin, plant saponin toxin, plant gelonin toxin, and pokeweed antiviral protein. .   92. Any of claims 82 to 91, wherein the vimentin binding component binds to a target cell surface and a therapeutic element is taken up to inhibit cell growth, compromise cell survival, or kill the cell. Some drugs.   A vaccine for treating or preventing a neoplasm comprising a vimentin polypeptide, or a partial sequence of the vimentin polypeptide, and at least one pharmaceutically acceptable vaccine component.   94. The vaccine of claim 93, wherein the vimentin polypeptide or polypeptide subsequence is the human vimentin polypeptide sequence of SEQ ID NO: 1.   94. The vaccine of claim 93, wherein the vimentin polypeptide subsequence is at least 8 amino acids long.   96. The vaccine of claim 95, wherein the vimentin polypeptide subsequence comprises a hapten.   94. The vaccine of claim 93, wherein the pharmaceutically acceptable vaccine component is an adjuvant.   The adjuvant is aluminum hydroxide, aluminum phosphate, calcium phosphate, oil suspension, bacterial product, whole inactivated bacteria, endotoxin, cholesterol, fatty acid, aliphatic amine, paraffin-like compound, vegetable oil, monophosphoryl lipid 98. The vaccine of claim 97, wherein said vaccine is selected from the group consisting of A, saponin, and squalene.   92. A method of treating or preventing a neoplasm in a subject, comprising administering a cell surface vimentin targeted therapeutic agent according to any one of claims 82 to 91.   The neoplasm is selected from the group consisting of breast cancer, ovarian cancer, myeloma, lymphoma, melanoma, sarcoma, leukemia, retinoblastoma, hepatoma, glioma, mesothelioma, and epithelial cancer, The method of paragraph 99.   99. The method of claim 99, wherein the subject is a human patient.   102. The method of claim 101, wherein the human patient suffers from a disease or disorder caused by the presence of multidrug resistant cells.   The neoplasm is derived from a tissue selected from the group consisting of blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, digestive tract, eye, breast, prostate, and ovary. 99. The method of claim 99, wherein:   99. A method of treating or preventing a neoplasm in a subject comprising administering a vimentin vaccine according to any one of claims 93 to 98.   The neoplasm is selected from the group consisting of breast cancer, ovarian cancer, myeloma, lymphoma, melanoma, sarcoma, leukemia, retinoblastoma, hepatoma, glioma, mesothelioma, and epithelial cancer, Item 104. The method according to Item 104.   105. The method of claim 104, wherein the subject is a human patient.   107. The method of claim 106, wherein the human patient suffers from a disease or disorder caused by the presence of neoplastic cells.   The neoplasm is derived from a tissue selected from the group consisting of blood, bone marrow, spleen, lymph node, liver, thymus, kidney, brain, skin, digestive tract, eye, breast, prostate, and ovary. 105. The method of claim 104.

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