HK1195621A - Detecting cancer with anti-ccl25 and anti-ccr9 antibodies - Google Patents

Abstract

Methods for detecting cancer in a subject are disclosed. The method includes detecting the level of expression of one or more cancer markers in a biological sample obtained from the subject; and comparing the level of expression of the one or more cancer markers in the biological sample to a normal level of expression of the one or more cancer markers. The one or more cancer markers comprise CCL25 or CCR9 or both CCL25 and CCR9.

Description

Detection of cancer with anti-CCL 25 and anti-CCR 9 antibodies

Priority is claimed for U.S. patent application No. 13/313,705 filed on 7/12/2011, U.S. patent application No. 13/248,904 filed on 29/9/2011, U.S. patent application No. 13/233,769 filed on 15/9/2011, and U.S. patent application No. 12/967,273 filed on 14/12/2010. This application also claims priority from U.S. patent application No. 13/312,343 filed on 6.12.2011. All of the above applications are hereby incorporated by reference in their entirety.

Technical Field

The present application relates generally to the detection of cancer (cancer). In particular, the invention relates to methods for detecting cancer by using anti-chemokine and/or anti-chemokine receptor antibodies.

Background

Cancer is one of the causes of death in the united states. Most cancers begin with only a single neoplastic cell. Neoplastic cells proliferate to form local "tumors". Tumors simply mean swelling; it is not necessarily cancerous. A tumor that grows only at its location or beginning and cannot spread far away is a benign tumor rather than cancer. However, tumors that have the ability to spread (whether actually yes or no) are referred to as malignant tumors or cancers. Cancer can spread to regional lymph nodes through the blood or lymphatic system and spread to distant sites through a process called metastasis. Metastatic cancer is more difficult to treat because it now spreads to many different tissues and organs. Early treatment has been shown to improve survival for many types of cancer, e.g., breast, colon, ovarian and prostate cancer.

Chemokines are a superfamily of small cytokine-like proteins that are resistant to hydrolysis, promote neovascularization or endothelial cell growth inhibition, induce cytoskeletal rearrangement, activate or inactivate lymphocytes, and modulate tropism through interaction with G protein-coupled receptors. Chemokines can regulate the growth and migration of host cells expressing their receptors.

Chemokine (C-C motif) ligand 25(CCL25), also known as thymus-expressed chemokine (TECK), is a small cytokine belonging to the CC chemokine family. CCL25 is chemotactic for thymocytes, macrophages, and dendritic cells. CCL25 exerts its effects by binding to the chemokine receptor CCR9 and is thought to play a role in T cell development. The human CCL25 produced was a protein precursor comprising 151 amino acids. The gene for CCL25 (scya25) is located on human chromosome 19.

Chemokine (C-C motif) receptor 9(CCR9), also known as GPR9-6, is highly expressed in the thymus (on immature and mature T cells) and less expressed in the lymph nodes and spleen. CCR9 is also abundant in the digestive tract, and its expression is associated with intestinal T cells. Note that the chemokine that binds protein D6 has been previously referred to as CCR9, but this molecule is a scavenger receptor and not a true (signaling) chemokine receptor.

Disclosure of Invention

One aspect of the invention relates to a method of detecting cancer in a subject. The method comprises the following steps: detecting the expression level of one or more cancer markers in a biological sample obtained from the subject; and comparing the level of expression of the one or more cancer markers in the biological sample to a normal level of expression of the one or more cancer markers, wherein a higher than normal level of expression of the one or more cancer markers in the biological sample indicates the presence of a cancer in the subject, wherein the normal level of expression of the one or more cancer markers is a predetermined value or is obtained from a control sample of known normal non-cancer cells of the same origin or type as the biological sample, wherein the cancer is blastoma, carcinoma (carcinoma), leukemia, lymphoma, melanoma, myeloma, sarcoma, or germ cell tumor, and wherein the one or more cancer markers comprises CCL25 or CCR9, or both CCL25 and CCR 9.

Another aspect of the invention relates to a method for detecting cancer in a subject. The method comprises the following steps: detecting the expression level of one or more cancer markers in a biological sample obtained from the subject; and comparing the level of expression of the one or more cancer markers in the biological sample to a normal level of expression of the one or more cancer markers, wherein a higher than normal level of expression of the one or more cancer markers in the biological sample indicates the presence of a cancer in the subject, wherein the normal level of expression of the one or more cancer markers is a predetermined value or is obtained from a control sample of known normal non-cancerous cells of the same origin or type as the biological sample, wherein the cancer is blastoma, carcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, or germ cell tumor, and wherein the one or more cancer markers comprise (1) one or more cancer markers selected from CCL25 and CCR 9; and (2) one or more cancer markers selected from CXCL13 and CXCR5 and/or one or more cancer markers selected from CXCL16 and CXCR 6.

Yet another aspect of the invention relates to a method of assessing the prognosis of a subject with cancer. The method comprises the following steps: determining the expression level of one or more cancer markers in a biological sample from the subject; and comparing the expression level of the one or more cancer markers in the biological sample to a control expression level of the one or more cancer markers, wherein a higher expression level of the one or more cancer markers in the biological sample relative to the control level indicates a poor prognosis of the subject, and wherein a lower or similar expression level of the one or more cancer markers in the biological sample relative to the control level indicates a good prognosis of the subject, wherein a poor prognosis indicates that the cancer is aggressive or invasive, wherein the cancer is blastoma, carcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, or germ cell tumor, and wherein the one or more cancer markers comprise CCL25 or CCR9, or both CCL25 and CCR 9.

Yet another aspect of the invention relates to a method of assessing the prognosis of a subject with cancer. The method comprises the following steps: determining the expression level of one or more cancer markers in a biological sample from the subject; and comparing the expression level of the one or more cancer markers in the biological sample to a control expression level of the one or more cancer markers, wherein a higher expression level of the one or more cancer markers in the biological sample relative to the control level indicates a poor prognosis of the subject, and wherein a lower or similar expression level of the one or more cancer markers in the biological sample relative to the control level indicates a good prognosis of the subject, wherein a poor prognosis indicates that the cancer is aggressive or invasive, wherein the cancer is blastoma, carcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, or germ cell tumor, and wherein the one or more cancer markers comprise: (1) one or more cancer markers selected from CCL25 and CCR 9; and (2) one or more cancer markers selected from CXCL13 and CXCR5 and/or one or more cancer markers selected from CXCL16 and CXCR 6.

Yet another aspect of the invention relates to a method of monitoring the course of cancer therapy in a subject. The method comprises the following steps: determining the expression level of one or more cancer markers in one or more biological samples obtained from the subject during or after the treatment; and comparing the expression level of the one or more cancer markers in the one or more biological samples to a control expression level of the one or more cancer markers, wherein the control level of the one or more cancer markers is a pre-treatment level of the one or more cancer markers in the subject, or a predetermined reference level, wherein treatment is deemed effective if the one or more cancer markers in the one or more biological samples is similar to or lower than the control level, wherein the cancer is blastoma, carcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, or germ cell tumor, and wherein the one or more cancer markers comprises CCL25 or CCR9, or both CCL25 and CCR 9.

Yet another aspect of the invention relates to a method of monitoring the course of cancer therapy in a subject. The method comprises the following steps: determining the expression level of one or more cancer markers in one or more biological samples obtained from the subject during or after the treatment; and comparing the expression level of the one or more cancer markers in the one or more biological samples to a control expression level of the one or more cancer markers, wherein the control level of the one or more cancer markers is a pre-treatment level of the one or more cancer markers in the subject, or a predetermined reference level, wherein treatment is deemed effective if the one or more cancer markers in the one or more biological samples is similar to or lower than the control level, wherein the cancer is blastoma, carcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, or germ cell tumor, and wherein the one or more cancer markers comprise: (1) one or more cancer markers selected from CCL25 and CCR 9; and (2) one or more cancer markers selected from CXCL13 and CXCR5 and/or one or more cancer markers selected from CXCL16 and CXCR 6.

Yet another aspect of the invention relates to a kit for detecting cancer. The kit comprises: a reagent for detecting the expression of CCL25 and/or CCR9 in a biological sample; and instructions for how to use the reagent, wherein the reagent comprises an anti-CCL 25 antibody, an anti-CCR 9 antibody, or both an anti-CCL 25 antibody and an anti-CCR 9 antibody.

Drawings

Figure 1 shows CCL25 expression in breast cancer tissue.

Figure 2 shows that CCL25 inhibited cisplatin-induced reduction in breast cancer cell line growth.

Figures 3A-B show that CCL25 protected breast cancer cells from cisplatin-induced apoptosis.

FIGS. 4A-B show PI3K and Akt activation resulting from CCL25-CCR9 interactions in breast cancer cell lines.

FIGS. 5A-B show phosphorylation of GSK-3 β and FKHR after CCL25 treatment of breast cancer cell lines.

FIG. 6 shows CCR9 and CCL25 expression in ovarian carcinoma tissues.

FIGS. 7A-B show an analysis of CCL25 expression in ovarian carcinoma tissues.

FIGS. 8A-B show analysis of CCR9 expression in ovarian cancer tissues.

FIGS. 9A-B show CCR9 and CCL25 expression of ovarian cancer cell lines.

FIGS. 10A-B show hypoxia-regulated CCR9mRNA and surface protein expression of ovarian cancer cells.

FIGS. 11A-B show hypoxia-mediated and CCL 25-mediated migration and invasion of SKOV-3 cells.

FIGS. 12A-B show CCL 25-induced collagenase expression by SKOV-3 cells.

FIGS. 13A-B show CCL 25-induced gelatinase expression of SKOV-3 cells.

FIGS. 14A-B show CCL 25-induced matrix degrading enzyme expression by SKOV-3 cells.

Figure 15 shows CCR9 expression by prostate cancer cells.

FIGS. 16A-D show CCR9 expression in prostate tissue.

Figures 17A-D show CCL25 expression in prostate cancer tissue.

Figure 18 shows serum CCL25 levels in normal healthy donors or patients with prostate disease.

FIGS. 19A-C show CCL25 expression by mouse bone marrow cells.

FIGS. 20A-B show CCR 9-mediated prostate cancer cell migration and invasion.

Figure 21 shows CCL 25-induced active MMP expression in prostate cancer cell lines.

FIGS. 22A-F show that CCR9 gene knockdown (knockdown) inhibits bone metastasis from the PC3 prostate cancer cell line.

Figure 23 shows serum CCL25 levels in lung cancer cell patients.

FIGS. 24A-C show CCR9 expression in non-tumor and lung cancer tissues.

FIGS. 25A-C show CCR9-CCL25 expression in colon cancer tissue.

Detailed Description

The following detailed description is presented to enable one skilled in the art to make and use the invention. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that these specific details are not required in order to practice the present invention. Descriptions of specific applications are provided only as exemplary embodiments. The present invention is not intended to be limited to the embodiments shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein.

Unless defined otherwise, scientific and technical terms used in connection with the present application shall have the meanings that are conventionally understood by those skilled in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

As used herein, the following terms shall have the following meanings:

the term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen. The term "antibody" is used in a broad sense and specifically includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity. By "specifically binds" or "immunoreacts with …," it is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with (i.e., bind to) or bind with very low affinity to other polypeptides. The term "antibody" also includes antibody fragments that comprise a portion of a full-length antibody, typically the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')2, and Fv fragments; a double body; a linear antibody; single chain antibody (scFv) molecules; and multispecific antibodies formed from antibody fragments. In certain embodiments of the invention, it is desirable, for example, to use antibody fragments, rather than whole antibodies, to enhance tumor penetration. In this case, it is desirable to use antibody fragments that have been modified by any means known in the art to increase their serum half-life.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from other species or belonging to other antibody classes or subclasses, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; and Morrison et al, Proc. Natl. Acad. Sci.81: 6851-6855(1984)), as well as fragments of these antibodies.

A "humanized" form of a non-human antibody is a chimeric antibody comprising minimal sequences derived from a non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) having the desired specificity, affinity and/or capacity (e.g., mouse, rat, rabbit or non-human primate). Methods for making humanized and other chimeric antibodies are known in the art.

A "bispecific antibody" is an antibody having binding specificity for at least two different antigens. In the present case, one of the binding specificities is for CXCL16 or CXCR 6. The second binding target is any other antigen and is advantageously a cell surface protein or a receptor or receptor subunit. Methods for making bispecific antibodies are known in the art.

The use of "non-homologously binding antibodies" is also within the scope of the present invention. The non-homologously bound antibody consists of two covalently bound antibodies. For example, such antibodies have been proposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980). It should be noted that antibodies can be prepared in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents.

The term "tumor" as used herein refers to a neoplasm or solid lesion formed by abnormal growth of cells. Tumors may be benign, premalignant or malignant.

A "primary tumor" is a tumor that occurs at a first location in a subject and is distinguished from a "metastatic tumor" that occurs in a subject at a location remote from the primary tumor.

The term "cancer" as used herein refers to or is to be interpreted as a physiological condition in a mammal that is typically characterized by unregulated cell growth. Exemplary cancers include: carcinomas, melanomas, sarcomas, lymphomas, leukemias, germ cell tumors, and blastomas. More specific examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric (gastrotic) or stomach (stomach) cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer (liver cancer), bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer (kidney or renal cancer), prostate cancer, vulval cancer, thyroid cancer, liver cancer (hepatoma), anal cancer, penile cancer, melanoma, multiple myeloma and B-cell lymphoma, brain and head and neck cancer, and related metastases.

The term "cancer" as used herein refers to an invasive malignancy consisting of altered epithelial cells or altered cells of unknown histological origin, but which has specific molecular or histological properties associated with epithelial cells, e.g., cytokeratin or intercellular bridge formation. Exemplary cancers of the present application include ovarian cancer, vaginal cancer, cervical cancer, uterine cancer, prostate cancer, anal cancer, rectal cancer, colon cancer, gastric cancer, pancreatic cancer, insulinoma, adenocarcinoma, adenosquamous cancer, neuroendocrine tumor, breast cancer, lung cancer, esophageal cancer, oral cancer, brain cancer, medulloblastoma, neuroectodermal tumor, glioma, pituitary tumor, and bone cancer.

The term "lymphoma" as used herein refers to a cancer of lymphocytes of the immune system. Lymphoma usually presents as a solid tumor. Exemplary lymphomas include: small lymphocytic lymphoma, lymphoplasmacytic lymphoma,Macroglobulinemia, splenic marginal zone lymphoma, plasmacytoma, extranodal marginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, Burkitt lymphoma, B cell chronic lymphocytic lymphoma, typical Hodgkin lymphoma, nodal lymphocytic major Hodgkin lymphoma, adult T cell lymphoma, extranodal NK/T cell lymphoma (rhinotype), enteropathy type T cell lymphoma, hepatosplenic T cell lymphoma, subattal NK cell lymphoma, mycosis, sezary syndrome, primary invasive skin CD30 positive T cell lymphoproliferative disorder, primary invasive skin anaplastic large cell lymphoma, human lymphomatoid lymphoma, human lymphoproliferative disorder of human skin positive T cells, primary invasive skin positive T cell lymphoproliferative disorder, human lymphomatoid tumor of human skin,Lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, non-specific peripheral T-cell lymphoma, and anaplastic large cell lymphoma. Exemplary forms of typical hodgkin lymphoma include: nodular sclerosing, mixed cell, lymphocyte-rich and lymphocyte-depleted or lymphocyte-non-depleted.

The term "sarcoma" as used herein is a cancer caused by variant cells in one of many tissues developed from embryonic mesoderm. Thus, sarcomas include tumors of bone, cartilage, fat, muscle, blood vessels, and hematopoietic tissues. For example, osteosarcoma is derived from bone, chondrosarcoma is derived from cartilage, liposarcoma is derived from fat, and leiomyosarcoma is derived from smooth muscle. Exemplary sarcomas include: askin's tumor, botryoid sarcoma, chondrosarcoma, Ewing's-PNET, malignant vascular endothelial cell tumor, malignant nerve sheath tumor, osteosarcoma, and soft tissue sarcoma. Subsets of soft tissue sarcomas include soft tissue alveolar sarcoma, angiosarcoma, phyllocystosarcoma, dermatofibrosarcoma, desmoplastic desmoid fibroma, desmoplastic small round cell tumor, epithelioid sarcoma, extraosseous chondrosarcoma, extraosseous osteosarcoma, fibrosarcoma, angiopericyte tumor, angiosarcoma, kaposi sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, and synovial sarcoma.

The term "leukemia" as used herein refers to a cancer of the blood or bone marrow characterized by an abnormal increase in leukocytes. Leukemia is a broad term covering a spectrum of diseases. Which in turn is part of a broader disease group called hematological neoplasms. Leukemias are subdivided into a number of major groups; the first group is between acute and chronic forms of leukemia. Acute leukemia is characterized by a rapid increase in the number of immature blood cells. The crowding caused by these cells prevents the bone marrow from producing healthy blood cells. Chronic leukemia is characterized by an excessive increase in relatively mature, but still abnormal, white blood cells. Typically developing over months or years, the cells are produced at a much faster rate than normal cells, resulting in the presence of many abnormal white blood cells in the blood. Leukemia is also subdivided by infected cells. This classification classifies leukemias either lymphoblastic or lymphocytic leukemia, as well as myeloid or myelogenous leukemia. In lymphoblastic or lymphocytic leukemias, cancerous changes occur in the type of myeloid cell that normally persists in the formation of lymphocytes. In myeloid leukemia or myelogenous leukemia, cancerous changes occur in the types of myeloid cells that normally continue to form red blood cells, some other types of white blood cells, and platelets. Combining these two classification methods provides all four main classifications. Within each of these four categories, there are generally several typical sub-categories. There are also rare types outside this classification. Exemplary leukemias include: acute Lymphoblastic Leukemia (ALL), Chronic Lymphocytic Leukemia (CLL), Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), Hairy Cell Leukemia (HCL), T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, juvenile myelomonocytic leukemia, B-cell prolymphocytic leukemia, Burkitt's leukemia, and adult T-cell leukemia.

The term "melanoma" as used herein is a cancer or malignancy of melanocytes. Melanocytes are cells that produce dark pigments, melanin, which are responsible for the color of the skin. They occur primarily in the skin, but are also found in other parts of the body, including the intestine and eyes. Melanomas are divided into the following types and subtypes: malignant lentigo, malignant lentigo melanoma, superficial spreading melanoma, acromelasma melanoma, mucosal melanoma, nodular melanoma, polypoidal melanoma, connective tissue proliferative melanoma, nonmelanoma, soft tissue melanoma, melanoma with small lentigo cells, melanoma with Spitz nevus characteristics, and pigmented layer melanoma.

The term "Germ Cell Tumor (GCT)" as used herein is a neoplasm obtained from germ cells. Germ cell tumors can be cancerous or non-cancerous tumors. Germ cells usually occur inside the gonads (ovary and testis). Germ cell tumors originating outside the gonads can be birth defects caused by errors in the development of the embryo. Germ cell tumors are roughly divided into two categories: germ cell tumors of germ cell tumor or seminoma and germ cell tumors of non-germ cell tumor or non-seminoma. Exemplary germ cell tumors or seminoma germ cell tumors include: germ cell tumors, dysgerminomas, and seminomas. Exemplary non-germ cell tumors or non-seminoma germ cell tumors include: embryonal carcinoma, endodermal sinus tumor or yolk sac tumor (EST, YST), choriocarcinoma, mature teratoma, dermatome cyst, immature teratoma, teratoma with malignancy, polyblastoma, gonadal blastoma and mixed GCT.

The term "metastasis" as used herein refers to the spread of a tumor or cancer from one organ or site to another, non-adjacent organ or site.

The term "biological sample" refers to a sample of biological material obtained from a mammalian subject (preferably, a human subject) and includes tissues, tissue samples, cell samples, tumor samples, stool samples, and biological fluids, e.g., blood, plasma, serum, saliva, urine, cerebral or spinal fluid, lymph fluid, and nipple aspirates. Biological samples can be obtained, for example, in the form of tissue biopsies, such as, for example, aspiration biopsies, brush biopsies, surface biopsies, needle biopsies, drill biopsies, resection biopsies, incisional biopsies, and endoscopic biopsies. In one embodiment, the biological sample is a blood, serum or plasma sample. In another embodiment, the biological sample is a saliva sample. In yet another embodiment, the biological sample is a urine sample.

An "isolate" of a biological sample (e.g., an isolate of a tissue or tumor sample) refers to a material or component (e.g., a biological material or component) that has been separated, obtained, extracted, purified, or isolated from the sample, and is preferably substantially free of undesired components and/or impurities or contaminants associated with the biological sample.

A "tissue sample" includes a part, slice, portion, piece, or fragment of tissue obtained or removed from a subject, preferably a human subject.

A "tumor sample" includes a part, slice, portion, piece, or fragment of a tumor, e.g., a tumor obtained or removed from a subject (preferably a human subject), e.g., a tumor removed or extracted from a tissue of a subject. Tumor samples can be obtained from primary tumors or metastatic tumors.

"mammal" for therapeutic purposes means any animal classified as a mammal, including humans, non-human primates, domestic and farm animals, zoo animals, sports animals, or pet animals, e.g., dogs, horses, cats, cows, etc., preferably, the mammal is a human.

The term "increased level" refers to a level above the normal or control level as generally defined or used in the relevant art. For example, the enhanced level of immunostaining in a tissue is a level of immunostaining that is considered by one of ordinary skill in the art to be higher than the level of immunostaining in a control tissue.

Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It will also be understood that there are many values disclosed herein, and that each value is also disclosed herein as "about" that particular value in addition to the value itself. For example, if the value "10" is disclosed, then "about 10" is also disclosed. It should also be understood that when a value is disclosed, then "less than or equal to" the value, "greater than or equal to the value," and possible ranges between values are also disclosed, as can be appreciated by those skilled in the art. For example, if the value "10" is disclosed, then "less than or equal to 10" and "greater than or equal to 10" are also disclosed. As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that include an antigen binding site that specifically binds to (immunoreacts with) an antigen.

Methods for detecting cancer by determining CCL25 and/or CCR9 expression or activity

CCL25 is a ligand for the CCR9 chemokine receptor. Both chemokines and receptors have been shown to modulate cancer metastasis and invasion. CCL25 and CCR9 are locally up-regulated in a variety of cancer tissue types (including ovarian, lung, breast, prostate, colon, bone, and pancreatic cancer) compared to normal tissue. CCL25 levels were also increased in the sera of subjects with those cancers. In addition, soluble CCL25 chemokines increased proliferation and metastasis of cancer cells in vivo and in vitro.

CCR9 is a member of the chemokine receptor family of G protein-coupled receptors (GPCRs) that can have multiple effects on the survival of cancer cells, presumably to protect them from the effects of chemotherapeutic drugs. We found that the interaction of CCR9 with CCL25 modulated Matrix Metalloproteinase (MMP) expression and increased the metastatic and invasive potential of cancer cells. This indicates that the CCR9-CCL25 interaction contributes to cancer cell metastasis and invasion. Therefore, blocking this axis is likely to inhibit cancer cell metastasis.

One aspect of the present application relates to a method of detecting the presence of cancer in a subject, the method comprising: detecting the expression level of one or more cancer markers in a biological sample obtained from the subject; and comparing the level of expression of one or more cancer markers in the biological sample to a normal level of expression of the one or more cancer markers, wherein a higher than normal level of expression of the one or more cancer markers in the biological sample indicates the presence of cancer in the subject, wherein the normal level of expression of the one or more cancer markers is a predetermined value or is obtained from a control sample of known normal non-cancerous cells of the same origin or type as the biological sample, and wherein the cancer is blastoma, carcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, or germ cell tumor, and wherein the one or more cancer markers comprises CCL25 or CCR9, or both CCL25 and CCR 9.

In one embodiment, the one or more cancer markers comprise: (1) CCL25 or CCR9, or both CCL25 and CCR9, and (2) CXCL13 or CXCR5, or both CXCL13 and CXCR 5. In another embodiment, the one or more cancer markers comprise: (1) CCL25 or CCR9, or both CCL25 and CCR9, and (2) CXCL16 or CXCR6, or both CXCL16 and CXCR 6.

In another embodiment, the one or more cancer markers comprise: (1) CCL25 or CCR9, or both CCL25 and CCR9, (2) CXCL13 or CXCR5, or both CXCL13 and CXCR5, and (3) CXCL16 or CXCR6, or both CXCL16 and CXCR 6. In another embodiment, the one or more cancer markers comprise: (1) CCL25 or CCR9, or both CCL25 and CCR9, and/or (2) CXCL13 or CXCR5, or both CXCL13 and CXCR5, and/or (3) CXCL16 or CXCR6, or both CXCL16 and CXCR6, and (4) one or more other cancer markers.

In yet another embodiment, the one or more additional cancer markers comprise: (1) CCL25 or CCR9, or both CCL25 and CCR9, and (2) a polypeptide selected from CXCL1, CXCL2, CXCL3, CXCL4, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCL 366372, CCL7, CCR7, a- α -binding protein (receptor binding protein), human chorion receptor binding protein (receptor) of human chorion receptor binding to human chorion receptor, human chorion receptor (receptor) receptor for human prostate receptor, androgen receptor, androgen receptor 7, androgen receptor (receptor for human, androgen receptor for human receptor, androgen receptor for human, androgen, Anti-p 53, osteopontin, ferritin, lysophosphatidylcholine, kinesin family member 4A (KIF4A), neurotrophin I (NPTX1), and fibroblast growth factor receptor 1 oncogene partner (FGFR1OP) protein.

In another embodiment, the cancer is breast cancer, and wherein the one or more cancer markers comprises (1) CCL25 or CCR9, or both CCL25 and CCR9, and (2) one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CXCR4, CXCR5, CXCR6, CX3CR1, HER2, RBM3, and CEA.

In another embodiment, the carcinoma is a prostate carcinoma, and wherein the one or more cancer markers comprise (1) CCL25 or CCR9, or both CCL25 and CCR9, and (2) one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CXCR4, CXCR5, CXCR6, CX3CR1, PSA, CEA, CGA, DHEA, NSE, PAP, prolactin, and B7-H3.

In yet another embodiment, the carcinoma is colorectal cancer, and wherein the one or more cancer markers comprise (1) CCL25 or CCR9, or both CCL25 and CCR9, and (2) one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CXCR4, CXCR5, CXCR6, CX3CR1, fibroblast activation protein alpha polypeptide, anti-p 53, osteopontin, and ferritin.

In yet another embodiment, the carcinoma is ovarian carcinoma, and wherein the one or more cancer markers comprise (1) CCL25 or CCR9, or both CCL25 and CCR9, and (2) one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CXCR4, CXCR5, CXCR6, CX3CR1, cancer antigen 125(CA-125), HE-4, OVX-1 macrophage colony stimulating factor (M-CSF), and lysophosphatidylcholine.

In yet another embodiment, the carcinoma is lung cancer, and wherein the one or more cancer markers comprise (1) CCL25 or CCR9, or both CCL25 and CCR9, and (2) one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL25, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CCR9, CXCR4, CXCR5, CXCR6, CX3CR1, kinesin family member 4A (KIF4A), neuropentraxin I (NPTX1), fibroblast growth factor receptor 1 oncogene partner (FGFR1OP) protein, and CEA.

In yet another embodiment, the cancer is pancreatic cancer or gastric cancer, and wherein the one or more cancer markers comprise (1) CCL25 or CCR9, or both CCL25 and CCR9, and (2) one or more cancer markers selected from CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CXCR4, CXCR5, CXCR6, CX3CR1, and CEA.

In yet another embodiment, the carcinoma is a brain cancer, a pituitary tumor, or a bone cancer, and wherein the one or more cancer markers further comprises one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CXCR4, CXCR5, CXCR6, and CX3CR 1.

In some other embodiments, the biological sample is a plasma sample, a saliva sample, or a urine sample.

In the context of the present application, the term "detection" is intended to include prediction and likelihood analysis. The present method is intended for clinical use in making decisions regarding cancer treatment regimens, including therapeutic interventions, diagnostic criteria such as disease stage (, and disease detection and monitoring.according to the present application, intermediate results may be provided to examine the condition of a subject.

Method of assessing prognosis of a subject with cancer

The methods of the present application for detecting cancer can also be used to assess the prognosis of a subject with cancer by comparing the expression level of one or more cancer markers in a biological sample obtained from the subject with the expression level of a reference sample.

Thus, another aspect of the present application relates to a method for assessing the prognosis of a subject suffering from cancer, the method comprising: determining the expression level of one or more cancer markers in a biological sample obtained from the subject; and comparing the expression level of the one or more cancer markers in the biological sample to a control expression level of the one or more cancer markers, wherein a higher level of expression of the one or more cancer markers in the biological sample relative to the control level is indicative of a poor prognosis for the subject, wherein a lower or similar expression level of the one or more cancer markers in the biological sample relative to the control level indicates a good prognosis of the subject, wherein poor prognosis means that the cancer is aggressive or invasive, wherein the cancer is blastoma, carcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, or germ cell tumor, and wherein the one or more cancer markers comprise CCL25 or CCR9, or both CCL25 and CCR 9.

In one embodiment, the one or more cancer markers further comprise CXCL13 or CXCR5, or both CXCL13 and CXCR 5. In another embodiment, the one or more cancer markers further comprise CXCL16 or CXCR6, or both CXCL16 and CXCR 6.

In another embodiment, the one or more cancer markers further comprise (1) CXCL13 or CXCR5, or both CXCL13 and CXCR5, and (2) CXCL16 or CXCR6, or both CXCL16 and CXCR 6.

In another embodiment, the one or more cancer markers further comprises one or more cancer markers selected from the group consisting of CXCL13, CXCR5, CXCL16, CXCR6, CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL27, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL12, CX3CL1, CCR2, CCR7, CCR8, CCR10, CXCR1, CXCR2, CXCR4, CXCR7, and CX3CR 1.

Alternatively, the level of one or more cancer markers in a biological sample can be determined over a spectrum of disease stages to assess the prognosis of the patient. An increase in the expression level of one or more cancer markers compared to a normal control level indicates a less desirable prognosis. A similar expression level of one or more cancer markers as compared to a normal control level indicates a more desirable prognosis for the patient.

In some other embodiments, the biological sample is a plasma sample, a saliva sample, or a urine sample.

Method for monitoring cancer treatment process

In certain embodiments, the level of one or more cancer markers is used to monitor the course of cancer therapy. In this method, the test biological sample is provided from a subject undergoing cancer therapy. Preferably, the plurality of test biological samples are obtained from the subject at different time points before, during or after treatment. The expression level of the cancer marker in the post-treatment sample can then be compared to the level of the cancer marker in the pre-treatment sample or to a reference sample (e.g., a normal control level). For example, if the marker level after treatment is lower than the marker level before treatment, one can conclude that the treatment is effective. Similarly, one can conclude that the treatment is effective if the marker level after treatment is similar or identical to the normal control marker level.

By "therapeutically effective" is meant that the treatment results in a decrease in the level of a cancer marker or a decrease in the size, prevalence, or metastatic capacity of the cancer in the subject. When treatment is applied prophylactically, "effective" means that the treatment retards or prevents the onset of cancer or slows the clinical symptoms of cancer. Cancer assessments can be made using standard clinical protocols. In addition, the effectiveness of the treatment can be determined in conjunction with any known method for diagnosing or treating cancer. For example, cancer is routinely diagnosed histopathologically or by identifying symptoms of abnormalities (e.g., weight loss and anorexia).

Thus, another aspect of the present application relates to a method for monitoring the course of cancer therapy in a subject, the method comprising: determining the expression level of the one or more cancer markers in one or more biological samples obtained from the subject during or after the treatment; and comparing the expression level of the one or more cancer markers in the one or more biological samples to a control expression level of the one or more cancer markers, wherein the control level of the one or more cancer markers is a pre-treatment level or a predetermined reference level of the one or more cancer markers in the subject, wherein the treatment is deemed effective if the one or more cancer markers in the one or more biological samples is similar to or below the control level, wherein the cancer is blastoma, carcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, or germ cell tumor, and wherein the one or more cancer markers comprises CCL25 or CCR9, or both CCL25 and CCR 9.

In one embodiment, the one or more cancer markers further comprise CXCL13 or CXCR5, or both CXCL13 and CXCR 5. In yet another embodiment, the one or more cancer markers further comprise CXCL16 or CXCR6, or both CXCL16 and CXCR 6.

In one embodiment, the one or more cancer markers further comprise (1) CXCL13 or CXCR5, or both CXCL13 and CXCR5, and (2) CXCL16 or CXCR6, or both CXCL16 and CXCR 6.

In yet another embodiment, the one or more cancer markers further comprises one or more cancer markers selected from the group consisting of CXCL13, CXCR5, CXCL16, CXCR6, CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL27, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL12, CX3CL1, CCR2, CCR7, CCR8, CCR10, CXCR1, CXCR2, CXCR4, CXCR7, and CX3CR 1.

Cancer markers

The term "cancer marker" as used herein refers to or describes a polypeptide or polynucleotide whose expression level, either alone or in combination with other polypeptides or polynucleotides, is correlated with cancer or the prognosis of cancer. Such a correlation may involve increased or decreased expression of the polypeptide or polynucleotide. For example, expression of a polypeptide or polynucleotide is indicative of cancer, or a lack of expression of a polypeptide or polynucleotide may be associated with poor prognosis in a cancer patient.

The term "expression level of a cancer marker" can be measured at the transcriptional level (in which case the presence and/or amount of a polynucleotide is determined), or at the translational level (in which case the presence and/or amount of a polypeptide is determined). Cancer marker expression can be characterized by using any suitable method.

Examples of such cancer markers include CCL25, CCR9 and other chemokines and chemokine receptors, e.g., CXCL1, CXCL2, CXCL3, CXCL4, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCR8, a-specific neuropeptide (fca), a, gcc α -receptor for a), a, CCR8, a, Osteopontin, ferritin, lysophosphatidylcholine, kinesin family member 4A (KIF4A), neurotrophin I (NPTX1), and fibroblast growth factor receptor 1 oncogene partner (FGFR1OP) protein.

In one embodiment, the cancer marker used in the present invention is selected from the group of melanoma markers comprising CCL25, CCR9, CXCL13, CXCR5, CXCL16, CXCR6, CCL27, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL12, CX3CL1, CCR10, CXCR1, CXCR2, CXCR4, and CX3CR 1. The markers in the melanoma group can be used to detect melanoma or to predict the prognosis of a subject with melanoma.

In one embodiment, the above cancer marker is selected from the group of cancer markers comprising CCL25, CCR9, CXCL13, CXCR5, CCL1, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL16, CCR7, CCR8, CXCR4, CXCR6 and CX3CR 1. The markers in the cancer marker panel can be used to detect cancer or to predict the prognosis of a subject with cancer.

In another embodiment, the above cancer marker is selected from the group of breast cancer markers consisting of CCL25, CCR9, CXCL13, CXCR5, CCL1, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL16, CCR7, CCR8, CXCR4, CXCR6, CX3CR1, HER2, RNA binding motif 3("RBM3"), and carcinoembryonic antigen (CEA). The markers in the breast cancer panel can be used to detect breast cancer or to predict the prognosis of a subject with breast cancer.

In another embodiment, the above cancer marker is selected from the group of prostate cancer markers consisting of CCL25, CCR9, CXCL13, CXCR5, CXCL16, CXCR6, CCL1, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CCR7, CCR8, CXCR4, CX3CR1, PSA, CEA, CGA, DHEA, NSE, PAP, prolactin, and B7-H3. The markers in the breast cancer panel may be used to detect prostate cancer or to predict the prognosis of a subject with prostate cancer.

In another embodiment, the one or more cancer markers are selected from the group of colorectal cancer markers comprising CCL25, CCR9, CXCL13, CXCR5, CXCL16, CXCR6, CCL1, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CCR7, CCR8, CXCR4, CX3CR1, fibroblast activation protein alpha polypeptide, anti-p 53, osteopontin, and ferritin. The markers in the group of colorectal cancers may be used to detect colorectal cancer or to predict the prognosis of a subject with colorectal cancer.

In another embodiment, the above cancer marker is selected from the group consisting of ovarian cancer markers consisting of CCL25, CCR9, CXCL13, CXCR5, CXCL16, CXCR6, CCL1, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CCR7, CCR8, CXCR4, CX3CR1, cancer antigen 125(CA-125), HE-4, OVX-1 macrophage colony stimulating factor (M-CSF), and lysophosphatidylcholine. The markers in the ovarian cancer panel can be used to detect ovarian cancer or to predict the prognosis of a subject with ovarian cancer.

In another embodiment, the above cancer marker is selected from the group of lung cancer markers consisting of CCL25, CCR9, CXCL13, CXCR5, CXCL16, CXCR6, CCL1, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CCR7, CCR8, CXCR4, CX3CR1, kinesin family member 4A (KIF4A), neurotropic pentameric protein I (NPTX1), fibroblast growth factor receptor 1 oncogene partner (FGFR1OP) protein, and CEA. The markers in the lung cancer panel can be used to detect lung cancer or predict the prognosis of a subject with lung cancer.

In another embodiment, the one or more cancer markers is selected from the group consisting of pancreatic cancer or gastric cancer markers comprising CCL25, CCR9, CXCL13, CXCR5, CXCL16, CXCR6, CCL1, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CCR7, CCR8, CXCR4, CX3CR1, and CEA. The markers in the pancreatic cancer group can be used to detect pancreatic cancer or gastric cancer, or to predict the prognosis of a subject with pancreatic cancer.

In another embodiment, the one or more cancer markers are selected from the group consisting of brain cancer, pituitary tumor, bone cancer, pancreatic cancer (pancratic cancer), or gastric cancer markers comprising one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CXCR4, CXCR5, CXCR6, and CX3CR 1.

Detection method

The expression of the cancer marker can be measured at the transcriptional level (i.e., the amount of mRNA) or the translational level (i.e., the amount of protein). In certain embodiments, the expression of the cancer marker is determined at the mRNA level by quantitative RT-PCR, northern blot, or other methods known to those skilled in the art. In other embodiments, expression of the cancer marker is determined at the protein level by ELISA, western blot, or other types of immunodetection methods using anti-cancer marker antibodies, such as anti-CCL 25 and anti-CCR 9 antibodies, anti-CXCL 13 and anti-CXCR 5 antibodies, and anti-CXCL 16 and anti-CXCR 6 antibodies.

In certain embodiments, the anti-CCL 25 and/or anti-CCR 9 antibody comprises an antibody that specifically binds to a CCL25 peptide or a CCR9 peptide. The CCL25 peptides include, but are not limited to, peptides consisting of one or more sequences selected from LAYHYPIGWAVL (SEQ ID NO:116), KRHRKVCGNPKSREVQRAMKLLDARNKVFAKLHH (SEQ ID NO:117), FEDCCLAYHYPIGWAVLRRA (SEQ ID NO:118), IQEVSGSCNLPAAIFYLPKRHRKVCGN (SEQ ID NO:119), AMKLLDAR (SEQ ID NO:120), KVFAKLHHN (SEQ ID NO:121), QAGPHAVKKL (SEQ ID NO:122), FYLPKRHRKVCGNP (SEQ ID NO:123) YLPKRHRKVCGNPK (SEQ ID NO:124), LPKRHRKVCGNPKS (SEQ ID NO:125), PKRHRKVCGNPKSR (SEQ ID NO:126), CGNPKSREVQRAMK (SEQ ID NO:127), GNPKSREVQRAMKL (SEQ ID NO:128), KFSNPISSSKRNVS (SEQ ID NO:129), PKSREV (SEQ ID NO:130), LHHNTQT (SEQ ID NO:131) and SSSN (SEQ ID NO:132), or peptides containing one or more sequences selected from LAYHYPIGWAVL (SEQ ID NO:116), KRHRKVCGNPKSREVQRAMKLLDARNKVFAKLHH (SEQ ID NO:117), FEDCCLAYHYPIGWAVLRRA (SEQ ID NO:118), IQEVSGSCNLPAAIFYLPKRHRKVCGN (SEQ ID NO:119), AMKLDAR (SEQ ID NO:120), KVFAKLHHN (SEQ ID NO:121), QAGPHAVKKL (SEQ ID NO:122), FYLPKRHRKVCGNP (SEQ ID NO:123), YLPKRHRKVCGNPK (SEQ ID NO:124), LPKRHRKVCGNPKS (SEQ ID NO:125), PKRHRKVCGNPKSR (SEQ ID NO:126), CGNPKSREVQRAMK (SEQ ID NO:127), GNPKSREVQRAMKL (SEQ ID NO:128), KFSNPISSSKRNVS (SEQ ID NO:129), PKSREV (SEQ ID NO:130), LHHNTQT (SEQ ID NO:131) and SSSKRN (SEQ ID NO: 132). Examples of CCR9 peptides include, but are not limited to, peptides consisting of one or more sequences selected from QFASHFLPP (SEQ ID NO:133), AAADQWKFQ (SEQ ID NO:134), TFMCKVVNSM (SEQ ID NO:135), IAICTMVYPS (SEQ ID NO:136) and VQTIDAYAMFISNCAVSTNIDICFQ (SEQ ID NO:137), or peptides comprising one or more sequences selected from QFASHFLPP (SEQ ID NO:133), AAADQWKFQ (SEQ ID NO:134), TFMCKVVNSM (SEQ ID NO:135), IAICTMVYPS (SEQ ID NO:136) and VQTIDAYAMFISNCAVSTNIDICFQ (SEQ ID NO: 137).

In another embodiment, the anti-CXCL 13 and/or anti-CXCR 5 antibody comprises an antibody that specifically binds to a CXCL13 peptide or a CXCR5 peptide. Examples of the CXCL13 peptide include, but are not limited to, peptides consisting of peptides selected from the group consisting of RSSSTLPVPVFKRKIP (SEQ ID NO:45), PRGNGCPRKEIIVWKK (SEQ ID NO:46), LPRGNGCPRKEIIVWK (SEQ ID NO:47), QILPRGNGCPRKEIIV (SEQ ID NO:48), ILPRGNGCPRKEIIVW (SEQ ID NO:49), RIQILPRGNGCPRKEI (SEQ ID NO:50), RGNGCPRKEIIVWKKN (SEQ ID NO:51), KRSSSTLPVPVFKRKI (SEQ ID NO:52), IQILPRGNGCPRKEII (SEQ ID NO:53), DRIQILPRGNGCPRKE (SEQ ID NO:54), RKRSSSTLPVPVFKRK (SEQ ID NO:55), RCRCVQESSVFIPRRF (SEQ ID NO:56), GNGCPRKEIIVWKKNK (SEQ ID NO:57), CVQESSVFIPRRFIDR (SEQ ID NO:58), IDRIQILPRGNGCPRK (SEQ ID NO:59), LRCRCVQESSVFIPRR (SEQ ID NO:60), FIDRIQILPRGNGCPR (SEQ ID NO:61), RCVQESSVFIPRRFID (SEQ ID NO:62), CRCVQESSVFIPRRFI (SEQ ID NO:63), QESSVFIPRRFIDRIQ (SEQ ID NO:64), RFIDRIQILPRGNGCP (SEQ ID NO:65), VQESSVFIPRRFIDRI (SEQ ID NO:66), ESSVFIPRRFIDRIQI (SEQ ID NO:67), SLRCRCVQESSVFIPR (SEQ ID NO:68), NGCPRKEIIVWKKNKS (SEQ ID NO:69), PQAEWIQRMMEVLRKR (SEQ ID NO:70), RRFIDRIQILPRGNGC (SEQ ID NO:71), LRKRSSSTLPVPVFKR (SEQ ID NO:72), VQESSVFIPRR (SEQ ID NO:73, EWIQRMMEVLRKRSSSTLPVPVFKRK (SEQ ID NO:74), KKNK (SEQ ID NO:75), RKRSSS (SEQ ID NO:76), RGNGCP (SEQ ID NO:77), VYYTSLRCRCVQESSVFIPRR (SEQ ID NO:78), DRIQILP (SEQ ID NO:79), RKEIIVW (SEQ ID NO:80) and KSIVCVDPQ (SEQ ID NO:81) or a peptide comprising one or more sequences selected from RSSSTLPVPVFKRKIP (SEQ ID NO:45), PRGNGCPRKEIIVWKK (SEQ ID NO:46), LPRGNGCPRKEIIVWK (SEQ ID NO:47), QILPRGNGCPRKEIIV (SEQ ID NO:48), ILPRGNGCPRKEIIVW (SEQ ID NO:49), RIQILPRGNGCPRKEI (SEQ ID NO:50), RGNGCPRKEIIVWKKN (SEQ ID NO:51), KRSSSTLPVPVFKRKI (SEQ ID NO:52), IQILPRGNGCPRKEII (SEQ ID NO:53), DRIQILPRGNGCPRKE (SEQ ID NO:54), RKRSSSTLPVPVFKRK (SEQ ID NO:55), RCRCVQESSVFIPRRF (SEQ ID NO:56), GNGCPRKEIIVWKKNK (SEQ ID NO:57), CVQESSVFIPRRFIDR (SEQ ID NO:58), IDRIQILPRGNGCPRK (SEQ ID NO:59), LRCRCVQESSVFIPRR (SEQ ID NO:60), FIDRIQILPRGNGCPR (SEQ ID NO:61), RCVQESSVFIPRRFID (SEQ ID NO:62), CRCVQESSVFIPRRFI (SEQ ID NO:63), QESSVFIPRRFIDRIQ (SEQ ID NO:64), RFIDRIQILPRGNGCP (SEQ ID NO:65), VQESSVFIPRRFIDRI (SEQ ID NO:66), ESSVFIPRRFIDRIQI (SEQ ID NO:67), SLRCRCVQESSVFIPR (SEQ ID NO:68), NGCPRKEIIVWKKNKS (SEQ ID NO:69), PQAEWIQRMMEVLRKR (SEQ ID NO:70), RRFIDRIQILPRGNGC (SEQ ID NO:71), LRKRSSSTLPVPVFKR (SEQ ID NO:72), VQESSVFIPRR (SEQ ID NO:73, EWIQRMMEVLRKRSSSTLPVPVFKRK (SEQ ID NO:74), NK (SEQ ID NO:75), RKRSSS (SEQ ID NO:76), RGNGCP (SEQ ID NO:77), VYYTSLRCRCVQESSVFIPRR (SEQ ID NO:78), DRIQILP (SEQ ID NO:79), RKEIIVW (SEQ ID NO:80) and KSIVCVDPQ (SEQ ID NO: 81). Examples of such CXCR5 peptides include, but are not limited to, peptides consisting of one or more sequences selected from TSLVENHLCPATE (SEQ ID NO:82), EGSVGWVLGTFLCKT (SEQ ID NO:83), LPRCTFS (SEQ ID NO:84), LARLKAVDNT (SEQ ID NO:85) and MASFKAVFVP (SEQ ID NO:86), or peptides containing one or more sequences selected from TSLVENHLCPATE (SEQ ID NO:82), EGSVGWVLGTFLCKT (SEQ ID NO:83), LPRCTFS (SEQ ID NO:84), LARLKAVDNT (SEQ ID NO:85) and MASFKAVFVP (SEQ ID NO: 86).

anti-CXCL 16 and/or anti-CXCR 6 antibodies include antibodies that specifically bind to a CXCL16 peptide or a CXCR6 peptide. Examples of the CXCL16 peptide include, but are not limited to, peptides consisting of peptides selected from the group consisting of AAGPEAGENQKQPEKN (SEQ ID NO:87), SQASEGASSDIHTPAQ (SEQ ID NO:88), STLQSTQRPTLPVGSL (SEQ ID NO:89), SWSVCGGNKDPWVQEL (SEQ ID NO:90), GPTARTSATVPVLCLL (SEQ ID NO:91), SGIVAHQKHLLPTSPP (SEQ ID NO:92), RLRKHL (SEQ ID NO:93), LQSTQRP (SEQ ID NO:94), SSDKELTRPNETT (SEQ ID NO:95), AGENQKQPEKNA (SEQ ID NO:96), NEGSVT (SEQ ID NO:97), ISSDSPPSV (SEQ ID NO:98), CGGNKDPW (SEQ ID NO:99), LLPTSPPISQASEGASSDIHT (SEQ ID NO:100), STQRPTLPVGSLSSDKELTRPNETTIHT (SEQ ID NO:101), SLAAGPEAGENQKQPEKNAGPTARTSA (SEQ ID NO:102), TGSCYCGKR (SEQ ID NO:103), DSPPSVQ (SEQ ID NO:104), RKHLRAYHRCLYYTRFQLLSWSVCGG (SEQ ID NO:105), WVQELMSCLDLKECGHAYSGIVAHQKHLLPTSPPISQ (SEQ ID NO:106), SDIHTPAQMLLSTLQ (SEQ ID NO:107), SEQ ID NO:108 (SEQ ID NO: 686) and 686 (SEQ ID NO:109), GKRISSDSPPSVQ (SEQ ID NO:110) and KDPWVQELMSCLDLKECGHAYSGIVAHQKH (SEQ ID NO:111) or a peptide comprising one or more sequences selected from AAGPEAGENQKQPEKN (SEQ ID NO:87), SQASEGASSDIHTPAQ (SEQ ID NO:88), STLQSTQRPTLPVGSL (SEQ ID NO:89), SWSVCGGNKDPWVQEL (SEQ ID NO:90), GPTARTSATVPVLCLL (SEQ ID NO:91), SGIVAHQKHLLPTSPP (SEQ ID NO:92), RKRLHL (SEQ ID NO:93), LTQRP (SEQ ID NO:94), SSDKELTRPNETT (SEQ ID NO:95), AGENQKQPEKNA (SEQ ID NO:96), NEGSVT (SEQ ID NO:97), ISSDSPPSV (SEQ ID NO:98), CGGNPW (SEQ ID NO:99), LLPTSPPISQASEGASSDIHT (SEQ ID NO:100), QS STQRPTLPVGSLSSDKELTRPNETTIHT (SEQ ID NO:101), SLAAGPEAGENQKQPEKNAGPTARTSA (SEQ ID NO:102), TGSCYCGKR (SEQ ID NO:103), DSPPSVQ (SEQ ID NO:104), RKHLRAYHRCLYYTRFQLLSWSVCGG (SEQ ID NO:105), WVQELMSCLDLKECGHAYSGIVAHQKHLLPTSPPISQ (SEQ ID NO:107), SEQ ID NO:107 (SEQ ID NO:107), RPTLPVGSL (SEQ ID NO:108), TAGHSLAAG (SEQ ID NO:109), GKRISSDSPPSVQ (SEQ ID NO:110) and KDPWVQELMSCLDLKECGHAYSGIVAHQKH (SEQ ID NO: 111). Examples of such CXCR6 peptides include, but are not limited to, peptides consisting of one or more sequences selected from HQDFLQFSKV (SEQ ID NO:112), AGIHEWVFGQVMCK (SEQ ID NO:113), PQIIYGNVFNLDKLICGYHDEAI (SEQ ID NO:114) and YYAMTSFHYTIMVTEA (SEQ ID NO:115) or peptides comprising one or more sequences selected from HQDFLQFSKV (SEQ ID NO:112), AGIHEWVFGQVMCK (SEQ ID NO:113), PQIIYGNVFNLDKLICGYHDEAI (SEQ ID NO:114) and YYAMTSFHYTIMVTEA (SEQ ID NO: 115).

In one embodiment, the antibody is bound to a solid support. By "solid support" is meant a non-aqueous matrix to which the antibodies of the present application can be attached or linked. Examples of solid phases included herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, silicones, and plastics (e.g., polystyrene, polypropylene, and polyvinyl alcohol).

Enzyme-linked immunosorbent assay

In certain embodiments, the cancer marker is detected by using an enzyme-linked immunosorbent assay (ELISA), which is typically performed by using an antibody-coated test plate or well. The ELISA assay conventionally used uses either sandwich immunoassay (sandwich immunoassay) or competitive binding immunoassay (competitive binding immunoassay).

Briefly, sandwich immunoassays are methods that use two antibodies that bind to different sites on an antigen or ligand. A first antibody having high specificity for an antigen is attached to a solid surface. The antigen is then added, followed by a second antibody, called the detection antibody. The detection antibody binds the antigen to a different epitope than the first antibody. As a result, the antigen is "sandwiched" between the two antibodies. The affinity of an antibody for an antigen is often the primary determinant of immunoassay sensitivity. As the concentration of antigen increases, the amount of detection antibody also increases, resulting in a higher measured response. The standard curve of the sandwich-binding assay has a positive slope. To quantify the extent of binding, different reporter factors may be used. Typically, the enzyme is attached to a second antibody which must be produced in a different species than the first antibody (i.e., if the first antibody is a rabbit antibody, the second antibody will be an anti-rabbit antibody from sheep, chicken, etc., rather than a rabbit antibody). The substrate for the enzyme is added to the reaction which forms a colorimetric reading (readout) as the detection signal. The signal generated is proportional to the amount of target antigen present in the sample.

The antibody-linked reporter factor used to determine the binding event determines the detection mode. A spectrophotometric plate reader (reader) may be used for colorimetric detection. Many kinds of reporter factors have been recently developed to increase the sensitivity of immunoassays. For example, chemiluminescent substrates have been developed which further amplify the signal and can be read on a luminescent plate reader. Furthermore, fluorescence readings in which the enzyme step of the assay is replaced with a fluorophor-labeled antibody are becoming very popular. This reading is then determined by using a fluorescence plate reader.

Competitive binding assays are based on the competition of labeled or unlabeled ligands for a limited number of antibody binding sites. Competitive inhibition assays are often used to measure smaller analytes. These assays are also used when the antibody and analyte pair is not present. The unique antibody was used in a competitive binding ELISA. This is due to steric hindrance if both antibodies attempt to bind to a very small molecule. A fixed amount of labeled ligand (tracer) and a variable amount of unlabeled ligand are incubated with the antibody. According to the law of mass action, the amount of labeled ligand is a function of the total concentration of labeled ligand and unlabeled ligand. As the concentration of unlabeled ligand increases, less labeled ligand binds to the antibody and the measured response decreases. Thus, the lower the signal, the more unlabeled analyte is present in the sample. The standard curve of the competitive binding assay has a negative slope.

Microbeads

In certain other embodiments, the cancer marker is detected by using antibody-coated microbeads. In some embodiments, the bead is a magnetic bead. In other embodiments, the beads are internally color-coded with a fluorescent dye, and the surfaces of the beads are labeled with an anti-cancer marker antibody (e.g., an anti-CCL 25 antibody or an anti-CCR 9 antibody) that can bind to a cancer marker in the test sample. In turn, the cancer marker is labeled directly with a fluorescent label or indirectly with an anti-marker antibody that binds to the fluorescent label. Thus, there are two sources of color, one from the bead and one from the fluorescent label. Alternatively, the beads may be internally coded in different sizes.

By using a mixture of different fluorescence intensities from the two dyes and different sized beads, the assay can measure up to hundreds of different cancer markers. During the assay, a mixture containing color/size encoded beads, fluorescently labeled anti-marker antibodies and sample are combined and injected into an instrument that uses sophisticated fluidic techniques to modulate the beads. The beads are then subjected to a laser and, based on their color or size, sorted or color intensity determined, which is processed to obtain quantitative data for each reaction.

When the sample is directly labeled with a fluorophore, the system can read or quantify the unique fluorescence on the beads without removing unbound fluorophore from the solution. The assay can be multiplexed by distinguishing between beads of different colors or sizes. Real-time testing is achievable when the sample directly requires an unlabeled sample. Standard assay procedures include incubating the sample with anti-marker antibody-coated beads, incubating with a biotin or fluorophore-labeled secondary antibody, and examining the fluorescent signal. The fluorescent signal can be visualized on the beads (by adding streptavidin-fluorophore conjugates to the biotinylated secondary antibody) and read by a bead analyzer. Bead-based immunoassays may be either sandwich-type or competitive-type immunoassays, relying on an anti-label immobilized on the bead surface.

Test strip

In some other embodiments, the cancer marker in the liquid biological sample is detected by using a test strip. The test strip typically includes a fluid impermeable housing and a fluid permeable "strip" having one or more detection regions. In one embodiment, each detection zone includes a dried binding reagent that binds to a cancer marker in the biological sample. In other embodiments, the dried binding reagent is a labeled binding reagent. In another embodiment, the test strip may further include a control region to indicate that the assay sample has been satisfactorily performed, that is, that the reagents are present in the test strip, and that they have become mobile and have been transported along the fluid path during the course of the experimental run. The control region also indicates that the reagents in the device are capable of immunochemical interaction, confirming the chemical integrity of the device. This is important when considering storage and transport of the apparatus under drying conditions within a certain temperature range. The control zone is typically placed downstream of the detection zone and may, for example, comprise an immobilized binding reagent for labeling the binding reagent. The labelled binding reagent may be present at a zone upstream of the mobile form of the control zone and the detection zone. The labeled binding reagent may be the same or different than the labeled binding reagent for the cancer marker.

In one embodiment, the test strip includes a fluid porous sample receptacle connected to and upstream of one or more flow paths. The porous sample receiver may be universal to all assay methods. In this way, a fluid sample for a conventional sample application zone of the device can flow along one or more flow paths to each detection zone. The porous sample receiver may be provided in a housing or may extend at least partially outside the housing and may be used, for example, to collect bodily fluids. The porous sample receiver may also act as a fluid reservoir. The porous sample receiving member is made of any absorbent material, porous material or fibrous material capable of rapidly absorbing liquid. The porosity of the material may be unidirectional (i.e., pores or fibers running wholly or predominantly parallel to the axis of the component) or multidirectional (omnidirectional, such that the component has an amorphous sponge-like structure). Porous plastic materials such as polypropylene, polyethylene (preferably of very high molecular weight), polyvinylidene fluoride, ethylene vinyl acetate, acrylonitrile and polytetrafluoroethylene may be used. Other suitable materials include fiberglass.

An absorbent "reservoir" may be provided at the distal end of the support material, if desired. The absorbent reservoir may comprise, for example, Walter door (Whatman) 3MM chromatography paper, and should provide sufficient absorbent capacity to allow any unbound labeled binding reagent to wash out of the test zone. As an alternative to this reservoir, it is sufficient to have a length of porous solid material extending beyond the detection zone.

After the binding reagent is applied to the detection zone, the residue of the porous solid phase material may be treated to block any remaining binding sites. Blocking may be achieved, for example, by treatment with a protein (e.g., bovine serum albumin or milk protein) or with polyvinyl alcohol or ethanolamine or a combination thereof. To assist in the free movement of the labeled binding reagent, the porous carrier may further include a sugar such as sucrose or lactose and/or other substances (e.g., polyvinyl alcohol (PVA) or polyvinyl pyrrolidone (PVP)) when wetted with the sample.

Alternatively, the porous carrier may not be closed at the time of manufacture; alternatively, the component for enclosing the porous carrier is comprised in the material upstream of the porous carrier. When wetting the test strip, the means for enclosing the porous carrier is moved and the enclosing means flows into and through the porous carrier, with the flow effecting an enclosure. Blocking components include proteins such as BSA and casein; and polymers, such as PVP, PVA; and sugars and detergents, such as Triton-X100. The closure member may be present in a macroporous support material.

The dry binding reagent may be provided on a porous support material disposed upstream of the porous support material comprising the detection zone. The upstream porous support material may be macroporous. The macroporous support material should be low protein-bound or non-protein-bound, or should be readily blockable by a reagent such as BSA or PVA, to minimize non-specific binding and to facilitate free movement of the labeling reagent after the macroporous body has been wetted with the liquid sample. If desired, the macroporous support material may be pretreated with a surfactant or solvent to make it more hydrophilic and to promote rapid absorption of the liquid sample. Suitable materials for the macroporous support include plastic materials such as polyethylene and polypropylene; or other materials such as paper or fiberglass. Where the label binding reagent is labeled with a detectable particle, the macroporous body may have a pore size at least 10 times greater than the maximum particle size of the particulate label. A larger pore size gives better release of the labeling agent. As an alternative to a macroporous support, the labelled binding reagent may be disposed on a non-porous substance disposed upstream of the detection zone, the non-porous substance forming part of the flow path. In another embodiment, the test strip may further comprise a sample receiving member for receiving a fluid sample. The sample receiving member may extend from the outer cover.

The housing may be constructed of a fluid impermeable material. The housing also desirably excludes ambient light. When penetrating from the outside of the device into the inside of the device, the housing is considered to substantially exclude ambient light if less than 10%, preferably less than 5%, and more preferably less than 1% of visible light is incident. Light-impermeable synthetic plastic materials, such as polycarbonate, ABS, polystyrene (polystyrene), polystyrene (polystyrol), high-density polyethylene or polypropylene, containing suitable light-blocking pigments, are suitable choices for constructing the housing. The opening may be disposed on an exterior of the housing in communication with an assay disposed within the interior space within the housing. Alternatively, the aperture may be used to extend the porous sample receiver from the housing to a position outside the housing.

Micro-lattice

In other embodiments, the cancer marker is detected by a protein microarray comprising immobilized cancer marker-specific antibodies on its surface. The microarray may be used in a "sandwich" assay, where antibodies on the microarray capture cancer markers in a test sample and the captured markers are detected with a labeled secondary antibody that specifically binds to the captured markers. In a preferred embodiment, the second antibody is biotinylated or enzyme-labeled. The detection is achieved by subsequent incubation with either streptavidin-fluorophore conjugates (for fluorescent detection) or enzyme substrates (for colorimetric detection).

Typically, microarray assays include multiple incubation steps, including incubation with a sample and incubation with various reagents (e.g., a first antibody, a second antibody, a reporter reagent, etc.). Repeated washes are also required between incubation steps. In one embodiment, the microarray assay is performed in a rapid assay format requiring only one or two incubations. It is also envisioned that the formation of detectable immune complexes (e.g., captured cancer marker/anti-marker antibody/indicator complexes) may be accomplished in a single incubation step by exposing the protein microarray to a mixture of the sample and all of the desired reagents. In one embodiment, the first and second antibodies are the same antibody.

In another embodiment, the protein array provides a competitive immunoassay. Briefly, a microarray comprising immobilized anti-marker antibodies is incubated with a test sample in the presence of labeled cancer marker standards. The labeled cancer marker competes with the unlabeled cancer marker in the test sample for binding to the immobilized antigen-specific antibody. In this competition mechanism, an increase in the concentration of the specific cancer marker in the test sample will result in a decrease in binding of the labeled cancer marker standard to the immobilized antibody, and thus a decrease in the intensity of the signal derived from the marker.

The microarray may be performed in a manual, semi-automatic or automatic mode. Manual mode refers to manual operation of the assay steps, including reagent and sample delivery onto the microarray, sample incubation, and microarray washing. Semi-automatic mode refers to manual operation to deliver samples and reagents to the microarray while automatically running incubation and washing steps. In the automatic mode, the three steps (sample/reagent delivery, incubation and washing) can be controlled by a computer with a keypad or an integrated experimental circuit board unit. For example, the microarray may be produced by the protein array Workstation (Perkinelmer Life Sciences, Boston, Mass.) or the Assay1200^TMThe workbation (Zyomyx, Hayward, Calif.). Scanners using fluorescence, colorimetry and chemiluminescence can be used for detectionThe microarray signals are measured and microarray images are captured. Microarray-based quantitation can also be achieved by other means, such as mass spectrometry and surface plasmon resonance (surface plasmon resonance). The captured microarray images may be analyzed by a separate image analysis software or using an image acquisition and analysis software package. For example, quantification of antigen microarrays can be achieved using a fluorescent PMT-based scanner- -ScanArray3000(General Scanning, Watertown, Mass.) or a colorimetric-based CCD scanner VisionSpot (Allied Biotech, Ijamsville, Md.). Typically, image analysis will involve data acquisition and preparation of an analysis report with a separate software package. To speed up the overall analysis process from capturing images to generating analysis reports, all analysis steps, including image capture, image analysis, and report generation, may be limited to and/or controlled by one software package. This unified control system will provide image analysis and generation of analysis reports in a user-friendly manner.

Implantable biosensor

In other embodiments, the cancer marker is detected by using an implantable biosensor. Biosensors are electronic devices that produce an electronic signal as a result of a biological interaction. In one embodiment, the biosensor uses antibodies, receptors, nucleic acids, or other components of a binding pair that bind to a cancer marker, which is typically the other component of the binding pair. Biosensors may be used with blood samples to determine the presence of cancer markers without the sample preparation and/or separation steps typically required for automated immunoassay systems.

In one embodiment, the sensor is a nanoscale device. The sensing system includes a biological recognition element coupled to the nanowire and a detector capable of determining a property associated with the nanowire. The biological recognition element is one component of a binding pair (e.g., a receptor for a cancer marker or an anti-cancer marker antibody), wherein the cancer marker being measured is the other component of the binding pair. Preferably, the nanowire sensor comprises a semiconductor nanowire having an outer surface formed thereon to form a gate; and a first end in electrical contact with the conductor to form a source electrode; and a second end in electrical contact with the conductor to form a drain. In one embodiment, the sensor is a field effect transistor comprising a substrate formed of an insulating material, a source, a drain, and a semiconductor nanowire disposed therebetween having a biological recognition element attached to a surface of the nanowire. When a binding event occurs between a biological recognition element and its specific binding partner, the detectable change occurs as a current-voltage characteristic of a field effect transistor.

In another embodiment, the sensing system includes a sensor array. One or more sensors in the array are coupled with a shield member that prevents interaction of the associated sensor with the surrounding environment. At a selected time, the guard member may be inactive, thus allowing the sensor to begin operating to interact with the surrounding fluid or tissue so that the biometric element may interact with the other member of its binding pair (if that counterpart member is present).

In another embodiment, the shield member is formed of an electrically conductive material that is oxidizable, biocompatible, bioabsorbable, and dissolvable in a solution such as blood upon application of an electrical potential. For example, the sensor may be formed in a hole of a substrate covered with a conductive material such as a biocompatible metal or an electroerosive polymer. In another embodiment, the shielding member is formed by using a material that dissolves within a predetermined period of time.

Mass spectrometry

In other embodiments, the cancer marker is detected by using Mass Spectrometry (MS), e.g., MALDI/TOF (time of flight), SELDI/TOF, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry, nuclear magnetic resonance spectroscopy, or tandem mass spectrometry (e.g., MS/MS, ESI-MS/MS, etc.).

Mass spectrometry is known in the art and has been used to quantify and/or identify biomolecules, such as proteins. Furthermore, mass spectrometry techniques have been developed to allow for at least partial resequencing of the isolated proteins. In certain embodiments, a gas phase ion spectrophotometer is used. In other embodiments, laser desorption/ionization mass spectrometry is used to analyze the sample. Modem laser desorption/ionization mass spectrometry ("LDI-MS") can be implemented in two main variations: matrix-assisted laser desorption/ionization ("MALDI") mass spectrometry and interface-enhanced laser desorption/ionization ("SELDI"). In MALDI, an analyte is mixed with a solution containing a matrix, and a drop of liquid is placed on the surface of a substrate. The matrix solution is then co-crystallized with the biomolecules. The substrate is inserted into the mass spectrometer. Laser energy is directed at the substrate surface where it desorbs and ionizes biomolecules without significantly damaging them. In SELDI, the substrate surface may be modified so that it becomes an active participant in the resolution process. In one embodiment, the substrate is derivatized with an adsorbent and/or capture reagent that selectively binds to the protein of interest. In another embodiment, the surface is derivatized with energy absorbing molecules that do not desorb when impinged with a laser. In another embodiment, the surface is derivatized with a molecule that binds to the protein of interest and includes a photolytic bond that is cleaved upon application of a laser. In each of these methods, the derivatizing reagents are typically localized to specific locations on the surface of the substrate to which the sample is applied. The two methods can be used in combination by: for example, a SELDI affinity surface is used to capture the analyte and a liquid comprising a matrix is added to the captured analyte to provide an energy absorbing material.

Detecting the presence of a cancer marker will typically comprise detecting signal intensity. This in turn can reflect the quantity and identity of the polypeptides bound to the substrate. For example, in certain embodiments, the signal intensities of the peaks from the spectra of the first and second samples may be compared (e.g., visually, by computer analysis, etc.) to determine the relative amounts of particular biomolecules. A software program such as the biomerker Wizard program (cipergen Biosystems, inc., Fremont, Calif.) may be used to assist in the analysis of mass spectra. The mass spectra and their techniques are known to the person skilled in the art.

It will be understood by those skilled in the art that any of the components of the mass spectrometer (e.g., resolving source, mass analyzer, detection, etc.) and various sample articles may be combined with other suitable components or articles described herein or known in the art. For example, in some embodiments, the control sample may include heavy atoms (e.g.,¹³C) allowing the test sample to be mixed with a control sample known in the same mass spectrometry run.

In a preferred embodiment, a laser desorption time of flight (TOF) mass spectrometer is used. In laser desorption mass spectrometry, a substrate with bound labels is introduced into an inlet system. The label is desorbed by laser light from an ionization source and ionized into a gas phase. The generated ions are collected by ion optics and then accelerated by a short high voltage field and drift into a high vacuum chamber in a time-of-flight mass analyser. At the far end of the high vacuum chamber, the accelerated ions hit the sensitive detector surface at different times. Since time of flight is a function of ion mass, the time elapsed between ion formation and ion detector impact can be used to identify the presence or absence of a particular mass molecule to obtain a charge ratio.

In some embodiments, the relative amount of one or more cancer markers present in the first or second sample is determined, in part, by executing an algorithm with a computer. The algorithm identifies at least one peak in the first mass spectrum and the second mass spectrum. The algorithm then compares the peak signal intensity of the first mass spectrum with the peak signal intensity of the second mass spectrum of the mass spectra. The relative signal intensity is indicative of the amount of cancer marker present in the first sample and the second sample. Standards comprising known amounts of cancer markers can be analyzed as a second sample to better quantify the amount of biomolecules present in the first sample. In certain embodiments, the identity of the cancer marker in the first sample and the second sample may also be determined.

Determination of Standard value, specificity and sensitivity

In the present invention, standard expression levels of cancer markers (e.g., blood concentration of CCL25) can be statistically determined. For example, the blood concentration of CCL25 in healthy individuals can be determined to statistically determine a standard blood concentration of CCL 25. When a statistically sufficient population can be collected, a value in the range from two or three times the standard deviation (s.d.) of the mean value is generally used as a standard value. Therefore, a value equivalent to the average value +2x.s.d. or the average value +3x s.d. can be used as the standard value. The standard values set as described theoretically include 90% and 99.7% of healthy individuals, respectively.

Alternatively, the standard value may also be set based on the actual expression level (e.g., CCL25 blood concentration) in a cancer patient. Generally, standard values for such methods are set to minimize the percentage of false positives, and are selected from a range of values that meet conditions that can maximize detection sensitivity. Here, the percentage of false positives refers to the percentage of patients in which the blood concentration of CCL25 is judged to be higher than a standard value in healthy individuals. In contrast, the percentage of patients in healthy individuals for whom the blood concentration of CCL25 is judged to be below the standard value indicates specificity. That is, the sum of false positives and specificities is always 1. The detection sensitivity refers to: the percentage of patients whose blood concentration of CCL25 is judged to be above a standard value among all patients in a population of individuals for whom the presence of cancer has been determined.

As used herein, the term "test sensitivity" is the ability of a screening assay to identify the true disease, and is also characterized by a high sensitivity test with fewer false negatives, and additionally, a test that is independent of the prevalence of the disease. The test sensitivity was calculated as true positive/total number of affected patients tested, expressed as a percentage.

The term "test specificity" is a screening experiment that is exactly negative in the absence of disease, has high specificity and fewer false positives, and is independent of disease prevalence. Test specificity was calculated as true negative/uninfected individuals tested, expressed as a percentage.

The term "PPV" (positive predictive value) is the percentage of patients who have a positive test for disease, and thus the reliability of the positive test is evaluated. And (3) calculating:

PPV = (true positive)/(true positive + false positive).

The term "NPV" (negative predictive value) refers to the percentage of patients that are negative to a test without disease, and thus the reliability of the negative test is evaluated. And (3) calculating:

NPV = (true negative)/(true negative + false negative).

As shown in the relationship shown above, each value of the sensitivity, the specificity, the positive predictive value, and the negative predictive value, which are indexes for evaluating the accuracy of detection, varies depending on the standard value for determining the blood concentration level of CCL 25.

The standard value is usually set so that false positives are low and sensitivity is high. However, as shown from the relationship shown above, there is a trade-off between false positive ratio and sensitivity. That is, if the standard value is decreased, the detection sensitivity is increased. However, since the false positive ratio also increases, it is difficult to meet the condition of having a "low false positive ratio". In view of these circumstances, for example, values given to the following prediction results may be selected as preferable standard values in the present invention: (1) a standard value for which the false positive ratio is 50% or less (that is, a standard value for which the specificity is not less than 50%); and (2) a standard value of not less than 20% in sensitivity.

The standard value is set by using a Receiver Operating Characteristic (ROC) curve. The ROC curve is a graph showing detection sensitivity on the vertical axis and false positive ratio (i.e., "1-specificity") on the horizontal axis. The ROC curve obtained after continuously changing the standard value of the high/low degree of blood concentration of the cancer marker (e.g., CCL25) was obtained by plotting changes in sensitivity and a false positive ratio.

The "standard value" used to obtain the ROC curve is a value temporarily used for statistical analysis. The "standard value" used to obtain the ROC curve can generally be varied continuously within a range that allows to cover all the selectable standard values. For example, the standard value may vary between the minimum and maximum measured blood CCL25 values in the analysis population.

Based on the obtained ROC curve, a preferable standard value to be used in the present invention can be selected from the range satisfying the above conditions. Alternatively, the standard value may be selected based on a ROC curve made by varying the standard value from a range that includes the vast majority of measured blood CCL 25.

Kit for detecting cancer

Another aspect of the present application relates to a kit for detecting cancer, comprising: reagents for determining CCL25 and/or CCR9 expression in a biological sample; and instructions for how to use the reagent, wherein the reagent comprises an anti-CCL 25 antibody, an anti-CCR 9 antibody, or both an anti-CCL 25 antibody and an anti-CCR 9 antibody.

In particular embodiments, the kit further comprises reagents for determining CXCL13 and/or CXCR5 expression in a biological sample; and instructions for how to use the agent, wherein the agent comprises an anti-CXCL 13 antibody, an anti-CXCR 5 antibody, or both an anti-CXCL 13 antibody and an anti-CXCR 5 antibody. In further particular embodiments, the kit further comprises reagents for determining CXCL16 and/or CXCR6 expression in a biological sample; and instructions for how to use the agent, wherein the agent comprises an anti-CXCL 16 antibody, an anti-CXCR 6 antibody, or both an anti-CXCL 16 antibody and an anti-CXCR 6 antibody.

In other particular embodiments, the kit further comprises reagents for determining CXCL16 and/or CXCR6 expression in a biological sample; and instructions for how to use the agent, wherein the agent comprises an anti-CXCL 16 antibody, an anti-CXCR 6 antibody, or both an anti-CXCL 16 antibody and an anti-CXCR 6 antibody.

The invention is further illustrated by the following examples, which should not be construed as limiting the application. The contents of all references, patents and published patent applications, and figures and tables cited in this application are hereby incorporated by reference.

Detailed Description

Example 1: in vitro analysis of CCL25 and CCR9 expression and activity in various carcinomas

As shown in fig. 1, breast cancer tissue expresses CCL 25. Breast cancer tissues were stained with isotype control or anti-CCL 25 antibody. Red-purple color shows CCL25 staining. An Aperio ScanScope CS system with a 40X objective lens captures digital images. A typical example of breast cancer shows the immunological strength of CCL 25.

Figure 2 demonstrates that CCL25 inhibits cisplatin-induced reduction in breast cancer cell line growth. With increasing cisplatin concentration, MDA-MB-231 cells were cultured for 24 hours with 0 or 100ng/ml CCL25 plus isotype control or anti-CCR 9 Ab. Cell proliferation was determined by BrdU pooling, and the assay was repeated 3 times and performed in triplicate. Asterisks indicate statistically significant differences between CCL 25-treated and untreated BrCa cells (p < 0.01).

Figures 3A-B show that CCL25 protected breast cancer cells from cisplatin-induced apoptosis. MDA-MB-231 cells were cultured for 24 hours with either 5mg/ml cisplatin alone or 0 or 100ng/ml CCL25 plus 1mg/ml anti-human CCR9 or isotype control (A). Cells were harvested and stained with dockerin (annexin V) and Propidium Iodide (PI). Apoptotic (dockerin positive) cells and living (non-fluorescent) cells and necrotic (PI positive) cells were distinguished by flow cytometry analysis of stained cells. Asterisks indicate statistically significant differences between CCL 25-treated and untreated breast cancer cells (p < 0.01). MDA-MB-231 cell lines were cultured for 24 hours (B) with 5mg/ml cisplatin or with 0 or 100ng/ml CCL25 plus 1mg/ml anti-human CCR9 or isotype control Ab. Detection of apoptotic cells was performed using the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. Apoptotic cells showed nuclear green fluorescence using standard fluorescence filtration cassettes (520. + -.20 nm). Asterisks indicate statistically significant differences between cisplatin CCL 25-treated and untreated breast cancer cell lines (p < 0.01).

FIGS. 4A-B show the PI3K and Akt activation of CCL25-CCR9 interaction in breast cancer cell lines. MDA-MB-231 cells were tested for their ability to activate PI3K and Akt after treatment with CCL25, cisplatin and specific kinase inhibitors (wortmannin and PF-573,228). In situ total and phosphorylated PI3K and Akt levels were quantified using a rapid activated cell based ELISA in the presence of cisplatin and kinase inhibitors, either before (0 min) or after (5 or 10 min) CCL25 stimulation. The ratio of activated (phosphorylated) PI3K (a) or akt (b) to total PI3K (a) or akt (b) ± SEM is provided in 3 independent experiments performed in triplicate. Asterisks indicate statistical differences between untreated and CCL 25-treated cells and CCL25+ cisplatin-treated cells.

FIGS. 5A-B show phosphorylation of GSK-3 β and FKHR after treatment with the breast cancer cell line CCL 25. MDA-MB-231 cells were tested for their ability to phosphorylate GSK-3 β and FKHR following treatment with CCL25, cisplatin and specific kinase inhibitors (wortmannin and PF-573,228). Total and phosphorylated GSK-3 β and FKHR levels in situ were quantified by rapid activated cell based ELISA in the presence of cisplatin and kinase inhibitors, either before (0 min) or after (5 or 10 min) CCL25 stimulation. The ratio of phosphorylated GSK-3 β (A) or FKHR (B) to total GSK-3 β (A) or FKHR (B) was provided as. + -. SE in 3 independent experiments performed in triplicate. Asterisks indicate statistical differences between untreated and CCL 25-treated cells and CCL25+ cisplatin-treated cells (p < 0.01).

FIG. 6 shows the expression of CCR9 and CCL25 in ovarian carcinoma tissues. Ovarian carcinoma tissues derived from non-tumor (n =8), serous adenocarcinoma (n =9), serous papillary cystadenoma (n =1), endometrioid adenocarcinoma (n =5), mucinous adenocarcinoma (n =2), cystadenoma (n =3), borderline mucinous adenocarcinoma (n =1), clear cell carcinoma (n =5), granulosa cell carcinoma (n =3), dysgerminoma (n =3), transitional cell carcinoma (n =3), bunner tumor (n =1), yolk sac tumor (n =4), adenocarcinoma (n =1) and fibroma (n =2) were stained with isotype control or anti-CCR 9 and CCL25 antibodies. Brown (DAB) color shows CCR9 staining and magenta color shows CCL 25. Digital images of each slide were captured with an Aperio ScanScope CS system with a 40X objective. Typical examples show the immunological strength of CCR9 and CCL 25.

FIGS. 7A-B show an analysis of CCL25 expression in ovarian carcinoma tissues. CCL25 expression was analyzed and presented using a modified boxplot (a). In the box lines, the lower line, the middle line, and the upper line represent the first quartile (Q1), the median (Q2), and the third quartile (Q3), respectively. The upper and lower whisker lines represent the median values. + -. 1.5 (Q3-Q1). Significant differences from non-tumors are indicated in the lower panel. Table (B) shows the respective p-values or significant differences between non-tumor tissue (NN) and Serous Adenocarcinoma (SA), endometrioid adenocarcinoma (EC), Mucinous Adenocarcinoma (MA), cystadenoma (C), borderline mucinous adenocarcinoma (MBA), Clear Cell Carcinoma (CCC), granulosa cell carcinoma (GCT), dysgerminoma (D), Transitional Cell Carcinoma (TCC), Breynoma (BT), Yolk Sac Tumor (YST), adenocarcinoma (a) and fibroma (F).

FIGS. 8A-B show analysis of CCR9 expression in ovarian cancer tissues. CCR9 expression was analyzed and presented using a modified box plot (box plot) (a). In the box lines, the lower line, the middle line, and the upper line represent the first quartile (Q1), the median (Q2), and the third quartile (Q3), respectively. The upper and lower whisker lines represent the median values. + -. 1.5 (Q3-Q1). Significant differences from non-tumors are indicated in the lower panel. Table (B) shows the respective p-values or significant differences between non-tumor tissue (NN) and Serous Adenocarcinoma (SA), endometrioid adenocarcinoma (EC), Mucinous Adenocarcinoma (MA), cystadenoma (C), borderline mucinous adenocarcinoma (MBA), Clear Cell Carcinoma (CCC), granulosa cell carcinoma (GCT), dysgerminoma (D), Transitional Cell Carcinoma (TCC), Breynoma (BT), Yolk Sac Tumor (YST), adenocarcinoma (a) and fibroma (F).

FIGS. 9A-B show CCR9 and CCL25 expression of ovarian cancer cell lines. Ovarian cancer cells were stained with Fluorescein (FITC) -conjugated anti-CCR 9 or FITC-conjugated isotype control antibody and analyzed by FACS (a). Ovarian carcinoma cells were stained with FITC-conjugated anti-CCR 9, intracellular CCL25 with Phycoerythrin (PE) -conjugated anti-CCL 25 antibody, and nuclei with Draq-5 (B). The merged data (merged data) shows CCR9 is expressed on the surface and CCL25 is expressed in the core.

FIGS. 10A-B show hypoxia-regulated CCR9mRNA and surface protein expression of ovarian cancer cells. Total RNA was isolated from SKOV-3 cell lines under normoxic and hypoxic conditions, or from normal primary ovarian tissue. Quantitative RT-PCR analysis of CCR9mRNA expression was performed in triplicate. The copy number of the transcript is expressed relative to the actual copy number of 18s rrna + SE (a). SKOV-3 cells in normoxia and hypoxia were stained with PE-bound isotype control antibody (Ab) (solid histogram) or PE-bound anti-CCR 9 monoclonal Ab (open histogram) and quantified by flow cytometry (B). The mean fluorescence intensity of PE positive cells is shown. The symbols represent the statistically significant (p <0.01) difference in CCR9 expression between normal tissue or isotype control and OvCa cells (), or between normoxic and hypoxic cells.

FIGS. 11A-B show SKOV-3 hypoxia-mediated and CCL 25-mediated migration and invasion. SKOV-3 cells were tested for their ability to migrate to the chemotactic gradient of CCL25 (a). During migration experiments, cells were co-cultured with 1.0 μ g/ml mouse anti-CCR 9 antibody (Ab) or isotype control Ab using 100ng/ml CCL25 under normoxic or hypoxic conditions. In addition, SKOV-3 cells were tested for their ability to invade or translocate through matrigel matrix in response to 100ng/ml of CCL25 under hypoxic conditions or normoxic conditions (B). During the invasion experiments, cells were co-cultured with 1.0 μ g/ml of monoclonal antibody against CCR9 using 100ng/ml CCL25 under normoxic or hypoxic conditions. The number of migrated or invaded cells (+ SE) is shown by a symbol indicating a significant (p <0.01) difference between CCL 25-treated and untreated normoxic cells (#), CCL 25-treated and untreated hypoxic cells (#), or similarly treated normoxic and hypoxic cells (#).

FIGS. 12A-B show CCL 25-induced collagenase expression from SKOV-3 cells. Cells were tested for their ability to express collagenase (MMP-1, MMP-8, and MMP-13) mRNA and active protein. SKOV-3 cells were cultured for 24 hours under normoxic or hypoxic conditions using either CCL25+1 μ g/ml isotype control antibody (Ab) at 100ng/ml, or mouse anti-CCR 9Ab at CCL25+1 μ g/ml. Total RNA was isolated and quantitative RT-PCR analysis was performed for collagenase mRNA expression and transcript copy number was expressed relative to the actual copy number of 18S rRNA (a). Active collagenase (B) was quantified by Fluorokine and Biotrak experiments in conditioned medium. The symbols represent significant (p <0.01) differences between CCL 25-treated and untreated normoxic cells (#), CCL 25-treated and untreated hypoxic cells (), or normoxic and hypoxic cells (#) treated similarly.

FIGS. 13A-B show CCL 25-induced gelatinase expression of SKOV-3 cells. Cells were tested for their ability to express gelatinase (MMP-2 and MMP-9) mRNA and active protein. SKOV-3 cells were cultured for 24 hours under normoxic or hypoxic conditions using either CCL25+1 μ g/ml isotype control antibody (Ab) at 100ng/ml, or mouse anti-CCR 9Ab at CCL25+1 μ g/ml. Total RNA was isolated and quantitative RT-PCR analysis was performed on mRNA expression of gelatinase, and transcript copy number was expressed relative to actual copy number of 18S rRNA (a). Active gelatinase (B) was quantified by Fluorokine and Biotrak experiments in conditioned medium. The symbols represent significant (p <0.01) differences between CCL 25-treated and untreated normoxic cells (#), CCL 25-treated and untreated hypoxic cells (), or normoxic and hypoxic cells (#) treated similarly.

FIGS. 14A-B show CCL 25-induced matrix degrading enzyme expression by SKOV-3 cells. Cells were tested for their ability to express matrix degrading enzyme (MMP-3, MMP-10, and MMP-11) mRNA and active proteins. SKOV-3 cells were cultured for 24 hours under normoxic or hypoxic conditions using either CCL25+1 μ g/ml isotype control antibody (Ab) at 100ng/ml, or mouse anti-CCR 9Ab at CCL25+1 μ g/ml. Total RNA was isolated and quantitative RT-PCR analysis was performed on mRNA expression of matrix degrading enzymes, and transcript copy number was expressed relative to actual copy number of 18S rRNA (a). Active matrix degrading enzymes (B) were quantified by Fluorokine and Biotrak experiments in conditioned medium. The symbols represent significant (p <0.01) differences between CCL 25-treated and untreated normoxic cells (#), CCL 25-treated and untreated hypoxic cells (), or normoxic and hypoxic cells (#) treated similarly.

FIG. 15 shows CCR9 expression for prostate cancer cell lines. Prostate cancer cell lines (C4-2B, LNCaP, and PC3) and normal prostate cells (RWPE-1) were stained with FITC-conjugated anti-human CCR9 (green) and 7AAD (nuclear stain; red). Positively stained cells were imaged and quantified by aminisimagestream. The right panel shows the mean fluorescence intensity of CCR9 staining.

FIGS. 16A-D show CCR9 expression in prostate tissue. Tissue Microarrays (TMAs) were obtained from National Institutes of Health (NIH), National Cancer Institute (NCI)) and university of alabama in birmingham and stained for CCR 9. An Aperio Scan Scope system with a 40X objective lens captures digital images of each slide. Representative examples of prostate cancer (CaP) (a), matched benign prostate tissue (MB) (B) and negative controls are indicated and the intensity of CCR9 was quantified for all tissues scanned and analyzed using ImageScope software (v.6.25). Figure 27D shows CCR9 (immunological) immunity between MB, Benign Prostatic Hyperplasia (BPH) and prostate cancer (PCa). Asterisks indicate significant (p <0.01) differences in immune intensity between MB, BPH and PCa tissues.

Figures 17A-D show CCL25 expression in prostate cancer tissue. Neuroendocrine differentiation of the endocrine-paracrine cell phenotype occurs frequently in prostate malignancies and has potential prognostic and therapeutic implications. The paracrine cell phenotype can be considered a post-mitotic subset of androgen insensitivity in prostate and prostate cancer. Figure 17A illustrates the expression of CCL25 in a paracrine pattern within prostate intraepithelial neoplasia. Double-headed arrows point to multiple paracrine cells producing CCL25 (red); brown arrows refer to cells expressing CCR9 (brown). Figure 17B shows cells staining CCL25 in red. The brown arrow indicates the cell NSE. Fig. 17A and C are high magnification views of fig. 17D and B, respectively.

Figure 18 shows serum CCL25 levels in normal healthy donors or patients with prostate disease. ELISA was used to quantify CCL25 in sera from normal healthy donors, prostate cancer (PCa), Prostatic Intraepithelial Neoplasia (PIN) and Benign Prostatic Hyperplasia (BPH). Asterisks indicate significant differences in CCL25 levels compared to normal healthy donors (p < 0.05).

FIGS. 19A-C show CCL25 expression by mouse bone marrow cells. Bone marrow cells from non-tumor bearing (a) and tumor bearing (B) mice were aspirated with an aspiration device and stained with FITC-conjugated anti-CCL 25 antibody. Positively stained cells were quantified with arnis ImageStream (C). Image-based analysis was performed using the IDEAS software and showed a 1.6-fold increase in CCL25 expression in bone marrow cells following prostate tumor challenge.

FIGS. 20A-B show CCR 9-mediated prostate cancer cell migration (A) and invasion (B). LNCaP cells, PC3 cells, and C4-2b cells were tested for their ability to migrate to CCL25 without addition (open column), 100ng/mL (hash bar), or 100ng/mL CCL25+1 μ g/mL anti-CCL 25 antibody (solid column). The number of cells migrating and invading (± SEM) in response to CCL25 (which was from the first 104 cells used to inoculate the migration and invasion chambers) showed that migration was CCL25 dependent and was blocked by anti-CCL 25 antibody. Asterisks indicate significant differences between no addition and CCL25 treated cells (p < 0.01).

Figure 21 shows CCL 25-induced active Matrix Metalloproteinase (MMP) expression from LNCaP, PC3, and C4-2b prostate cancer cell lines. Cells were cultured for 24 hours without (open box) or with 100ng/mL CCL25 (solid box). The protein levels of MMP-1, MMP-2, MMP-3, MMP-9, MMP-10 and MMP-11 in the culture supernatant were determined by MMP activity assay. Asterisks indicate the significance of MMP secretion (P <0.05) increased or decreased in CCL 25-treated cell lines compared to untreated cell lines.

FIGS. 22A-F show the inhibition of bone metastasis of the PC3 prostate cancer cell line by CCR9 gene knockdown (knockdown). Mice were challenged with a PC3 cell line expressing luciferase-and doxycycline (doxycycline) -inducible CCR 9-specific shRNA (A, D). Mice were challenged with this cell line by intracardiac injection. Subsequently, the mice received no or doxycycline (0.2mg/mL) added to the beverage for 21 days. Metastasis and tumor growth were monitored using the Caliper Xenogen100 in vivo imaging system. There was no change at 24 hours post challenge (B, E), but three weeks post challenge, CCR9 gene knockdown PC3(F) cells grew as significantly less bone metastases compared to CCR9 positive PC3 cells (C).

Figure 23 shows serum CCL25 levels in lung cancer patients. A CCL25ELISA was performed to quantify CCL25 levels in sera from patients diagnosed with adenocarcinoma (AdenoCa; n =14), squamous cell carcinoma (SSC; n =17) and normal healthy donors (controls; n = 9). The ELISA was able to detect >5pg/mL of CCL 25. Filled circles represent serum CCL25 levels of individuals, while lines represent median concentrations for each group. Asterisks indicate significant differences between control and lung cancer groups (p < 0.01).

FIGS. 24A-D show CCR9 expression in non-tumor and lung cancer tissues. From non-tumors (n =8) (a), adenocarcinoma (n =54) (B) and squamous cell carcinoma (n =24) (C) were stained with isotype control or anti-CCR 9 antibodies. Brown (DAB) color shows CCR9 staining. The Aperio ScanScope CS system with a 40X objective lens captures digital images of each slide.

FIGS. 25A-D show CCR9-CCL25 expression in colon cancer tissues. Colon tissues from lung tumors (n =8) and adenocarcinomas (n =16) were stained with isotype control (a), anti-CCR 9(B) or anti-CCL 25(C) antibodies. Brown (DAB) staining indicated CCR9 positive, while magenta staining indicated CCL25 positive. An Aperio ScanScope CS system with a 40X objective lens captures digital images.

Example 2: detection of chemokine expression levels using real-time PCR analysis

Primer design

CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR5a, CXCR5b, CXCR6, CXCR7, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCR6, CCL6, XCL6, CCL6, xccl 6, xc 6, CCR6, xc 6. Primers were designed using the beacon j2.0 computer program. Thermodynamic analysis of the primers was performed using computer programs Primer PremierJ and MIT Primer 3. The resulting primer sets were compared against the entire human genome to determine specificity.

Real-time PCR analysis

Cancer cell lines (ATCC, Rockville, Md.) were cultured in RMPI-1640 (complete medium) supplemented with non-essential amino acids, L-glutamic acid and sodium pyruvate, containing 10% fetal bovine serum. Primary tumors and normal paired matched tissues were obtained from clinical isolates (Clinomics Biosciences, Frederick, MD and UAB Tissue procuring, Birmingham, AL). Messenger RNA (mRNA) was isolated from 106 cells using TriReagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's instructions. Potential genomic DNA contamination was removed from these samples by treatment with 10U/Fl RNase-free DNase (Invitrogen, San Diego, Calif.) for 15 minutes at 37 ℃. The RNA was then pelleted and resuspended in RNA Secure (Ambion, Austin, TX). Approximately 2. mu.g of total RNA was reverse transcribed by using Taqman7 reverse transcription reagent (Applied Biosystems, Foster City, Calif.) according to the manufacturer's instructions. Subsequently, cDNA was amplified using SYBR7Green PCR master mix reagent (applied biosystems) with specific human cDNA primers for CXCL, CXCR5, CXCR, CCL-1, CCL-2, CCL, CCR, CCL, XCR, CCR, XCL, XCR, CX3CR, or CX CL3 according to the manufacturer's instructions. The copy levels of these target mrnas were evaluated by real-time PCR analysis using a BioRad Icycler and software (Hercules, CA).

Using CXCL-, CXCR-, CCL-and CCL-2-, (CCL-, CCL-and CCL-2-), CCL27-, CCL28-, CCR1-, CCR2-, CCR3-, CCR4-, CCR5-, CCR6-, CCR7-, CCR8-, CCR9-, CCR10-, CCR11-, XCL1-, XCL2-, XCR1-, CX3CR 1-or CX3CL 1-specific primer sets. The primers produce amplicon products of different sizes relative to the polymorphisms leading to CXCR5a versus CXCR5b and CCL25, CCL25-1 versus CCL 25-2. To this end, RT-PCR analysis of adenoma, carcinoma, leukemia, lymphoma, melanoma and/or myeloma cell lines and tumor tissues shows that cancer cells differentially express chemokines and chemokine receptors.

Example 3: anti-chemokine and anti-chemokine receptor antibodies inhibit tumor cell growth in vitro and in vivo

Antiserum preparation

15 amino acid peptide syntheses (SigmaGenosys, The Woodlands, TX) from CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL12, CXCR5a, CXCR5b, CXCL13, CXCR6, CXCL16, CCL16, CCL25, CCL25-1, CCL25-2, CCR9, CX3CR1 and CX3CL1(SEQ ID NOS:1-SEQ ID NO:21) and bind hen egg lysozyme (Pierce, Rockford, IL) to generate antigens for subsequent immunization to produce antisera preparation or monoclonal antibodies. Endotoxin levels of chemokine peptide conjugates were quantified by a chromogenic limulus amoebocyte lysate assay (Cape Cod, inc., Falmouth, MS) and shown to be <5 EU/mg. For the first immunization, 100. mu.g of antigen was used as immunogen, in a final volume of 1.0ml, together with the complete Freund's adjuvant Ribi Adjuvant System (RAS). The mixture was administered subcutaneously in 100ml aliquots onto two locations on the back of rabbits and 400ml was administered intramuscularly into each hind leg muscle. Three to four weeks later, the rabbits received 100 μ g of antigen in addition to incomplete freund's adjuvant for the subsequent 3 immunizations. Antiserum is collected when anti-CXCR 1 antibody, anti-CXCR 2 antibody, anti-CXCL 1 antibody, anti-CXCL 2 antibody, anti-CXCL 3 antibody, anti-CXCL 5 antibody, anti-CXCL 6 antibody, anti-CXCL 7 antibody, anti-CXCL 8 antibody, anti-CXCL 12 antibody, anti-CXCR 5a antibody, anti-CXCR 5b antibody, anti-CXCL 13 antibody, anti-CXCR 6 antibody, anti-CXCL 16 antibody, anti-CCL 16 antibody, anti-CCL 25 antibody, anti-CCL 25-1 antibody, anti-CCL 25-2 antibody, anti-CCR 9 antibody, anti-CX 3CR1 antibody, and anti-CX 3CL1 antibody titer reaches 1:1,000,000. Subsequently, normal or antisera were heat inactivated and diluted 1:50 in PBS.

Monoclonal antibody product

15 amino acid peptides from CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL12, CXCR5a, CXCR5b, CXCL13, CXCR6, CXCL16, CCL16, CCL25, CCL25-1, CCL25-2, CCR9, CX3CR1 and CX3CL1 were synthesized (Sigma Genosys) and bound to hen egg lysozyme (Pierce) to produce "antigens" for subsequent immunization to produce antisera preparations or monoclonal antibodies. Endotoxin levels of chemokine peptide conjugates were quantified by a chromogenic limulus amoebocyte lysate assay (Cape Cod, inc., Falmouth, MS) and shown to be <5 EU/mg. For the first immunization, 100. mu.g of antigen was used as immunogen, in a final volume of 200ml, together with the complete Freund's adjuvant Ribi Adjuvant System (RAS). The mixture was administered subcutaneously in 100ml aliquots to two locations on the back of rats, mice or immunoglobulin-humanized mice. After two weeks, the animals received 100 μ g of antigen in addition to incomplete freund's adjuvant for the subsequent 3 immunizations. Serum was collected and when anti-CXCR 1 antibody, anti-CXCR 2 antibody, anti-CXCL 1 antibody, anti-CXCL 2 antibody, anti-CXCL 3 antibody, anti-CXCL 5 antibody, anti-CXCL 6, anti-CXCL 7 antibody, anti-CXCL 8 antibody, anti-CXCL 12 antibody, anti-CXCR 5a antibody, anti-CXCR 5b antibody, anti-CXCL 13 antibody, anti-CXCR 6 antibody, anti-CXCL 16 antibody, anti-CCL 16 antibody, anti-CCL 25 antibody, anti-CCL 25-1 antibody, anti-CCL 25-2 antibody, anti-CCR 9 antibody, anti-CX 3CR1 antibody, and anti-CX 3CL1 antibody titers reached 1:2,000,000, the host was sacrificed and splenocytes were isolated to produce hybridomas. Briefly, B cells from the spleen or lymph nodes of an immunized host are fused with a immortal myeloma cell line (e.g., YB 2/0). The hybridomas are then isolated after selective culture conditions (i.e., HAT supplemented medium) and dilution methods that define hybridoma clones. Cells producing antibodies with the desired specificity were selected using ELISA. Hybridomas from normal rats or mice are humanized using commonly used molecular biology techniques. After cloning of high affinity and high yield hybridomas, antibodies were isolated from ascites or culture supernatants and adjusted to titers of 1:2,000,000 and diluted 1:50 in PBS.

Antiserum or monoclonal antibody treatment

Immunodeficient nude NIH-III mice (8 to 12 weeks old, Charles River Laboratory, Wilmington, MA) (lacking T cells, B cells and NK cells) received 1X10 subcutaneously⁶Cancer cells, thereby establishing a tumor. The established solid tumor is then removed from the host for immediate transplantation or stored in liquid nitrogen for subsequent transplantation. Tumor groups freshly isolated or liquid nitrogen frozen using surgeryTissue (1g) was transplanted in intestinal adipose tissue to generate tumors. Once the xenograft tumors reached 5mm size, NIH-III mice received 200 μ l of intraperitoneal injection of antisera or monoclonal antibodies every three days and were monitored for progression and regression of tumor growth.

Data analysis

Statistical significance of the data was analyzed and confirmed using SigmaStat2000(Chicago, IL) software. Subsequently, the data was analyzed by the Steton's t test (Student's t-test) using a two-factor unpaired test. In this assay, the treated sample is compared to an untreated control. Significance level was set at p < 0.05.

In vitro growth study

Adenomas, carcinomas, leukemias, lymphomas, melanomas and/or myeloma cell lines are grown in complete medium in the presence or absence of antibodies specific for CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6CXCL7, CXCL8, CXCR4, CXCL12, CXCR5a, CXCR5b, CXCL13, CXCR6, CXCL16, CCL16, CCR9, CCL25, CCL25-1, CCL25-2, CX3CR1 or CX3CL 1. Antibodies to CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, or CXCL8 inhibit the growth of cancer cell lines expressing CXCR1 and/or CXCR 2. Likewise, antibodies to CXCR4 or CXCL12 inhibit the growth of cancer cell lines expressing CXCR 4. Antibodies to CXCR5a, CXCR5b, or CXCL13 inhibit the growth of cancer cell lines expressing CXCR5a or CXCR5 a. Antibodies to CXCR6 or CXCL16 inhibit proliferation of cancer cell lines expressing CXCR 6. Antibodies to CCR9, CCL25, CCL25-1 or CCL25-2 inhibit the growth of cancer cell lines expressing CCR 9. The antibodies to CX3CR1 or CXC3L1 inhibit the proliferation of CX3CR 1-expressing cancer cell lines. Of interest are antibodies against soluble ligands, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL12, CXCL13, CXCL16, CCL16, CCL25, CCL25-1, CCL25-2 or CX3CL1, which are more effective in growth inhibition against membrane receptors.

In vitro angiogenesis study

In an in vitro assay for angiogenesis (BD-Biocoat, Hercules, CA), microvascular endothelial cells (Cell Systems, Kirkland, WA) are grown and formed into microvascular venules according to the instructions of the supplier in the presence or absence of antibodies specific for CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCR4, CXCL12, CXCR5a, CXCR5b, CXCL13, CXCR6, CXCL16, CCL16, CCR9, CCL25, CCL25-1, CCL25-2, CX3CR1 or CX3CL 1. Antibodies against CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCR4, CXCL12, CXCR6, or CXCL16 inhibit angiogenesis.

In vivo growth study

Cancer cell lines or primary tumor tissue inheritance (adoptively) were transferred into NIH-III mice and allowed to develop the desired xenograft tumors. Antibodies directed against CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCR4, CXCL12, CXCR5a, CXCR5b, CXCL13, CXCR6, CXCL16, CCL16, CCR9, CCL25, CCL25-1, CCL25-2, CX3CR1, or CX3CLl differentially affect the progression and regression of tumor size. In certain instances, antibodies directed to CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCR4, CXCL12, CXCR6, or CXCL16 are effective to cause regression of tumor growth and prevent progression of tumor growth. Antibodies directed against CXCR4, CXCL12, CXCR5a, CXCR5b, CXCL13, CCL16, CCR9, CCL25, CCL25-1, CCL25-2, CX3CR1, or CX3CL1 effectively inhibit the increase in tumor size.

The protein sequences of the chemokines used herein are recorded in the NIH-NCBI gene bank as follows: (1) CXCR1(ACCESSION # NP000625), SEQ ID NO:1, (2) CXCR2(ACCESSION # NP001548), SEQ ID NO:2, (3) CXCL1(ACCESSION # NP001502), SEQ ID NO:3, (4) CXCL2(ACCESSION # NP002080), SEQ ID NO:4, (5) CXCL3(ACCESSION # NP002081), SEQ ID NO:5, (6) CXCL5(ACCESSION # NP002985), SEQ ID NO:6, (7) CXCL6 (ACCESON # NP002984), SEQ ID NO:7, (8) CXCL7(ACCESSION # 002695), SEQ ID NO:8, (9) CXCL8(IL-8, ACCESSION # NP 000707), SEQ ID NO:9, (10) CXCR4(ACCESSION # 003575, SEQ ID NO: 00311, SEQ ID NO: 00611 (ACCESSION # NP) CXCR # 4611, SEQ ID NO: 00611 (SEQ ID NO: 00611) CXCR # 4611, SEQ ID NO:11 (CXCL 11611) CXCR # 11, SEQ ID NO:11 NP11 (SEQ ID NO: 4611) (SEQ ID NO: 11), 15, (16) CXCL16(ACCESSION # NP071342), 16, (17) CCL16(ACCESSION # NP004581), 17, (18) CCL25(ACCESSION # NP-005615.2), 18, (19) CCL25-1(ACCESSION # NP005615), 19, (20) CCL25-2(ACCESSION # NP 683), 20, (21) CX3CR1(ACCESSION # NP001328), 21, and (22) CX3CL1(ACCESSION # NP002987), 22.

The cDNA sequence is known and available in the NIH-NCBI Genbank under the following accession numbers: (23) CXCR1(ACCESSION # NM000634), SEQ ID NO:23, (24) CXCR2(ACCESSION # NM001557), SEQ ID NO:24, (25) CXCL1(ACCESSION # NM001511), SEQ ID NO:25, (26) CXCL2(ACCESSION # NM002089), SEQ ID NO:26, (27) CXCL3(ACCESSION # NM002090), SEQ ID NO:27, (28) CXCL5(ACCESSION # NM002994), SEQ ID NO:28, (29) CXCL6(ACCESSION # 002993), SEQ ID NO:29, (30) CXCL7(ACCESSION # 002704), SEQ ID NO:30, (31) CXCL8(IL-8, ACCESSION # 00031), SEQ ID NO:31, (32) CXCR4(ACCESSION # NM # 00300331, SEQ ID NO: 00632), (SEQ ID NO: 00648) CXCR # 4633, SEQ ID NO:29,29, SEQ ID NO:29,29, 37, (38) CXCL16(access # NM022059), 38, (39) CCL16(access # NM004590), 39, (40) CCL25(access # NM _005624.3), 40, (41) CCL25-1(access # NM005624), 41, (42) CCL25-2(access # NM148888), 42, (43) CX3CR1(access # NM001337), 43, and (44) CX3CL1(access # NM002996), 44.

As shown in the table below, the specific chemokines expressed by most all tumors can vary. The methods of the present application may be specific to a particular patient, depending on the chemokine that is over-expressed by the patient's own tumor. The methods of the present application can be used to identify specific chemokines that are overexpressed in a tumor and to administer antibodies against the overexpressed chemokines. Tailored therapy for cancer patients is novel and of particular value for use.

Table 1 shows the different amounts of specific chemokines overexpressed in the specific tumors studied.

Example 4: CCR9-CCL 25-induced anti-apoptosis and/or survival signaling associated with PCa chemoresistance

LNCaP (hormone responsive, wild type p53 expression), PC3 (hormone refractory, p53null) and DU145 (hormone refractory, p53 mutated) cell lines were grown for 4,8, 12 and 24 hours with or without doxorubicin (1. mu.M/2. mu.M/4. mu.M), etoposide (20. mu.M/40. mu.M), estramustine (4. mu.M/10. mu.M) or docetaxel (10nM/20nM/40 nM). Cell survival, pro-apoptotic, and anti-apoptotic signals (Akt, Src, CamKII, FAK, FKHR, FOXO, CREB, NF-. kappa.B, Myc, Fos, Jun Apaf1, Bax, Bcl2, BclX) were evaluated by real-time PCR and Western blotting_LBaK, Bad, Bik, Bim, TP53, caspase-3, caspase-6, caspase-8, caspase-9, survivin, vitronectin, beta-catenin) and molecules responsible for drug resistance or metabolism (Twist-1, Snail-1, glutathione-S-transferase-pi (GST-pi), p53, topoisomerase I, II alpha, II beta, and ABC drug transporter). Briefly, following cell treatment, changes in gene expression were tested using real-time PCR. In addition, phosphorylation-specific antibodies (i.e., western blot analysis) were used to test for activation of the signal molecules. To further confirm the effect of the activated signaling molecule, after CCL25 treatment, expression or activity of candidate molecules was inhibited using chemical inhibitors or sirnas, and the target gene was analyzed by real-time PCR. Subsequently, by VyThe brant apoptosis assay (Molecular probes) kit evaluates the response of treated cells to chemotherapeutic drugs.

RNA isolation and real-time PCR

Using Trizol^TM(Invitrogen) method Total RNA was isolated and quantified by UV spectrophotometry. RNA quality was analyzed by electrophoresis. Using iScript^TMcDNA synthesis kit (BioRad) cDNA synthesis was performed according to the manufacturer's instructions. Use of IQ according to manufacturer's instructions^TMSYBR green supermix (BioRad) and specific to FAK, FKHR, FOXO, Apaf1, Bax, Bcl2, BclX_LReal-time PCR was performed using primers designed for BaK, Bad, Bid, XIAP, Bik, Bim, TP53, cytochrome C, caspase-3, caspase-6, caspase-8, caspase-9, survivin, lamin, CamKII, vitronectin, β -catenin, cadherin, Twist-1, Snail-1, CREB, NF-. kappa.B, Myc, Fos, Jun, β -actin and GAPDH. Results were calculated by Δ Ct to quantify fold change in mRNA compared to untreated group.

Western blotting method

Cells were collected and resuspended in lysis buffer to extract total protein. Lysis buffer contained 50mM Tris-HCl, pH7.4, 150mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 5mM EDTA with protease inhibitor added, 1mM phenylmethylsulfonyl fluoride, 1mM benzamidine, 10. mu.g/mL soybean trypsin inhibitor, 50. mu.g/mL leupeptin, 1. mu.g/mL pepstatin, and 20. mu.g/mL aprotinin. Cell lysates were kept on ice for 30 min, centrifuged (14000xg) for 20 min at 4 ℃, and supernatants were used for western blot analysis of genes, demonstrating significant modulation at mRNA level. Likewise, phosphor-specific antibodies were used to test for changes in phosphorylation levels of Akt1/2/3, mTOR, FAK, FKHR, FOXO, and GSK-3 β. In addition, activation of cleaved caspase and PARP was evaluated using specific antibodies. The results obtained after chemiluminescence detection of the protein bands by ECL plus reagent (Pharmecia) on X-ray films were normalized for β -actin and/or GAPDH using Image J Image analysis software (NIH).

Detection of cytochrome C Release

The cells were harvested, washed in PBS and resuspended in a solution containing 220mM mannitol, 68mM sucrose, 50mM PIPES-KOH, pH7.4, 50mM KCl, 5mM EGTA, 2mM MgCl₂1mM DTT, and protease inhibitor. After 30 min incubation on ice, the cells were homogenized using a Glass-Teflon homogenizer and the homogenate would be spun at 14,000g for 15 min. The cytosolic extract was used for western blot analysis using an anti-cytochrome C monoclonal antibody (PharMingen).

siRNA transfection, chemical inhibitors, and apoptosis assays

Prostate cancer cell lines were transfected with gene-specific and non-specific control sirna (dharmacon) using LipofectAMINE2000 (Invitrogen). Optimal gene knockdown times and siRNA concentrations were confirmed by western blot analysis and cell survival was further assessed with or without drug treatment with CXCL16, a control antibody, and/or an anti-CXCR 6 antibody. Assays evaluating living cells, apoptotic cells and necrotic cells were as follows: using FACScan flow cytometer and CellQuest^TMThe software (BD Pharmingen) tested for cell survival using Vybrant apoptosis according to the manufacturer's instructions. Changes in downstream gene expression following gene knockdown were tested using real-time PCR and western blotting.

Cells treated with CCL25 showed increased cell survival and expression of drug transporter proteins, which showed a difference in their expression patterns in hormone-responsive and non-responsive cells. anti-CCL 25Abs effectively reversed the effect of CCL25 in PCa cells. Doxorubicin, estramustine, etoposide and docetaxel induced apoptosis of PCa cells in the absence of CCL25 treatment (or CCR9 blocking).

Example 5: CCR9-CCL25 induced alteration of ABC drug transporter

LNCaP cells, PC3 cells and DU145 cells were grown for 4 hours, 8 hours, 12 hours or 16 hours with or without CCL25, anti-CCL 25 antibody, control antibody and/or anti-CCR 9 antibody, with or without doxorubicin, estramustine, etoposide or docetaxel, as described previously. After treatment, the variation in ABC transporter and Twist-1mRNA expression was quantified by real-time PCR using specific primers for ABC and Twist-1 cDNAs, as described above. Genes that demonstrate significant changes in mRNA expression are further tested by western blot analysis. Nuclear extracts of the treated cells were evaluated by chromatin immunoprecipitation (ChIP) assay to determine whether the transcription factor induced by CXCL16 bound to ABC transporter and promoter regions of Twist-1.

Chromatin immunoprecipitation (ChIP)

The results of example 4 provide information on the genes that are modulated as well as genes that can modulate transcription factors activated by the CCR9-CCL25 interaction. Based on these results, the target transcription factor and gene are selected. Specific PCR primers were designed for the promoter regions of these genes containing the binding sites for transcription factors. PCR primers were used to amplify the DNA precipitated with the transcription factor. Cells were harvested by trypsinization in the presence of 20mM butyrate. 50,000 cells were resuspended in 500. mu.l PBS/butyrate. Proteins and DNA were crosslinked with 1% formaldehyde for 8 minutes at room temperature and crosslinking was stopped with 125mM glycine within 5 minutes. Cells were centrifuged at 470g for 10 min in a swing head using a gentle deceleration setting at 4 ℃ and washed twice by vortexing followed by centrifugation in 0.5ml ice-cold PBS/butyrate. The cells were lysed by addition of lysis buffer (50mM Tris-HCl, pH8, 10mM EDTA, 1% SDS, protease inhibitor cocktail (Sigma-Aldrich), 1mM PMSF, 20mM butyrate, vortexing and then centrifugation. this procedure is known to produce 500bp chromatin fragments. the sonicated lysates were diluted 8-fold in RIPA buffer containing protease inhibitor cocktail, 1mM PMSF and 20mM butyrate (RIPA ChIP buffer). RIPA ChIP buffer (330. mu.l) was added to the pellet and mixed by vortexing Cross-linking reversal and proteinase K digestion. DNA was extracted using phenol chloroform isoamyl alcohol, ethanol precipitated in the presence of acrylamide carrier (Sigma-Aldrich), and dissolved in TE. Immunoprecipitated DNA from 3-4 independent ChIPs was analyzed by real-time PCR. Real-time PCR data is expressed as the percentage of precipitated (antibody-bound) DNA (± SD) relative to the added DNA in three independent replicates of the ChIP assay.

Phosphorylation and activation of transcription factors such as CREB, Fos, Jun and NFkB via the CCR9-CCL25 signaling pathway subsequently leads to increased expression of ABC transporters and Twist-1. If negative regulatory elements are present in the same promoter, a decrease in gene expression is observed. Because hormone-dependent and non-responsive PCa cells have different expression of these intracellular signaling molecules, they show changes in the genes that are to be modulated by hormone-dependent and non-responsive states. Modulation of gene expression shows the difference between treatment with drug in the presence of CCL25 and in the absence of CCL25 treatment.

Example 6: in vivo evaluation of CCL 25-directed therapy

Male nude mice were challenged subcutaneously with androgen sensitive (LNCaP-Luc) and non-sensitive (PC3-Luc) cells expressing luciferase. Tumor progression is determined non-invasively by using an in vivo imaging system. After measurable tumor establishment, mice were divided into treatment groups (A, B, C, D and E) and control groups (F, G, H, I, J and K). Group "A" received CCL25 neutralizing antibody (12.5 mg/kg/day) every other day and control (group F) received isotype control antibody (12.5 mg/kg/day). Group "B", "group" C "," group "D" and "group E" received CCL25 neutralizing antibody (12.5 mg/kg/day) with intraperitoneal injections of doxorubicin (5 mg/kg/day on days 1-3 followed by administration on days 15-17), intravenous injection of etoposide (10 mg/kg/day; on days 1, 5, 9, 14, 19 and 24), intravenous injection of estramustine (4 mg/kg/day on days 1-5 and days 26-31), or intraperitoneal injection of docetaxel (8 mg/kg/day, 4 weeks, 2 times per week), respectively. Controls from these treated groups received these drugs using an isotype control antibody (12.5 mg/kg/day) using similar concentrations and injection protocols. Group "K" received PBS and served as a control. Tumor growth and regression in treatment and control were assessed by non-invasive imaging in vivo. Tumors from treated and untreated controls were isolated and evaluated by immunohistochemistry for changes in cell survival and drug resistant proteins. In the context as used herein, the term "CCL 25 neutralizing antibody" refers to an anti-CCL 25 antibody and/or an anti-CCR 9 antibody.

Statistics (significance) and sample size

Sample size (or magnification) calculations are relevant to preliminary study design and determine the requirements for the proposed trial. Significance tests and statistical analysis are also important in order to explain our results. The statistical significance of this study was evaluated using the conventional alpha-value, i.e., p = 0.01. The proposed trial would require a minimum of 10 mice per group. Data are expressed as mean ± SEM and compared by using either a two-tailed paired (or unpaired) steton's t-test for a conventionally distributed sample or an unpaired Mann Whitney U-test for an unconventional distributed sample. Results were analyzed using a SYSTAT (SYSTAT software Inc.) statistical program. One-and two-factor ANOVA analysis was used for evaluation and subgroups, respectively. Thus, if the p-value is <0.05, the results are considered statistically significant.

Animal(s) production

Adult male nude mice, six to eight weeks old, were injected subcutaneously with PCa cells. Briefly, 5x10⁶Individual luciferase-expressing PC3 cells were resuspended in 100 μ l sterile PBS and injected into the flanks of nude mice under isoflurane anesthesia. LNCaP cells expressing luciferase: (5x10⁶Cells) were mixed with 50% matrigel (Becton Dickinson) and injected into the flanks of nude mice under isoflurane anesthesia.

In vivo tumor growth analysis

Tumor bearing nude mice received 150 mg/kgD-luciferin (Xenogen) by intraperitoneal injection 15 minutes prior to imaging using a25 x5/8 "metering needle. Mice were imaged using the IVIS100 in vivo imaging system and the results were in photons/sec/cm²And/sr. Tumor volume was measured by using a caliper and by the formula (larger diameter) x (smaller diameter)²x 0.5.

Cell survival, apoptosis and drug resistance gene expression analysis

Three days after completion of the treatment protocol, all groups of tumors were excised. Tumors were fixed in 4% PFA and embedded in paraffin. Paraffin sections (7 μm thick) were placed on slides, deparaffinized, and rehydrated (5 min xylene treatment; 1 min each of pure, 95% and 70% ethanol). Rehydrated sections were used for drug transporter, PI3K, Akt, FAK, FKHR, FOXO, Apaf1, Bax, Bcl2, BclX_LPeroxidase-based immunohistochemical staining of BaK, Bad, Bid, XIAP, Bik, Bim, TP53, cytochrome C, caspase-3, caspase-6, caspase-8, caspase-9, survivin, lamin, CamKII, vitronectin, β -catenin, cadherin, Twist-1, CREB, NF- κ B, Myc, Fos, Jun, CCR9, and CCL 25. After staining, slides were scanned with the Aperio scanscope (Aperio) system and analyzed.

Neutralization of CCL25 resulted in decreased cell survival in response to the drug, thereby decreasing tumor volume. However, this response also changes in tumors formed by hormone sensitive (LNCaP) and hormone refractory (PC3 cells). In addition, chemotherapeutic drugs have lower efficacy in tumors with a functional CCR9-CCL25 axis (which can increase the expression of ABC proteins known to transport these drugs out of the cell).

The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention and is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is to be understood, however, that such obvious modifications and variations are included within the scope of the present application, which is defined by the following claims. The claims are to be understood to cover any sequence of components or steps which is effective to meet the objectives intended herein, unless the context specifically indicates the contrary. All references cited in the application documents are hereby incorporated by reference in their entirety.

Claims (26)

1. A method of detecting the presence of cancer in a subject, the method comprising:

detecting the expression level of one or more cancer markers in a biological sample obtained from the subject; and

comparing the expression level of the one or more cancer markers in the biological sample to a normal expression level of the one or more cancer markers,

wherein a higher than normal expression level of the one or more cancer markers in the biological sample indicates the presence of cancer in the subject,

wherein the normal expression level of the one or more cancer markers is a predetermined value or is obtained from a control sample of known normal non-cancerous cells of the same origin or type as the biological sample, and

wherein the cancer is blastoma, carcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma or germ cell tumor, and

wherein the one or more cancer markers comprise CCL25 or CCR9, or both CCL25 and CCR 9.

2. The method of claim 1, wherein said one or more cancer markers further comprises CXCL13 or CXCR5, or both CXCL13 and CXCR 5.

3. The method of claim 2, wherein said one or more cancer markers further comprises CXCL16 or CXCR6, or both CXCL16 and CXCR 6.

4. The method of claim 1, wherein said one or more cancer markers further comprises CXCL16 or CXCR6, or both CXCL16 and CXCR 6.

5. The method of claim 1, wherein said one or more cancer markers further comprises one or more cancer markers selected from the group consisting of CXCL13, CXCR5, CXCL16, CXCR6, CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL27, CXCL1, CXCL2, CXCL3, CXCL7, CXCL8, CXCL12, CX3CL1, CCR2, CCR7, CCR8, CCR10, CXCR1, CXCR2, CXCR4, CXCR7, and CX3CR 1.

6. The method of claim 1, wherein the cancer is a carcinoma.

7. The method of claim 6, wherein said carcinoma is breast cancer, and wherein one or more cancer markers further comprises one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CXCR4, CXCR5, CXCR6, CX3CR1, HER2, RBM3, and CEA.

8. The method of claim 6, wherein said carcinoma is prostate cancer, and wherein one or more cancer markers further comprises one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CXCR4, CXCR5, CXCR6, and CX3CR 1.

9. The method of claim 6, wherein the carcinoma is brain, pituitary, or bone cancer, and wherein one or more cancer markers further comprises one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CXCR4, CXCR5, CXCR6, and CX3CR 1.

10. The method of claim 6, wherein said carcinoma is colorectal cancer, and wherein one or more cancer markers further comprises one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CXCR4, CXCR5, CXCR6, CX3CR1, fibroblast activation protein a polypeptide, anti-p 53, osteopontin, and ferritin.

11. The method of claim 6, wherein the carcinoma is ovarian carcinoma and wherein one or more cancer markers further comprises one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CXCR4, CXCR5, CXCR6, CX3CR1, cancer antigen 125(CA-125), HE-4, OVX-1 macrophage colony stimulating factor (M-CSF), and lysophosphatidylcholine.

12. The method of claim 6, wherein the carcinoma is lung cancer, and wherein one or more cancer markers further comprises one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL25, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CCR9, CXCR4, CXCR5, CXCR6, CX3CR1, kinesin family member 4A (KIF4A), neurotropic pentameric protein I (NPTX1), fibroblast growth factor receptor 1 oncogene partner (FGFR1OP) protein, and CEA.

13. The method of claim 6, wherein said carcinoma is pancreatic cancer or gastric cancer, and wherein one or more cancer markers further comprises one or more cancer markers selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL25, CXCL12, CXCL13, CXCL16, CX3CL1, CCR2, CCR7, CCR8, CCR9, CXCR4, CXCR5, CXCR6, CX3CR1, and CEA.

14. The method of claim 1, wherein the biological sample is plasma, saliva, or urine.

15. A method of assessing prognosis of a subject having cancer, the method comprising:

determining the expression level of one or more cancer markers in a biological sample from the subject; and

comparing the expression level of the one or more cancer markers in the biological sample to a control expression level of the one or more cancer markers,

wherein a higher expression level of said one or more cancer markers in said biological sample indicates a poor prognosis of said subject,

wherein a lower or similar expression level of the one or more cancer markers in the biological sample relative to the control level indicates a good prognosis of the subject,

wherein poor prognosis means that the cancer is aggressive or invasive, wherein the cancer is blastoma, carcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, or germ cell tumor, and

wherein the one or more cancer markers comprise CCL25 or CCR9, or CCL25 and CCR 9.

16. The method of claim 15, wherein said one or more cancer markers further comprise (1) CXCL13 or CXCR5, or CXCL13 and CXCR5, and/or (2) CXCL16 or CXCR6, or CXCL16 and CXCR 6.

17. The method of claim 15, wherein said one or more cancer markers further comprises one or more cancer markers selected from the group consisting of CXCL13, CXCR5, CXCL16, CXCR6, CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL27, CXCL1, CXCL2, CXCL3, CXCL7, CXCL8, CXCL12, CX3CL1, CCR2, CCR7, CCR8, CCR10, CXCR1, CXCR2, CXCR4, CXCR7, and CX3CR 1.

18. The method of claim 15, wherein the biological sample is plasma, saliva, or urine.

19. A method for monitoring the course of cancer therapy in a subject, the method comprising:

determining the expression level of one or more cancer markers in one or more biological samples obtained from the subject during or after treatment; and

comparing the expression level of the one or more cancer markers in the one or more biological samples to a control expression level of the one or more cancer markers,

wherein the control level of the one or more cancer markers is a pre-treatment level, or a predetermined reference level, of the one or more cancer markers in the subject,

wherein the treatment is considered effective if the one or more cancer markers in the one or more biological samples are similar to or below the control level,

wherein the cancer is blastoma, carcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma or germ cell tumor, and

wherein the one or more cancer markers comprise CCL25 or CCR9, or CCL25 and CCR 9.

20. The method of claim 19, wherein said one or more cancer markers further comprise (1) CXCL13 or CXCR5, or CXCL13 and CXCR5, and/or (2) CXCL16 or CXCR6, or CXCL16 and CXCR 6.

21. The method of claim 19, wherein said one or more cancer markers further comprises one or more cancer markers selected from the group consisting of CXCL13, CXCR5, CXCL16, CXCR6, CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL27, CXCL1, CXCL2, CXCL3, CXCL7, CXCL8, CXCL12, CX3CL1, CCR2, CCR7, CCR8, CCR10, CXCR1, CXCR2, CXCR4, CXCR7, and CX3CR 1.

22. The method of claim 19, wherein the biological sample is plasma, saliva, or urine.

23. A kit for detecting cancer, the kit comprising:

reagents for determining the expression of CCL25 and/or CCR9 in a biological sample; and instructions for how to use the reagents,

wherein the reagent comprises an anti-CCL 25 antibody, an anti-CCR 9 antibody, or both an anti-CCL 25 antibody and an anti-CCR 9 antibody.

24. The kit of claim 23, comprising:

(1) reagents for determining the expression of CXCL13 and/or CXCR5 in a biological sample; and instructions for how to use the reagents,

wherein the agent comprises an anti-CXCL 13 antibody, an anti-CXCR 5 antibody, or both an anti-CXCL 13 antibody and an anti-CXCR 5 antibody.

25. The kit of claim 24, comprising:

reagents for determining the expression of CXCL16 and/or CXCR6 in a biological sample; and instructions for how to use the reagents,

wherein the agent comprises an anti-CXCL 16 antibody, an anti-CXCR 6 antibody, or both an anti-CXCL 16 antibody and an anti-CXCR 6 antibody.

26. The kit of claim 23, comprising:

reagents for determining the expression of CXCL16 and/or CXCR6 in a biological sample; and instructions for how to use the reagents,

wherein the agent comprises an anti-CXCL 16 antibody, an anti-CXCR 6 antibody, or both an anti-CXCL 16 antibody and an anti-CXCR 6 antibody.