Methods of forecasting and prediction of cancer and control on therapy of cancer

FIELD: medicine.

SUBSTANCE: immunological detection and quantitative analysis of sequential changes in protein levels of VEGF-165 in obtained patient's samples taken in course of time are conducted, where increasing levels of the protein VEGF-165 in course of time indicate progression of disease or adverse reaction to the therapy, and where decreasing levels of protein VEGF-165 in course of time indicate remission of the disease or a positive reaction to the therapy.

EFFECT: method enables to conduct noninvasive analysis of levels of circulating VEGF-165 which serves as a valuable prognostic indicator of disease outcome.

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The technical field

This invention relates to biomarkers and biomarkers for prognosis and prediction of cancer, as well as the use of biomarkers to monitor the efficacy of anticancer therapy. More specifically this invention relates to the use of VEGF-165 as a biomarker for multikinase inhibitors.

Prior art

Receptors of vascular endothelial growth factor (VEGFR) and their ligands, vascular endothelial growth factors (VEGF) play a critical role in the migration and proliferation of endothelial cells. System VEGFR/VEGF includes three receptor (VEGFR-1, VEGFR-2 and VEGFR-3) and four ligand (VEGF-A, b, C, D and e, and placental growth factor). VEGF-A is additionally consists of four isoforms of VEGF-121, VEGF-165, VEGF-185 and VEGF-204, obtained by alternative transcription of the gene VEGF-A. Receptors are proteins associated with the plasma membrane (plasma membrane-spanning proteins with intracellular tyrosinekinase domains. As for other protein kinases, activation of VEGFR is a key mechanism of control signals for proliferation of endothelial cells, and it is believed that abnormalities VEGFR/VEGF contribute to abnormal angiogenesis in many human diseases, such as psoriasis and cancer.

In embryogenesis system VEGFR/VEGF is needed in order to the proper development of the vascular system. In adults VEGFR/VEGF is important in the healing of wounds, inflammation and angiogenesis.

Non-invasive analysis of circulating levels of VEGF-165 patients before treatment drugs is a potentially important addition to making therapeutic decisions. Although the analysis of total VEGF-A is used in humans as a prognostic indicator of the disease, up to the present description is not reported on a comparison between the levels of VEGF-165 patients before chemotherapy and the outcome of treatment. Therefore, VEGF-165 can serve as a valuable prognostic indicator and an indicator for monitoring the effectiveness of treatment multikinase inhibitor.

Summary of invention

This invention relates to biomarkers and biomarkers for prognosis and prediction of cancer, as well as the use of biomarkers to monitor the efficacy of anticancer therapy. More specifically this invention relates to the use of VEGF-165 as a biomarker for multikinase inhibitors (for example, Sorafenib).

In one embodiment, this invention relates to the use of quantitative immunoassays for the measurement of protein levels of VEGF-165 in the fluids of the human body to treat multikinase inhibitor (e.g., Sorafenib). These levels are especially useful as lights the RA capacity for suffering of cancer patients, treated multikinase inhibitor (e.g., Sorafenib), the benefits of such therapy.

Measurement of the levels of VEGF-165 before treatment can be applied clinically as a therapeutic adjunct for the choice of therapy the patient, to monitor the status of preneoplastic/neoplastic disease in a patient, and/or for monitoring how a patient suffering from preneoplastic/neoplastic disease responds to treatment. In one embodiment, the levels of VEGF-165 can be used to aid in the choice of therapy of the patient and when making decisions regarding the optimal method of treatment of the patient.

Levels of VEGF-165 can be measured in the patient samples, such as, but not limited to, blood, serum, plasma, urine, saliva, seminal fluid, exudate from breast cancer, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosole, ascites, pleural effusions, amniotic fluid, flushing fluid bladder and bronchoalveolar lavage.

In another embodiment, this invention relates to the use of immunoassay as a means of selecting patients who are likely to benefit from treatment multikinase inhibitor (e.g., Sorafenib), through the measurement of the levels of VEGF-165 to treatment in a patient-derived sample is x, and the assessment of the possible outcome based on nomograms possible result for the patient to levels of VEGF-165.

Method of monitoring the status of the diseases associated with activated by VEGF-165 patients may also be predictive for the disease, where the levels of total protein VEGF-165 in patient-derived samples are indicative for better or worse treatment outcome for the patient. The forecast can be clinical result selected from the group comprising the reaction rate (SRS), complete response (CR), partial response (PR), stable disease (Sz), clinical benefits [including complete response (CR), partial response (PR) and stable disease (Sz)], time to disease progression (vdps), survival without progression (EBP) and overall survival (S).

These methods can have standard formats, for example, immunoassay in the form of a sandwich immunoassay, such as sandwich enzyme-linked immunosorbent assay (ELISA)or equivalent analysis. In such immunoassays can be used monoclonal antibodies such as anti-VEGF-165 monoclonal antibodies. In addition, the monoclonal antibody can be biotinylation.

Another variant embodiment of the invention relates to a quantitative immunoassay for measuring incremental changes in the levels of total Bel is and VEGF-165 in patient-derived samples, as a method of selecting a therapy for a patient suffering from a disease, for example, preneoplastic/neoplastic disease.

As an example, one such method of selection of therapy may include stages:

(a) immunological detection and quantitative analysis of total protein VEGF-165 in the sample taken from control group;

(b) immunologically detecting and quantifying levels of total protein VEGF-165 in a sample taken from a patient over time; and

(c) determine whether the use of conventional therapy and/or therapy multikinase inhibitor (e.g., Sorafenib) for the treatment of the patient based on the protein level of VEGF-165 in the patient samples.

For example, if you found that the protein level of VEGF-165 in the patient sample is more than 70 PG/ml, it can be concluded that the patient suffers illness, managed VEGF, and can be decided on the application of therapy multikinase inhibitor (e.g., Sorafenib) for the treatment of a patient either alone or in combination with one or more other therapies.

Therapy aimed at the path of VEGF-165, may include multikinase inhibitors, tyrosine kinase inhibitors, bis-arelatively, antisense inhibitors of VEGFR-2 or monoclonal antibody therapy, or the like, for Example, therapy directed the military on the path of VEGF-165, may include bis-allocatio Sorafenib, which is an inhibitor of angiogenesis, as well as a tyrosine kinase inhibitor, or a tyrosine kinase inhibitor STI571 (also known as imatinib mesilate or Gleevec®).

Another variant of implementation of the present invention relates to the use of quantitative immunoassay for determining changes in the levels of VEGF-165 in combination with the levels of one or more protein(s). This additional protein(s) may include, for example, inhibitors (e.g., tissue inhibitor of metalloproteinases-1 (TIMP-1)), oncoproteins (e.g., HER-2/neu, ras p21), receptors, growth factor (e.g., the receptor for epidermal growth factor (EGFR), alpha receptor for platelet-derived growth factor (DERIVED-α)), proteins metastases (for example, plasminogen activator urokinase type (Ira)), tumor markers (e.g., carcinoembryonic antigen (CEA)and tumor suppressors (e.g. p53). Such methods can be applied, for example, as diagnostic/prognostic tools, choice of therapy for patients suffering from a disease, monitoring disease in a patient and for monitoring how a patient suffering from a disease which responds to directed on the path of VEGF or other therapy. It is preferable to test patients (e.g. cancer patients) on serial changes in total VEGF-165 and additional proteins, such as proteins, which can activate the path VEGF-165, as a means to extend the clinical perspective, therapeutic resources, and diagnostic/prognostic parameters to select the optimal therapeutic combinations for the most promising treatment results.

In another embodiment, the present invention presents an analytical kit for monitoring the efficacy of therapy on the patient sample containing the specific protein to the antibody. In some embodiments, implementing the set also includes instructions for use of kit. In certain embodiments of the implementation of the set may also include solutions for suspension or fixation of the cells, detectable tag or label, the solutions for the visualization of the polypeptide that is sensitive to the binding of the antibody solutions for lizirovania cells or solutions for the purification of polypeptides. In yet another embodiment, the antibody is specific to VEGF-165.

Description of the drawings

Figure 1 shows the mean levels of VEGF-165 in groups of patients for a stable and progressive disease.

Figure 2 shows the average reduction of the tumor measured in groups of patients for a stable and progressive disease.

Detailed description of the invention

It should be clear that this izopet the tion is not limited to the described specific methodology, protocols, colonies of cells, species or genera of animals, constructs, and reagents, as such may vary. Should also be understood that the terminology used here is given for descriptive purposes only particular embodiments and is not intended to limit the scope of the present invention, which is limited only by the claims.

It should be noted that in this description and the claims the singular includes reference to the plural, unless the context clearly indicates to the contrary. Thus, for example, reference to "a gene" is a reference to one or more genes and includes cash equivalents, known to experts in the art, and so forth.

If not stated otherwise, all technical and scientific terms used herein have the same values that are generally accepted and understood by professionals in this area of technology to which belongs the invention. Although in practice or testing of the present invention can be applied to any methods, devices and materials similar or equivalent to those described hereinafter described the preferred methods, devices, and materials.

All publications and patents mentioned herein are included here by reference for purposes of describing and disclosing, for example, constructs and m is centered methodology, which are described in the publications, which can be used in connection with the invention described here. Publications listed above and hereinafter, are provided solely for their description before the date of filing of this application. Nothing here should be construed as an assumption that the authors of the present invention does not have the right to date this description by virtue of the preceding inventions.

Definition

For convenience, the meaning of certain terms and phrases used in the description, the examples and the claims presented below.

The term "isolated from the patient sample" in this description means a sample taken from the patient. The sample can be any biological tissue or fluid. The sample can be a sample obtained from the patient. Such samples include, but are not limited to, blood, serum, plasma, urine, saliva, seminal fluid, exudate from breast cancer, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosole, ascites, pleural effusions, amniotic fluid, flushing fluid bladder and bronchoalveolar lavage, blood cells (e.g., white cells), tissue samples or biopsy (for example, a biopsy of the tumor), or cells from them. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes.

The term "bi is the token" covers a wide range of intra - and extracellular events, as well as physiological changes in the body. Biomarkers can be represented virtually every aspect of cellular function, such as, but not limited to, levels or the rate of formation of signaling molecules, transcription factors, metabolites, gene transcripts, as well as posttranslational modifications of proteins. Biomarkers can include a complete genomic analysis of transcript levels or full proteomic analysis of protein levels and/or modification.

The term biomarker can also refer to a gene or gene product with Overdrive or a down-regulation in treated patient connection cell of a patient suffering from the disease, compared with non-treated diseased cells. That is, the gene or gene product is quite specific in relation to the treated cell that it can be used, optionally with other genes or gene products, to identify, predict or determine the efficacy of a small molecule. Thus, the biomarker is a gene or gene product, which is characteristic of the effectiveness of the compound in the patient cell or reaction of the specified patient cells for processing the connection.

The term "cancer" includes, but is not limited to, solid tumors such as breast cancer, respiratory put the th, brain, reproductive organs, digestive tract, urinary tract, eye, liver, skin, head and neck, thyroid, parathyroid and their distant metastases. The term also includes lymphoma, sarcoma, and leukemia.

Examples of breast cancer include, but are not limited to, invasive chatter-orbital carcinoma, invasive lobular carcinoma, Doctorow carcinoma in situ and lobular carcinoma in situ.

Examples of cancer of the respiratory tract include, but are not limited to, small cell and non-small cell carcinoma of the lung and bronchial adenoma and plavalaguna blastoma.

Examples of brain cancer include, but are not limited to, glioma, brain stem and hypothalamus, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, and neuroectodermal tumour and tumor of the pineal region.

Tumors of the male reproductive organs include, but are not limited to, prostate cancer and ovarian cancer. Tumors of the female reproductive organs include, but are not limited to, cancer of the endometrium, cervix, ovary, vagina and vulva, as well as sarcoma of the uterus.

Tumors of the digestive tract include, but are not limited to, colorectal cancer, colon cancer, colon and rectum, esophagus, gall bladder, stomach, pancreas, rectum, small intestine and salivary glands.

tumors of the urinary tract include, but not limited to, cancer of the bladder, penis, kidney, renal pelvis, ureter and urethra.

Cancer of the eye includes, but is not limited to, intraocular melanoma and retinoblastoma.

Examples of liver cancer include, but are not limited to, hepatocellular cancer (carcinoma of the liver cells with or without fibrolamellar option), cholangiocellular carcinoma (carcinoma intrahepatic bile ducts) and mixed hepatocellular cholangiocarcinoma.

Skin cancer includes, but is not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, cancer cells Merkel and non-melanoma skin cancer.

Head and neck cancer includes, but is not limited to, cancer of the larynx/hypoglossi/nasopharyngeal/oropharyngeal cancer lip and oral cavity.

Lymphomas include, but are not limited to, AIDS-related lymphoma, nahodkinskuju lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease and lymphoma of the Central nervous system.

Sarcomas include, but are not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.

Leukemias include, but are not limited to, acute myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia and hairy-cell leukemia.

The term "patient" or "bject" in this description includes mammals (for example, human and animal).

This invention relates to the quantitative immunoassays that measure the protein levels of VEGF-165 in patient-derived samples. Such analyses can be useful when selecting a therapy for a patient suffering from a disease associated with by VEGF-165. In this description of "the way of VEGF-165" is defined as the path VEGF-165, activated or excessive expression or mutation of the protein VEGF-165 and, as such, covers the way VEGF-165 with increasing regulation and/or induced mutation.

Examples of neoplastic diseases associated with activated by VEGF-165, and also precancerous lesions, leading to neoplastic diseases, are the following: metastatic medulloblastoma, gastrointestinal stromal tumors (IXO), dermatofibrosarcoma wybuchowa (DFSW), chronic myeloproliferative diseases (HMPS)cancer, colorectal cancer, colon cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, acute myelogenous leukemia, thyroid cancer, pancreatic cancer, bladder cancer, kidney cancer, melanoma, breast cancer, prostate cancer, cancer ovarian cancer, cervical cancer, head and neck cancer, brain tumor, hepatocellular carcinoma, and hematologic malignancy. Thus, the protein levels of VEGF-165, individually or in the Oceania with the levels of other proteins (for example, other oncoproteins), can be used to predict clinical outcome and/or as an aid when choosing therapy.

Thus, the present invention is described and claimed the use immunoassay for the quantitative measurement of the levels of VEGF-165 obtained from the patient sample (e.g. the levels of circulating VEGF-165) to assess the likelihood of suffering from cancer patient will benefit from treatment multikinase inhibitor (e.g., Sorafenib).

In one embodiment of the present invention the protein VEGF-165 quantify in patient-derived samples taken during diagnosis or to treatment. Such patient-derived samples can be blood, serum, plasma, urine, saliva, seminal fluid, exudate from breast cancer, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosole, ascites, pleural effusions, amniotic fluid, flushing fluid bladder and bronchoalveolar lavage, among other body fluids. Patient-derived samples may be fresh or frozen and can be treated with heparin, citrate or add.

Examples of immunoassays which can be used in methods in accordance with this invention, is a sandwich ELISA. However, it should be clear that other methods, dopolnenie described here, can be used for quantitative analysis of protein VEGF-165 in patient-derived samples. In addition, a variety of detection methods can be used to visualize the protein VEGF-165, such as a fluorescent label.

Many formats can be adapted for use in accordance with the methods of the present invention. For example, the detection and quantitative analysis of the protein VEGF-165 in patient-derived samples can be performed using enzyme-linked immunosorbent assays, radioimmunoassays, sandwich assays using double antibody, agglutination assays, fluorescent immunoassays, immunoelectron and scanning microscopy, among other analyses, widely known in the art. Quantitative analysis of the protein VEGF-165 in such assays can be adapted by conventional methods known in the art. In one embodiment, the serial changes in circulating levels of the protein VEGF-165 can be identified and quantitatively analyzed using sandwich-analysis, in which the immobilized antibody immobilized with conventional techniques on a substrate.

Suitable substrates include, for example, synthetic polymeric substrate, such as polypropylene, polystyrene, substituted polystyrene, polyaki the amides (such as polyamides and polyvinyl chloride), glass beads, agarose and cellulose nitrate.

In the example of a sandwich ELISA immunoassay that can be used in methods in accordance with this invention can be applied purified mouse monoclonal antibody against human VEGF-165 as the immobilized antibody and biotinylated goat polyclonal antibody against human VEGF-165 as detector antibodies. Immobilized monoclonal antibody immobilized in the wells of microtiter plate. Diluted samples of human serum/plasma or standards VEGF-165 (for example, a recombinant protein VEGF-165 wild type) incubated in the wells for binding antigen VEGF-165 with immobilized monoclonal antibody. After washing the wells immobilized antigen VEGF-165 is treated with biotinylated detector antibody, after which the wells are again washed. Then add the conjugate of streptavidin - horseradish peroxidase. After the last wash the wells add the Blue TMB substrate Substrate to determine the binding activity of peroxidase. The reaction is stopped by the addition of 2.5 N. sulfuric acid and the optical density measured at 450 nm. The correlation values of the optical density of the samples with the standards of VEGF-165 allows to determine the quantitative value of VEGF-165 PG/ml of serum or plasma.

Should be understand the but what other proteins (e.g., inhibitors, oncoproteins, receptors, growth factors, angiogenic factors, metastatic proteins, tumor markers, tumor suppressor proteins, associated with the path VEGF) can be used for detection and quantitative analysis in combination with VEGF-165. For example, other proteins, suitable for testing together with VEGF-165, include tissue inhibitor of metalloproteinases-1 (TIMP-1), HER-2/neu, ras p21, the receptor for epidermal growth factor (EGFR), alpha receptor for platelet-derived growth factor, vascular endothelial growth factor (VEGF), plasminogen activator urokinase type (Ira), the carcinoembryonic antigen (CEA) and p53. These other proteins can be determined using assays that are known to specialists in this field of technology. For example, immunoassays for the quantitative analysis of HER-2/neu and TIMP-1 are commercially available, for example, Oncogene Science TIMP-1 ELISA (Oncogene Science, Cambridge, MA (USA)), which can detect ng/ml levels of TIMP-1 in human serum or plasma.

Monitoring levels of VEGF-165 to treatment may be an indicator of clinical outcome after treatment multikinase inhibitor (e.g., Sorafenib).

One way to assess clinical outcome is to evaluate the reaction rate (SRS), complete response (CR), partial response (PR), stable disease (Sz), clinical on what ESI (including complete response (CR), partial response (PR) and stable disease (Sz)), time to disease progression (vdps), survival without progression (EBP) and overall survival (S).

The term "antibody" herein is used in its broadest sense and specifically covers monoclonal antibodies (including full-size monoclonal antibodies), polyclonal antibodies, multispecific antibodies (for example, bespecifically antibodies) and antibody fragments. Antibodies used in the methods in accordance with this invention, can be obtained using conventional methodologies and/or techniques of genetic engineering. For example, antibodies in accordance with this invention include antibodies that bind to VEGF-165.

"Antibody fragments" comprise a portion of a full-sized antibodies, usually antigennegative plot or variable domain. Examples of fragments of antibodies include Fab, Fab', F(ab')2and Fv fragments; dyatel; linear antibodies; single-chain molecule antibodies; bespecifically antibodies; and multispecific antibodies derived from fragments of antibodies.

The term "monoclonal antibody" in the present description refers to an antibody obtained from a population essentially homogeneous antibodies, that is, the individual antibodies comprising an identical population, except for the possible nature is the breaking of mutations, which may be present in minor amounts. Monoclonal antibodies are highly specific, that is directed against a single antigenic site. In addition, unlike conventional drugs (polyclonal) antibodies, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single antigen determinants. The definition of "monoclonal" indicates the character of the antibody as being obtained from almost homogeneous population of antibodies, and should not be construed as requiring receipt of antibodies specific way. For example, the monoclonal antibodies used in accordance with this invention, can be obtained hybridoma technology, first described by Kohler, et al., (Nature 256: 495, 1975), or may be obtained by the methods of recombinant DNA (see, for example, U.S. patent No. 4816567). Monoclonal antibodies can also be isolated from phage libraries of antibodies using techniques described, for example, Clackson, et al., (Nature 352: 624-628,1991) and Marks et al., (J. Mol. Biol. 222: 581-597, 1991).

The monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins)in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived inconcrete species or belonging to a particular class or subclass of antibody, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from other species or belonging to another class or subclass antibodies as well as fragments of such antibodies, provided that they possess the desired biological activity (see, for example, U.S. patent No. 4816567; and Morrison, et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855, 1984).

"Humanized" forms of inhuman (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from a nonhuman immunoglobulin. The most part, humanized antibodies are human immunoglobulins (recipient antibody)in which residues hypervariable regions of the recipient replaced the remnants of the hypervariable regions of the nonhuman species (donor antibody)such as mouse, rat, rabbit or non-human primates, with the desired specificity, affinity and capacity. In some cases, remnants of the frame region (CR) of human immunoglobulin can be replaced with the appropriate inhuman remnants. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. Such modifications carried out to further improve e the efficiency of antibody. In General, humanitariannet antibody can contain almost all of the at least one or typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of non-human immunoglobulin and all or substantially all of the TO are those of the sequence of a human immunoglobulin. Humanitariannet antibody, if necessary, may also contain at least part of a constant region of immunoglobulin (Fc), typically a human immunoglobulin. The review is presented in Jones, et al., (Nature 321:522-525, 1986); Reichmann, et al., (Nature 332:323-329,1988); and Presta, (Curr. Op. Struct. Biol. 2: 593-596, 1992).

"Single-chain Fv" or "sFv" antibody fragments contain VHand VLdomains of antibodies, where these domains are present in a single polypeptide chain. Usually Fv polypeptide includes polypeptide linker between domains VHand VLwhich enables the sFv to form the desired structure for binding to the antigen. The review presents Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol.113, Rosenburg and Moore eds. Springer-Veriag, New York, pp.269-315, 1994).

The term "diately" ("diabodies"refers to small fragments of antibodies with two antihistamine areas where the fragments contain the variable domain of the heavy chain (VH)associated with the variable domain of the light chain (VLin one the e polypeptide chain (V H-VL). Through the use of a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains of another chain and create two antigenspecific plot. Diately described in more detail, for example, in EP 404097; WO 93/11161; and Hollinger et al., (Proc. Natl. Acad. Sci. USA 90: 6444-6448, 1993).

The expression "linear antibodies" refers to antibodies described in Zapata et al., (Protein Eng. 8(10): 1057-1062, 1995). Briefly, these antibodies have a pair of tandem Fd segments (VH-CH1-VH-CH1), which form a pair antigenspecific areas. Linear antibodies can be bespecifically or monospecific.

Typical monoclonal antibodies used in accordance with this invention include mouse monoclonal antibodies against human VEGF-165, such as are in the kit sandwich ELISA kit from Oncogene Sciences, intended for measurement of human VEGF-165. Monoclonal antibodies used in accordance with this invention, serve to identify proteins VEGF-165 in various laboratory prognostic tests, such as clinical samples.

Common sources that describe additional molecular biological techniques used here, including antibodies, include Berger andKimmel (Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol.152, Academic Press, Inc.); Sambrook, et al., (Molecular Cloning: A Laboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol.1-3); Current Protocols in Molecular Biology (F. M. Ausabel et al. [Eds.], Current Protocols, a joint venture between Green Publishing Associates, Inc. and John Wiley & Sons, Inc. (amended in 2000)); Harlow et al., (Monoclonal Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988), Paul [Ed.]); Fundamental Immunology, (Lippincott Williams & Wilkins (1998)); and Harlow, et al., (Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1998)).

Antibodies used in accordance with this invention for the identification of proteins VEGF-165, may be marked by any standard method. An example of a label is horseradish peroxidase, and an example of a method of labeling antibodies is the use of complexes of Biotin-streptavidin.

Appropriately, the antibodies used in immunoassays in accordance with this invention, which are used as indicators, can be marked in any way, directly or indirectly, that gives the signal, which can be seen or reproduced visually. Defined marker substances include radionuclides, such as3H,125I and131I; fluorescent substances such as isothiocyanate fluorescein and other fluorochromes, ficobiliproteins, FIKO-aretin, chelates of rare earth metals, theheavy red, dansyl and rhodamine; colorimetric reagents (Chromogens); opaque to electron materials (elecron-opaque materials), such as colloidal gold; bioluminogenic; chemiluminogenic; dyes; enzymes such as horseradish peroxidase, alkaline phosphatase, glucose oxidase, glucose-6-phosphatedehydrogenase, acetylcholinesterase, alpha-, beta-galactosidase, among others; coenzymes; enzyme substrates, enzyme cofactors; enzyme inhibitors; enzyme subunit; metal ions; free radicals; or any other immunologically active or inert substances, which provide a means for measuring or detecting the presence or quantity of the formed immunocomplex. Examples of combinations of enzyme substrates are horseradish peroxidase and tetramethylbenzidine (TMB), and alkaline phosphatase and paranitrophenylphosphate (pNPP).

Other detection systems and quantitative analysis in accordance with this invention give fluorescent signals bioluminescene (NL) or chemiluminescent (CHL). In chemiluminescence (CHL) or bioluminescence (NL) analyses the intensity of the total light emission is measured and correlated with the concentration of the unknown analyte. Light can be measured quantitatively using a luminometer (as detector photomultiplier tube) or on the device charge-coupled, or qualitatively with the use of photographic or x-ray film. The main advantages of the mi application of such tests is their simplicity and analytical sensitivity, allowing the detection and/or quantitative analysis of very small quantities of analyte.

Examples of fluorescent labels include esters of acridine, sulfonyl-carboxamide acridine, luminal, umbelliferon derived isoluminol, pocobelli, such as acorin, and luciferase fireflies, marine bacteria, Vargulla and Renilla. Luminal may be used if necessary, with molecules of a reinforcing agent such as 4-itfinal or 4-hydroxyurea acid. Typically, the signal PI is obtained by treatment with oxidizing agent under alkaline conditions.

Additional fluorescent detection systems include those in which the signal (detectable marker) is the enzyme reaction on the substrate. Definition schema HL and BL designed for the analysis of labels in the form of alkaline phosphatase (alkaline phosphatase, glucose oxidase, glucose-6-phosphate dehydrogenase, horseradish peroxidase (HRP) and xanthine oxidase, among others. Alkaline phosphatase and HRP are the two enzyme labels, which can be quantified using a series of reactions of CR and NL. For example, alkaline phosphatase can be used with a substrate such as a substituted 1,2-deoxyadenylate substrate (such as AMPPD or CSPD; Kricka, L.J., "Chemiluminescence and Bioluminescence, Analysis by, "Molecular Biology and Biotechnology: A Comprehensive Desk Reference (ed. R.A. Meyers) (VCH Publishers; N.Y., N.Y.; 1995)); for example, the disodium salt of 4-methoxy-4-(3-forfatter)Spiro[1,2-dioxetane-3,2'-hell is the'antan], with or without molecules reinforcing agent such as dichloride, 1-(trioctylphosphine methyl)-4-(tributylphosphine methyl)benzene. HRP can be used with substrates, such as 2',3',6'-triperformance-10-metrocream-9-carboxylate.

PI and NL reactions may be adapted to analyze not only enzymes, but other substrates, cofactors, inhibitors, metal ions and the like. For example, the reaction lyuminola, luciferase fireflies and luciferase marine bacteria are indicator reactions for the formation or acquisition of peroxide, ATP and NADPH, respectively. They can be combined with other reactions, including oxidase, kinase and dehydrogenase, and can be used to measure any component coupled reactions (enzyme, substrate, cofactor).

Detectable marker may be directly or indirectly connected with the antibody used in the analysis in accordance with this invention. An example of indirect communication detectable label is the use of binding pairs between the antibody and the marker, or the application of the system to amplify the signal.

Examples of binding pairs that can be used to link the antibody to detectable markers include Biotin/avidin, streptavidin or antibodies against Biotin; avidin/antibodies against avidin; thyroxine/thyroxine binding globulin; antigen/EN is Italo; antibody/intential; carbohydrate/lectin; hapten/anticarcinoma antibody; dyes and hydrophobic molecules/hydrophobic binding sites of the protein; enzyme inhibitor, coenzyme or cofactor/enzyme; poliolefinovoy acid/homologous sequence poliolefinovoy acid; fluorescein/antibody against fluorescein; dinitrophenol/antibodies against dinitrophenol; vitamin b 12/internal factor; cortisone, cortisol/cortisol-binding protein; and ligands for a specific protein receptor/specific receptor proteins associated with the membrane.

Various methods of binding labels, directly or indirectly, with antibodies known in the art. For example, the label can be bound either covalently or ecovalence. Examples of methods of conjugation of the antibodies described in Avarmeas, et al.. Scan. J. Immunol. 8 (Suppl.7): 7, 1978); Bayer et al., Meth. Enzymol. 62: 308. 1979; Chandler, et al., J. Immunol. Meth. 53: 187. 1982; Ekeke and Abuknesha, J. Steroid Biochem. 11: 1579, 1979; Engvall and Perlmann, J. Immunol. 109: 129, 1972; Geoghegan, et al., Immunol. Comm. 7:1, 1978; Wilson and Nakane, Immunofluorescence and Related Techniques, Elsevier/North Holland Biomedical Press, Amsterdam (1978).

Depending on the nature of the label, various techniques can be used for detection and quantitative analysis of the label. For fluorescent substances used many system. For chemiluminogenic apply luminometry or film. For enzymes, fluorescent, chemiluminescent ntny or colored product can be defined or measured fluorometrically, lumino-metrically, spectrophotometrically or visually.

Different types of chemiluminescent compounds having acadeniy, Ben-screenjelly or acridology type heterocyclic ring systems are other examples of tags. Examples of esters of acridine include compounds having heterocyclic rings or ring systems that contain a heteroatom in a positive oxidation state, including such ring systems, as the cation acridine, benzo[a]acridine, benzo[b]acridine, benzo[C]acridine, benzimidazole, chinoline, skinline, chinoline, cyclic substituted chinoline, phenanthridine and honokalani.

The indicator can be obtained by joining the selected antibody, directly or indirectly, reactive functional groups present in the complex air acridine or benzacridine, as is well known to experts in the art (see, for example. Weeks, et al., Clin. Chem. 29 (8): 1474-1479, 1983). Examples of the compounds include esters of acridine and benzacridine with the outgoing aryl ring group and a reactive functional group that is either para-or metaprogram aryl rings (see, for example, U.S. patent No. 4745181 and WO 94/21823).

In this description, "aimed at the way VEGF therapy" includes any therapy that focused the s on the path of VEGF, including the inhibition of the expression of VEGF protein (e.g., antisense oligonucleotides), warning, membrane localization is essential for the activation of VEGFR, or inhibition following effectors VEGFR (e.g., Rafc/trionychidae). Aimed at the way VEGF therapies include multikinase inhibitors, tyrosinekinase inhibitors, monoclonal antibodies and bis-arelatively.

An example of a kinase inhibitor is bis-allocating Sorafenib, a small molecule and a new dual-action inhibitor as Raf (protein-serine/trionychinae)and VEGFR (the receptor for vascular endothelial growth factor receptor tyrosinekinase), and, therefore, the inhibitor and proliferation of tumor cells, and angiogenesis (Onyx Pharmaceuticals, Richmond, CA, and Voeg Pharmaceuticals Corporation, West Haven, CT (USA); Lyons, et al., Endocrine-Related Cancer 8: 219-225, 2001). In addition, it was found that Sorafenib inhibits some other receptor tyrosine kinase involved in tumor growth and neovascularization, including DERIVED-β, Flt-3 and c-KIT. PD 166285 (Pfizer, Groton, CT), total tyrosinekinase inhibitor, may cause the counter and PDGF, and FGF-2-mediated responses (Bansai, et al., J. Neuroscience Res.74 (4): 486-493, 2003).

Other examples of therapies that focus on the path of VEGF include: Sutent/SUl 1248, RTC 787, MLN518, RKS-412, CDP860 and XL9999. Sutent/SUl1248 (sunitinib malate; indolin-2-one) (Pfizer, Groton, CT) aims to cocktail recipes. what percent slapshot-sincinaty (RTC), including DERIVED, with antiangiogenic and antitumor activity. DERIVED plays a significant role in angiogenesis through the regulation of proliferation and migration of pericytes, cells that support blood vessels and believe that Sutent/SU11248 inhibits angiogenic effect DERIVED.

RTC 787 (Novartis, Basel, Switzerland and Schering AG, Berlin, Germany) is an oral low-molecular antiangiogenesis agent (anyinflate), active anti-DERIVED, as well as against VEGFR and c-Kit tyrosinekinase receptors (see, e.g., Garcia-Echevera and Fabbro, Mini Reviews in Medicinal Chemistry 4(3):273-283, 2004).

MLN518 (formerly known as ST; Millenium Pharmaceuticals, Cambridge, MA) is an oral small molecule compound, designed for in-generowania type III receptor tyrosinekinase (RTK), including DERIVED, FLT3 and C-Kit.

RKS-412 [midostaurin; N benzoyltartaric (derived staurosporine, the product of the bacterium Streptomyces); Novartis, Basel, Switzerland) inhibits DERIVED, VEGF-165R and numerous protein kinase C, "which makes it particularly attractive for patients with wild-type KIT with mutations in DERIVED" (SW 412-An Interview with Charles Blanke, MD, FACP (www.gistsupport.org/pkc412.html); see also Reichardt, et al., J. Clin. Oncol. 23 (16S): 3016, 2005).

XL999 (one of several Spectrum Selective Kinase Inhibitors™ (SSKI) from Exelixis South San Francisco, CA, USA) inhibits VEGF-165R, and other RTK, such as DERIVED-beta, FGFR1 and FLT3.

EXAMPLES

Patterns, materials, compositions, and str is ordinary, described here are typical examples of this invention, and it should be understood that the scope of the present invention is not limited to the scope of the examples. Specialists in the art will understand that the invention can be carried out with variations of the described structures, materials, compositions and methods, and such variations are assumed to be included in the scope of this invention.

Example 1. Solid-phase microtiter sandwich ELISA drugs samples human serum and plasma

Suitable samples for analysis of VEGF-165 ELISA include human plasma treated with heparin, citrate or add, and human serum. Because of the possible confounding factors, special attention should be given to the preparation and analysis of human serum and plasma. Any coagulating material must be removed from the samples by microentrepreneurial to breeding. The original concentration of the sample serum or plasma should be about 12-13% (1:8 dilution of the sample in the sample diluent). For example, 40 μl of the sample can be diluted with 280 ál of sample diluent and 100 μl added to the wells of the microplate.

The method of analysis

The following ELISA Protocol used for sandwich ELISA (Oncogene Science, Cambridge, MA) for measurement of human VEGF-164 in human plasma or serum.

1. Get the working of rest is p (IX) Platewash (available as part of a set for analysis).

2. Add pre-diluted samples and controls, and each of the six standards of soluble VEGF-165 (from 0 to 8000 PG/ml) in duplicate pipette 100 ál into the appropriate wells using a clean pipette tips for each sample and standard. Add standard 0 in one additional hole, which will be used to determine a control sample substrate.

3. Cover wells clean plastic wrap or a device for sealing tablets. Incubate microtiter plate for 1.5 hours at 37°C.

4. Carefully remove plastic wrap or a device for sealing tablets. Rinse the wells with application of 300 µl per well of buffer Platewash (rinsed three cycles, rotate the tablet 180° and washed three cycle).

5. Pipette add 100 ál of detection antibody to all wells except wells with the control sample substrate, which is left empty. Cover the hole with a new piece of plastic wrap. Incubate microtiter plate for 1 hour at 37°C.

6. To prepare the conjugate dilution of the appropriate volume of concentrate conjugate (dilution 1:50) in conjugate diluent to.

7. Wash the wells as in stage 4. Immediately go to step 8.

8. Pipette add 100 ál of working conjugate is about all wells except wells with the control sample substrate, which leave blank. Cover the hole with a new piece of plastic wrap. Incubate the microtiter plate at room temperature (20-27°C) for 1 hour.

9. To prepare working substrate by combining equal parts of solution a and solution Century. Six ml of each substrate solution will give 12 ml of working substrate, sufficient to process a single microtiter plate. To increase the amount of working substrate based on the number of strips used. Carefully stir.

10. To distribute the work substrate in a cell with pure reagent to reach room temperature.

11. Wash the wells as in stage 5. Caution: do not allow the plates to dry. Immediately go to step 12.

12. Pipette add 100 μl of working substrate to all wells and cover the tablet with a plastic wrap or a device for sealing tablets. Incubate the microtiter plate at room temperature (20-27°C) for 45 minutes.

13. Pipette add 100 ál of storeevent to all wells.

14. Measure the optical density in each well using spectrophotometric tablet reader at a wavelength of 650 nm. Wells must be read within 30 minutes after adding storeevent.

Standard curves

Quantitative analysis is performed by constructing a standard curve with applied the eat standard VEGF-165 (recombinant human VEGF-165) in 6 different concentrations 0, 150, 1000, 3000, 5000 and 8000 PG/ml

Samples of human serum and plasma

Frozen plasma samples obtained from patients with confirmed nemelka-cell lung cancer to treatment with Sorafenib.

Example 2. Plasma of patients suffering from non-small cell lung carcinoma

Double samples used for measuring the level of VEGF-165 using Oncogene Science VEGF-165 ELISA (Oncogene Science, Cambridge; MA) according to manufacturer's instructions. The average value of two measurements is determined for each patient. The average level of VEGF-165 for 31 patients in this analysis are presented in table 1. Table 2 shows the average decrease of the tumor, measured radiologically, the appropriate group of patients. The results show that the average level of VEGF-165 patients, which then in response to treatment with Sorafenib showed stable disease, is to 67.9 PG/ml Patients who show progressive disease despite treatment with Sorafenib, had a medium level of VEGF-165 227,2 PG/ml Patients demonstrating stable disease had a mean decrease in tumor by 5.1%, and patients with advanced disease had an average tumor growth by 20.6%. These results are presented graphically in figure 1 and 2.

Table 1
VEGF-165
Stable diseaseProgressive disease
The average VEGF-165 (PG/ml)67,9227,2
n238

Table 2
The decrease in tumor
Stable diseaseProgressive disease
Average % reduction in tumor- 5,120,6
n228
Standard error of the mean (SEM)- 3,17,6

The description above of a variant of implementation of the present invention is presented for the purpose of illustration and description. It is not exhaustive and does not limit the invention to the described specific forms, and many possible modifications and variations apparent in the light of presented what about the descriptions. Embodiments of the chosen and described in order to explain the principle of this invention and its practical application that will allow other experts in the art to apply the invention in various embodiments, implementation and with various modifications required for a particular intended application. Everything mentioned here references incorporated by reference.

1. Method of monitoring related disease by VEGF-165, activated or excessive expression or mutation of the protein VEGF-165 in the patient and/or monitoring how a patient with a specified disease responds to treatment, providing immunological detection and quantitative analysis of serial changes in the protein levels of VEGF-165 in patient-derived samples taken over time, where increasing the protein levels of VEGF-165 over time indicate disease progression or negative reaction to a specified therapy and where decreasing the protein levels of VEGF-165 over time indicate remission of the disease or a positive response to the indicated therapy.

2. The method according to claim 1, where the specified therapy choose from multikinase inhibitors, tyrosinekinase inhibitors, monoclonal antibodies and bis-allocation.

3. The method according to claim 1, where the specified therapy is aimed at p is th VEGF-165 therapy.

4. The method according to claim 3, where the specified aimed at the path of VEGF-165 therapy is tyrosinekinase inhibitor imatinib mesilate or bis-allocating Sorafenib.

5. The method according to claim 1, where these are taken from the patient sample is a fluid of an organism selected from the group comprising blood, serum, plasma, urine, saliva, seminal fluid, exudate from breast cancer, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosole, ascites, pleural effusions, amniotic fluid, flushing fluid bladder and bronchoalveolar lavage.

6. The method according to claim 5, where a specified liquid body is serum or plasma.

7. The method according to claim 1, where the specified immunological detection and quantitative analysis is performed using immunoassay in the form of a sandwich ELISA or equivalent analysis.

8. The method according to claim 7, where the sandwich ELISA or equivalent analysis involves the application of one or more monoclonal antibodies that selectively bind to the protein VEGF-165.

9. The method of selecting a therapy for a human patient suffering from the disease, including:
(a) immunological detection and quantitative analysis of the average protein level of VEGF-165 in the control samples taken from individuals in the control group;
(b) immunologically detecting and quantifying serial from the of ineni the protein levels of VEGF-165 in equivalent taken from patient samples obtained from the patient over time;
(c) comparing the protein levels of VEGF-165 in the patient samples with an average protein level of VEGF-165 in the control samples and
(d) determining whether the use of conventional therapy and/or therapy directed on the path of VEGF-165, activated or excessive expression or mutation of the protein VEGF-165, for the treatment of the patient on the basis of the difference between the protein levels of VEGF-165 in patient-derived samples and an average protein level of VEGF-165 in the control samples and taking into account the successive changes of protein levels of VEGF-165 in patient-derived samples.

10. The method according to claim 9, in which the patient sample obtained from a patient suffering from cancer, with no response to treatment.

11. Diagnostic method for detecting diseases associated with by VEGF-165, activated or excessive expression or mutation of the protein VEGF-165, in a patient, including:
(a) immunological detection and quantitative analysis of the average protein level of VEGF-165 in the control samples taken from individuals from the control group;
(b) immunologically detecting and quantifying serial changes of the protein VEGF-165 in equivalent taken from patient samples over time and
(c) comparing the protein levels of VEGF-165 in patient-derived samples with medium b is the left main coronary artery VEGF-165 in the control samples;
where the protein level of VEGF-165 in patient-derived samples, exceeding the average protein level of VEGF-165 in the control sample, is indicative of activated VEGF-165 path and the presence of diseases associated with activated by VEGF-165, patient.

12. The method according to claim 11, where the above stage (a) and (b) immunological detection and quantitative analysis is performed using immunoassay in the form of a sandwich ELISA or equivalent analysis.

13. The method according to any one of claims 1, 9 and 11, which is also prognostic for the specified disease, where these protein levels of VEGF-165 in the patient samples are an indication of better or worse prognosis for the patient.

14. The method according to item 13, where the specified forecast is the clinical result selected from the group comprising: the reaction rate (SRS), complete response (CR), partial response (PR), stable disease (Sz), time to disease progression (vdps), survival without progression (EBP), overall survival (S) and clinical benefit, which includes the complete response (CR), partial response (PR) and stable disease (Sz).

15. The method according to item 13, where increased levels of VEGF-165 is indicative of a higher likelihood of early relapse or metastasis.

16. The method according to any one of claims 1,9 or 11, where the specified condition is PR the neoplastic/neoplastic disease.

17. The method according to clause 16, where the specified preneoplastic/neoplastic disease is selected from the group consisting of metastatic Protocol, gastrointestinal stromal tumors, dermatofibrosarcoma wybuchowa, colorectal cancer and colon, colon cancer, lung cancer, non-small cell lung cancer, small cell lung cancer, chronic myeloproliferative disorders, acute myelogenous leukemia, thyroid cancer, pancreatic cancer, bladder cancer, kidney cancer, melanoma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, head and neck cancer, brain tumors, hepatocellular carcinoma, hematological malignant tumors and precancerous lesions, leading to the above-mentioned cancer.

18. The method according to any one of claims 1,9 or 11, also involving the application of immunoassay for detecting or detecting and quantifying levels of one or more other proteins in the patient samples.

19. The method according to p where specified other protein is or these other proteins are selected from the group consisting of inhibitors, oncoproteins, receptors, growth factors, angiogenic factors, metastatic proteins, tumor markers and tumor suppressors.

20. The method according to claim 19, where the specified inhibitor assessedby inhibitor of metalloproteinases-1 (TIMP-1), these oncoproteins are selected from the group consisting of HER-2/neu and ras p21, said receptors of growth factors selected from the group consisting of a receptor for epidermal growth factor (EGFR) and alpha-receptor platelet-derived growth factor (DERIVED-α)specified angiogenic factor is vascular endothelial growth factor (VEGF), specified metastatic protein is plasminogen activator urokinase type (Ira)indicated a tumor marker is the carcinoembryonic antigen (CEA) and the specified tumor suppressor is p53.

21. The method according to PP and 11, where these are taken from the patient sample is a fluid of an organism selected from the group comprising blood, serum, plasma, urine, saliva, seminal fluid, exudate from breast cancer, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosole, ascites, pleural effusions, amniotic fluid, flushing fluid bladder and bronchoalveolar lavage.

22. The method according to PP and 11, where a specified liquid body is serum or plasma.



 

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