# Patent

## Title
Anti-CXCL13 and anti-CXCR5 antibodies for the prevention and treatment of cancer and cancer cell migration

## Abstract

Methods for prevention or inhibition of the growth or metastasis of cancer cells in a subject are disclosed. One method comprises the step of administering to the subject a therapeutically effective amount of an antibody to the chemokine CXCL13 and/or the chemokine receptor CXCR5. Another method comprises the step of administering to the subject a therapeutically effective amount of an expression vector that expresses an antibody to the chemokine CXCL13 and/or the chemokine receptor CXCR5.

## Background
This application is a Continuation-In-Part of U.S. patent application Ser. No. 13/233,769, filed on Sep. 15, 2011, which is a Continuation-In-Part of U.S. patent application Ser. No. 12/967,273, filed Dec. 14, 2010, which is a continuation of U.S. patent application Ser. No. 10/712,398, filed on Nov. 14, 2003, now U.S. Pat. No. 7,919,083, which claims priority of U.S. Provisional Patent Application No. 60/426,347, filed Nov. 15, 2002. The entirety of all of the aforementioned applications is incorporated herein by reference.


FIELD
This application generally relates to the prevention and treatment of cancer. In particular, the invention relates to the use of anti-chemokine and/or anti-chemokine receptor antibodies for the inhibition or prevention of the growth and/or migration of cancer cells.
BACKGROUND
Despite recent advances in cancer research, the development of cell-specific therapies for the treatment of malignancies remain elusive. The many and complex factors that enable malignant cells to undergo mutations, evade immune protection and promote angiogenesis to deliver nutrients to the rapidly growing cells complicate the development of targeted treatment modalities. Current therapies have multiple untoward side effects. For example, chemotherapy results in multiple painful and sometimes lethal side effects. Advances in biotechnology have promoted the development of targeted biologicals with fewer side effects.
Host cells have surface receptors that associate with ligands to signal and cause host cell activities. The epidermal growth factor receptor helps control cell growth and metastasis. Many tumor cells express higher numbers of epidermal growth factor receptors than normal cells. A new treatment designated IMC-225 was specifically designed to target and block epidermal growth factor receptors, thus preventing cell division and repair. Recently, trastuzumab, which is a HER-2-specific monoclonal antibody, has proven effective at treating metastatic breast cancers. This antibody blocks interactions on cancer cells that inhibit cell growth. HER-2, however, is only found on about 25 to 30 percent of breast cancer cells.
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 mediate chemotaxis through interactions with G-protein coupled receptors. Chemokines can mediate the growth and migration of host cells that express their receptors.
CXCL13 is a small cytokine belonging to the CXC chemokine family. As its name suggests, this chemokine is selectively chemotactic for B cells belonging to both the B-1 and B-2 subsets, and elicits its effects by interacting with chemokine receptor CXCR5. CXCL13 and CXCR5 control the organization of B cells within follicles of lymphoid tissues. CXCR5 is expressed highly in the liver, spleen, lymph nodes, and gut of humans. CXCR5 plays an essential role in B cell migration.
In T-lymphocytes, CXCL13 expression may reflect a germinal center origin of the T-cell. Hence, expression of CXCL13 in T-cell lymphomas, such as Angioimmunoblastic T-cell Lymphoma, is thought to reflect a germinal center origin of the neoplastic T-cells.
SUMMARY
One aspect of the present invention relates to a method for treating melanoma, sarcoma, blastoma, carcinoma, lymphoma, myeloma or leukemia in a subject. In one embodiment, the method comprises the step of administering to the subject a therapeutically effective amount of an anti-CXCL13 antibody, or an anti-CXCR5 antibody, or a combination thereof. In another embodiment, the method comprises the step of immunizing the subject with an effective amount of CXCL13 and/or CXCR5 immunogen(s) as protein, peptide or encoded gene to induce antibodies that inhibit the biological activity of CXCL13 and/or CXCR5. In another embodiment, the method comprises the step of administering to the subject an expression vector that expresses an anti-CXCL13 antibody, or an anti-CXCR5 antibody, or a combination thereof in said subject. In another embodiment, the method comprises the step of administering to the subject an effective amount of an expression vector that expresses an agent capable of (1) inhibiting the expression of CXCL13 and/or CXCR5, (2) inhibiting the interaction between CXCL13 and CXCR5, or (3) inhibiting a biological activity of CXCL13 and/or CXCR5.
Another aspect of the present invention relates to a method for prevention or inhibition of the migration or metastasis of cancer cells with elevated expression of CXCL13 and/or CXCR5 in a subject. In one embodiment, the method comprises the step of administering to the subject a therapeutically effective amount of an anti-CXCL13 antibody, an anti-CXCR5 antibody, or a combination thereof. In another embodiment, the method comprises the step of immunizing the subject with an effective amount of CXCL13 and/or CXCR5 immunogen(s) as protein, peptide or encoded gene to induce antibodies that inhibit the biological activity of CXCL13 and/or CXCR5. In another embodiment, the method comprises the step of administering to the subject an expression vector that expresses an anti-CXCL13 antibody, or an anti-CXCR5 antibody, or a combination thereof in said subject. In another embodiment, the method comprises the step of administering to the subject an effective amount of an expression vector that expresses an agent capable of (1) inhibiting the expression of CXCL13 and/or CXCR5, (2) inhibiting the interaction between CXCL13 and CXCR5, or (3) inhibiting a biological activity of CXCL13 and/or CXCR5.
Another aspect of the present invention relates to a method for treating cancer in a subject. The method comprises the steps of detecting a level of CXCL13 expression and/or CXCR5 expression in a biological sample from said subject and, if an elevated level of CXCL13 expression and/or CXCR5 expression is detected in said biological sample, administering to the subject (1) a therapeutically effective amount of an antibody to CXCL13 and/or an antibody to CXCR5 or (2) an expression vector that expresses an anti-CXCL13 antibody, or an anti-CXCR5 antibody, or a combination thereof in said subject. In another embodiment, the method comprises the steps of detecting a level of CXCL13 expression and/or CXCR5 expression in a biological sample from said subject and, if an elevated level of CXCL13 and/or CXCR3 expression is detected in said biological sample, immunizing the subject with an effective amount of CXCL13 and/or CXCR5 immunogen to elicit an antibody response to inhibit the biological activity of CXCL13 and/or CXCR5. In another embodiment, the method comprises the step of detecting a level of CXCL13 expression and/or CXCR5 expression in a biological sample from said subject and, if an elevated level of CXCL13 expression and/or CXCR5 expression is detected in said biological sample, administering to the subject an effective amount of an expression vector that expresses an agent capable of (1) inhibiting the expression of CXCL13 and/or CXCR5, or (2) inhibiting the interaction between CXCL13 and CXCR5, or (3) inhibiting a biological activity of CXCL13 and/or CXCR5.
Another aspect of the present invention relates to a method for enhancing the effect of chemotherapy. The method comprises administering to a subject who is under chemotherapy for a cancer, an effective amount of an anti-CXCL13 antibody, or an anti-CXCR5 antibody, or a combination thereof. In another embodiment, the method comprises the step of immunizing the subject+who is under chemotherapy for a cancer with an effective amount of CXCL13 and/or CXCR5 immunogen(s) as protein, peptide or encoded gene to induce antibodies that inhibit the biological activity of CXCL13 and/or CXCR5. In another embodiment, the method comprises the step of administering to the subject who is under chemotherapy for a cancer an expression vector that expresses an anti-CXCL13 antibody, or an anti-CXCR5 antibody, or a combination thereof. In another embodiment, the method comprises the step of administering to the subject an effective amount of an expression vector that expresses an agent capable of (1) inhibiting the expression of CXCL13 and/or CXCR5, or (2) inhibiting the interaction between CXCL13 and CXCR5, or (3) inhibiting a biological activity of CXCL13 and/or CXCR5
Another aspect of the present invention relates to a method for treating cancer in a subject by immunizing the subject with an effective amount of one or more of CXCL13 and CXCR5 immunogens and an effective amount of one or more of CXCL16 and CXCR6 immunogens, wherein said cancer is melanoma, lymphoma, leukemia, sarcoma, blastoma, or carcinoma.
Another aspect of the present invention relates to a method for treating cancer in a subject by immunizing the subject with an effective amount of a CXCL16 immunogen and/or a CXCR6 immunogen to induce antibodies that inhibit the biological activity of CXCL16 and/or CXCR6, wherein the cancer is melanoma, lymphoma, leukemia, sarcoma, blastoma, or carcinoma, wherein the CXCL16 immunogen is a peptide comprising one or more sequences 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), RPTLPVGSL (SEQ ID NO:108), TAGHSLAAG (SEQ ID NO:109), GKRISSDSPPSVQ (SEQ ID NO:110) and KDPWVQELMSCLDLKECGHAYSGIVAHQKH (SEQ ID NO:111), and wherein the CXCR6 immunogen is a peptide comprising one or more sequences selected from the group consisting of HQDFLQFSKV (SEQ ID NO:112), AGIHEWVFGQVMCK (SEQ ID NO:113), PQIIYGNVFNLDKLICGYHDEAI (SEQ ID NO:114) and YYAMTSFHYTIMVTEA (SEQ ID NO:115).



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-B show expression of DOCK2 by prostate cancer (PCa) cell lines.
FIG. 2 shows that CXCL13:CXCR5 interaction promotes LNCaP and PC3 cell invasion independent of DOCK2.
FIG. 3 shows CXCL13 regulation of Akt and ERK1/2 activation.
FIG. 4 shows that CXCL13 induces JNK activation through DOCK2 in PC3 cells.
FIG. 5 shows CXCL13 regulation of prostate cancer (PCa) cell proliferation through JNK and DOCK2.
FIG. 6 shows that JNK inhibition and DOCK2 knockdown lead to reduction of PCa cell proliferation not due to cell death by apoptosis.
FIG. 7 shows CXCL13 modulation of signaling cascades in PCa cell lines.
FIGS. 8A-C show the expression of α subunit isoforms of G-protein by prostate cancer cell lines.
FIGS. 9A-B show G-protein β and γ subunit isoform expression of by prostate cancer cell lines.
FIGS. 10A-D show expression of CXCR5 and associated G proteins in prostate cancer cell lines treated with or without CXCL13.
FIGS. 11A-B depict validation of Gq/11 and Gαi2 protein association with CXCR5 by immunoprecipitation.
FIGS. 12A-C show the identification of CXCR4 and CXCR5 coupled to Gα13 following CXCL13 stimulation.
FIG. 13 depicts a hypothetical model of CXCR5 interactions in prostate cancer cells.
FIG. 14 depicts how CXCL13 regulates key molecules involved in the cell cycle.
FIG. 15 depicts how CXCL13 regulates key molecules involved in cell migration.
FIG. 16 depicts how CXCL13 regulates key molecules involved in cell survival and growth.
FIG. 17 depicts the top Canonical pathways regulated by CXCL13 in PC3 cells.
FIG. 18 shows CXCL13 mediation of differential phosphorylation of proteins belonging to the PI3K/Akt and SAPK/JNK signaling pathways.
FIG. 19 is a summary diagram of signaling pathways modulated by CXCL13-CXCR5 interactions.
FIG. 20 shows confirmation of major CXCL13-CXCR5 cell signaling cascades.
FIG. 21 shows CXCL13-CXCR5 signaling events required for AKT activation.
FIGS. 22A-B depicts CXCL13-mediated CXCR5 ligation and translocation to nuclei.
FIGS. 23A-B show that anti-CXCL13 antibody treatment inhibits prostate cancer progression and bone metastasis.
FIGS. 24A-B show that CXCL13 blockade inhibits prostate tumor growth in bone.
FIGS. 25A-B show that CXCL13 blockade abrogates osteolytic prostate tumor growth in bone.
FIGS. 26A-B show that CXCL13 blockade inhibits loss of bone mineral density (BMD) induced by prostate cancer metastasis to bone.
FIG. 27 depicts the levels of CXCL13 in serum of normal healthy controls and lung cancer subjects.
FIG. 28 shows CXCR5 expression by non-neoplastic lung and lung cancer tissue.
FIG. 29 shows CXCR5 expression by non-neoplastic mammary and breast cancer tissue.
FIG. 30 is a depiction of CXCR5 and CXCL13 expression by colon cancer tissue relative to non-neoplastic controls.
FIG. 31 is a depiction of CXCR5 and CXCL13 expression by ovarian cancer tissue relative to non-neoplastic controls.



DETAILED DESCRIPTION
The following detailed description is presented to enable any person 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. However, it will be apparent to one skilled in the art that these specific details are not required to practice the invention. Descriptions of specific applications are provided only as representative examples. 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 otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
Definitions
As used herein, the following terms shall have the following meanings:
The terms “treat,” “treating” or “treatment” as used herein, refers to a method of alleviating or abrogating a disorder and/or its attendant symptoms. The terms “prevent”, “preventing” or “prevention,” as used herein, refer to a method of barring a subject from acquiring a disorder and/or its attendant symptoms. In certain embodiments, the terms “prevent,” “preventing” or “prevention” refer to a method of reducing the risk of acquiring a disorder and/or its attendant symptoms.
As used herein, the term “antibody” 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 the broadest sense and specifically covers 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 bind” or “immunoreacts with” is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react (i.e., bind) with other polypeptides or binds at much lower affinity with other polypeptides. The term “antibody” also includes antibody fragments that comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody (scFv) molecules; and multispecific antibodies formed from antibody fragments. In certain embodiments of the invention, it may be desirable to use an antibody fragment, rather than an intact antibody, to increase tumor penetration, for example. In this case, it may be desirable to use an antibody fragment that has been modified by any means known in the art in order to increase its serum half life.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
“Humanized” forms of non-human antibodies are chimeric antibodies which contain minimal sequence derived from 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) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and/or capacity. Methods for making humanized and other chimeric antibodies are known in the art.
“Bispecific antibodies” are antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for CXCL16 or CXCR6. The second binding target is any other antigen, and advantageously is a cell-surface protein or receptor or receptor subunit. Methods for making bispecific antibodies are known in the art.
The use of “heteroconjugate antibodies” is also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents.
The present invention also contemplates the use of “immunoconjugates” comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y, and 186Re.
In a pharmacological sense, in the context of the present invention, a “therapeutically effective amount” of an antibody refers to an amount effective in the prevention or treatment of a disorder for the treatment of which the antibody is effective. A “disorder” is any condition that would benefit from treatment with the antibody, including carcinoma and chemoresistance. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.
The term “tumor” as used herein refers to a neoplasm or a solid lesion formed by an abnormal growth of cells. A tumor can be benign, pre-malignant or malignant.
The term “cancer” is defined as a malignant neoplasm or malignant tumor and is a class of diseases in which a group of cells display uncontrolled growth, invasion that intrudes upon and destroys adjacent tissues, and sometimes metastasis, or spreading to other locations in the body via lymph or blood. These three malignant properties of cancers differentiate them from benign tumors, which do not invade or metastasize. Exemplary cancers include: carcinoma, melanoma, sarcoma, lymphoma, leukemia, germ cell tumor, and blastoma.
The term “carcinoma” as used herein refers to an invasive malignant tumor consisting of transformed epithelial cells or transformed cells of unknown histogenesis, but which possess specific molecular or histological characteristics that are associated with epithelial cells, such as the production of cytokeratins or intercellular bridges. Exemplary carcinomas of the present invention include ovarian cancer, vaginal cancer, cervical cancer, uterine cancer, prostate cancer, anal cancer, rectal cancer, colon cancer, stomach cancer, pancreatic cancer, insulinoma, adenocarcinoma, adenosquamous carcinoma, neuroendocrine tumor, breast cancer, lung cancer, esophageal cancer, oral cancer, brain cancer, medulloblastoma, neuroectodermal tumor, glioma, pituitary cancer, and bone cancer.
The term “lymphoma” as used herein is a cancer of lymphatic cells of the immune system. Lymphomas typically present as a solid tumor. Exemplary lymphomas include: small lymphocytic lymphoma, lymphoplasmacytic lymphoma, Waldenström 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, classical Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, adult T cell lymphoma, nasal type extranodal NK/T cell lymphoma, enteropathy-type T cell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma, mycosis fungoide, Sezary syndrome, primary cutaneous CD30-positive T cell lympho-proliferative disorders, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cell lymphoma, unspecified peripheral T cell lymphoma, and anaplastic large cell lymphoma. Exemplary forms of classical Hodgkin lymphoma including: nodular sclerosis, mixed cellularity, lymphocyte-rich, and lymphocyte-depleted or not depleted
The term “sarcoma” as used herein is a cancer that arises from transformed cells in one of a number of tissues that develop from embryonic mesoderm. Thus, sarcomas include tumors of bone, cartilage, fat, muscle, vascular, and hematopoietic tissues. For example, osteosarcoma arises from bone, chondrosarcoma arises from cartilage, liposarcoma arises from fat, and leiomyosarcoma arises from smooth muscle. Exemplary sarcomas include: Askin's tumor, botryodies, chondrosarcoma, Ewing's-PNET, malignant Hemangioendothelioma, malignant Schwannoma, osteosarcoma, soft tissue sarcomas. Subclases of soft tissue sarcomas include: alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcomadesmoid tumor, desmoplastic small round cell tumor, epithelioid sarcomaextraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcomal, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, and synovial sarcoma.
The term “leukemia” as used herein is a cancer of the blood or bone marrow characterized by an abnormal increase of white blood cells. Leukemia is a broad term covering a spectrum of diseases. In turn, it is part of the even broader group of diseases called hematological neoplasms. Leukemia is subdivided into a variety of large groups; the first division is between acute and chronic forms of leukemia. Acute leukemia is characterized by a rapid increase in the numbers of immature blood cells. Crowding due to such cells makes the bone marrow unable to produce healthy blood cells. Chronic leukemia is characterized by the excessive build up of relatively mature, but still abnormal, white blood cells. Typically taking months or years to progress, the cells are produced at a much higher rate than normal cells, resulting in many abnormal white blood cells in the blood. Leukemia is also subdivided by the blood cells affected. This split divides leukemias into lymphoblastic or lymphocytic leukemias and myeloid or myelogenous leukemias. In lymphoblastic or lymphocytic leukemias, the cancerous change takes place in a type of marrow cell that normally goes on to form lymphocytes. In myeloid or myelogenous leukemias, the cancerous change takes place in a type of marrow cell that normally goes on to form red blood cells, some other types of white cells, and platelets. Combining these two classifications provides a total of four main categories. Within each of these four main categories, there are typically several subcategories. There are also rare types outside of this classification scheme. 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 leukemia, and adult T-cell leukemia.
The term “melanoma” as used herein is a cancer or malignant tumor of melanocytes. Melanocytes are cells that produce the dark pigment, melanin, which is responsible for the color of skin. They predominantly occur in skin, but are also found in other parts of the body, including the bowel and the eye. Melanoma is divided into the following stereotypes and subtypes: lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft-tissue melanoma, melanoma with small nevus-like cells, melanoma with features of a Spitz nevus, and uveal melanoma.
The term “germ cell tumor (GCT)” as used herein is a neoplasm derived from germ cells. Germ cell tumors can be cancerous or non-cancerous tumors. Germ cells normally occur inside the gonads (ovary and testis). Germ cell tumors that originate outside the gonads may be birth defects resulting from errors during development of the embryo. Germ cell tumors are broadly divided in two classes: germinomatous or seminomatous and nongerminomatous or nonseminomatous germ cell tumors. Exemplary germinomatous or seminomatous germ cell tumors include: germinoma, dysgerminoma, and seminoma. Exemplary nongerminomatous or nonseminomatous germ cell tumors include: Embryonal carcinoma, endodermal sinus tumor or yolk sac tumor (EST, YST), choriocarcinoma, mature teratoma, dermoid cyst, immature teratoma, teratoma with malignant transformation, polyembryoma, gonadoblastoma, and mixed GCT.
The term “metastasis” as used herein refers to the spread of a cancer or carcinoma from one organ or part to another non-adjacent organ or part.
“Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, non-human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
The term “inhibits” is a relative tem, an agent inhibits a response or condition if the response or condition is quantitatively diminished following administration of the agent, or if it is diminished following administration of the agent, as compared to a reference agent. Similarly, the term “prevents” does not necessarily mean that an agent completely eliminates the response or condition, so long as at least one characteristic of the response or condition is eliminated. Thus, a composition that reduces or prevents an infection or a response, such as a pathological response, can, but does not necessarily completely eliminate such an infection or response, so long as the infection or response is measurably diminished, for example, by at least about 50%, such as by at least about 70%, or about 80%, or even by about 90% of (that is to 10% or less than) the infection or response in the absence of the agent, or in comparison to a reference agent.
The term “increased level” refers to a level that is higher than a normal or control level customarily defined or used in the relevant art. For example, an increased level of immunostaining in a tissue is a level of immunostaining that would be considered higher than the level of immunostaining in a control tissue by a person of ordinary skill in the art.
The term “CXCL13 immunogen” and “CXCR5 immunogen” refers to an immunogenic composition comprising (1) an immunogenic peptide derived from CXCL13 or CXCR5 and/or (2) an expression vector that encodes, and is capable of expressing, an immunogenic peptide derived from CXCL13 or CXCR5. The immunogenic peptide derived from CXCL13 or CXCR5 may be fused to another moiety to enhance its immunogenicity. Examples of the CXCL13 immunogenic peptides include, but are not limited to, peptides consisting of, or comprising, one or more sequences 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). Examples of the CXCR5 immunogenic peptides include, but are not limited to, peptides consisting of, or comprising, one or more sequences selected from the group consisting of 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).
The term “CXCL16 immunogen” and “CXCR6 immunogen” refers to an immunogenic composition comprising (1) an immunogenic peptide derived from CXCL16 or CXCR6 and/or (2) an expression vector that encodes, and is capable of expressing, an immunogenic peptide derived from CXCL16 or CXCR6. The immunogenic peptide derived from CXCL16 or CXCR6 may be in the form of a fusion protein to enhance its immunogenicity. Examples of the CXCL16 immunogenic peptides include, but are not limited to, peptides consisting of, or comprising, one or more sequences 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), RPTLPVGSL (SEQ ID NO:108), TAGHSLAAG (SEQ ID NO:109), GKRISSDSPPSVQ (SEQ ID NO:110), KDPWVQELMSCLDLKECGHAYSGIVAHQKH (SEQ ID NO:111). Examples of the CXCR6 immunogenic peptides include, but are not limited to, peptides consisting of, or comprising, one or more sequences selected from the group consisting of HQDFLQFSKV (SEQ ID NO:112), AGIHEWVFGQVMCK (SEQ ID NO:113), PQIIYGNVFNLDKLICGYHDEAI (SEQ ID NO:114) and YYAMTSFHYTIMVTEA (SEQ ID NO:115).
The term “biological sample,” as used herein, refers to material of a biological origin, which may be a body fluid or body product such as blood, plasma, urine, saliva, spinal fluid, stool, sweat or breath. Biological sample also includes tissue samples and cell samples.
Ranges may 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 is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed 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 is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed.
CXCL13-CXCR5 Binding Mediates DOCK2-Dependent Chemotaxis
CXCL13, also known as B lymphocyte chemoattractant (BLC), is a ligand for the CXCR5 chemokine receptor. Both the chemokine and the receptor appear to play a role in the regulation of metastasis and invasion of cancer. Both CXCL13 and CXCR5 are locally up-regulated in multiple carcinoma tissue types compared to normal tissues, including ovarian, lung, breast, prostate, bone and pancreatic cancers. CXCL13 levels are also increased in the serum of patients with those cancers. Additionally, soluble CXCL13 chemokine enhances both in vivo and in vitro proliferation and migration of cancer cells.
CXCR5 (CD185), also known as Burkett lymphoma receptor 1 (BLR1) is a member of the chemokine receptor family of G protein coupled receptors (GPCRs) that may have a diverse role in cancer cell survival that presumably supports protection against chemotherapeutic drugs. Interaction of CXCR5 with CXCL13 activates DOCK2 (Dedicator of cytokenesis 2), which binds to the DOCK-binding protein ELMO1 (Engulfment and cell motility protein 1), allowing DOCK2-mediated Rae (Ras-related C3 botulinum toxin substrate proteins, a family of signaling G proteins that is a subfamily of the Rho family of GTPases) activation in lymphocytes. DOCK2 binds both the Rac1 and Rac2 isoforms and DOCK2-dependent Rac activation regulates neutrophil NADPH oxidase and is important for chemotaxis in neutrophils. In the present invention, the term “CXCR5” is inclusive of the transcription variants of CXCR5, such as CXCR5a (CXCR5 transcription variant 2) and CXCR5b (CXCR5 transcription variant 1).
Methods for Treating or Preventing Cancer Using Anti-CXCL13, Anti-CXCR5, Anti-CXCL16 and/or Anti-CXCR6 Antibodies
One aspect of the present invention relates to methods for treating or preventing cancer using an anti-CXCL13 antibody and/or an anti-CXCR5 antibody. The method comprises administering to a subject in need of such treatment, a therapeutically effective amount of an anti-CXCL13 antibody, an anti-CXCR5 antibody, or a combination thereof. In one embodiment, the cancer is melanoma, lymphoma, myeloma, leukemia, sarcoma, blastoma or a carcinoma. Examples of carcinoma include, but are not limited to, acinic cell carcinoma, adenoid cystic carcinoma, adenocarcinoma, adenosquamous carcinoma, adrenocortical adenoma, adrenocortical carcinoma, anaplastic carcinoma, apudoma, basal cell carcinoma, carcinoid, carcinosarcoma, clear cell carcinoma, cylindroma, cystadenocarcinoma, ductal carcinoma, gastrinoma, giant cell carcinoma, glioma, glucagonoma, Hurthle cell carcinoma, insulinoma, large cell carcinoma, lobular carcinoma, medulloblastoma, medullary carcinoma, mucinous cystadenoma, mucoepidermoid carcinoma, neuroectodermal tumor, oncocytoma, papillary hidradenoma, papilloma, pleomorphic carcinoma, pulmonary blastoma, sarcomatoid carcinoma, serous cystadenoma, Signet ring cell carcinoma, small cell carcinoma, somatostatinoma, spindle cell carcinoma, squamous cell carcinoma, thymoma, verrucous carcinoma, and of organs or tissues that line the inner or outer surfaces of the body originating from endodermal, extodermal, or epithelial cells. These organs and tissues include, but are not limited to: bone, breast, central nervous system, cervix, colon, endometrium, esophagus, fallopian tube, gastrointestinal tract, kidney, lung, lymphoid, mammary gland, oral cavity, ovary, pancreas, pituitary gland, prostate, rectum, reproductive tract, respiratory tract, stomach, sweat gland, thymus, thyroid, uterus, vagina.
In another embodiment, the subject is diagnosed with a cancer that results in elevated CXCL13 and/or CXCR5 expression in the cancer cells. Examples of such cancer include, but are not limited to, melanoma, lymphoma, myeloma, leukemia, sarcoma, blastoma and carcinoma. In one embodiment, the subject is diagnosed with brain cancer. In another embodiment, the subject is diagnosed with prostate cancer. In another embodiment, the method comprises the step of immunizing the subject with an effective amount of CXCL13 and/or CXCR5 immunogen(s) as protein, peptide or encoded gene to induce antibodies that inhibit the biological activity of CXCL13 and/or CXCR5. In another embodiment, the subject is diagnosed with bone cancer. In another embodiment, the subject is diagnosed with pituitary cancer. In yet another embodiment, the subject is diagnosed with ovarian cancer. In another embodiment, the subject is diagnosed with lung cancer. In another embodiment, the subject is diagnosed with breast cancer. In another embodiment, the subject is diagnosed with colon cancer. In another embodiment, the subject is diagnosed with lymphoma or myeloma. In another embodiment, the subject is diagnosed with leukemia.
In another embodiment, the method comprises the step of immunizing the subject with an effective amount of CXCL16 and/or CXCR6 immunogen(s) as protein, peptide or encoded gene to induce antibodies that inhibit the biological activity of CXCL16 and/or CXCR6.
In another embodiment, the method comprises the step of immunizing the subject with an effective amount of one or more of CXCL13 and CXCR5 immunogens and an effective amount of one or more of CXCL16 and CXCR6 immunogens.
Another aspect of the present invention relates to methods for inhibiting or preventing metastasis of a cancer by treatment of a subject in need thereof with an anti-CXCL13 antibody and/or an anti-CXCR5 antibody. In one embodiment, an anti-CXCL13 antibody and/or an anti-CXCR5 antibody inhibits or prevents invasion of a tissue by a cancer. In another embodiment, the method comprises the step of immunizing the subject with an effective amount of CXCL13 and/or CXCR5 immunogen(s) as protein, peptide or encoded gene to induce antibodies that inhibit the biological activity of CXCL13 and/or CXCR5. In some embodiments, the cancer is prostate cancer. In other embodiments, the cancer is melanoma. In some embodiments, said tissue is bone.
Another aspect of the present invention relates to methods for treatment of a subject in need thereof with an anti-CXCL13 antibody and/or an anti-CXCR5 antibody causing regression of an established tumor. In certain embodiments, the method comprises the step of immunizing the subject with an effective amount of CXCL13 and/or CXCR5 immunogen(s) as protein, peptide or encoded gene to induce antibodies that inhibit the biological activity of CXCL13 and/or CXCR5. In particular embodiments, the established tumor is an advanced tumor. In some embodiments, the established tumor is a metastatic tumor.
Another aspect of the present invention relates to methods for treatment of a subject in need thereof with an anti-CXCL13 antibody and/or an anti-CXCR5 antibody to prevent or inhibit osteolytic growth of a tumor in bone. In some embodiment, the method comprises the step of immunizing the subject with an effective amount of CXCL13 and/or CXCR5 immunogen(s) as protein, peptide or encoded gene to induce antibodies that inhibit the biological activity of CXCL13 and/or CXCR5. In some other embodiments, the tumor is a melanoma, lymphoma, sarcoma, blastoma or carcinoma. In some embodiments, the tumor is a carcinoma. In a particular embodiment, the tumor is prostate cancer. In some embodiments, the treatment with an anti-CXCL13 antibody and/or an anti-CXCR5 antibody prevents osteolysis or bone resorption by a tumor.
In another embodiment, the method further comprises determining the level of CXCL13 and/or CXCR5 expression in a tissue from the subject, and, if an increased level of CXCL13 and/or CXCR5 is detected, administering to the subject a therapeutically effective amount of an anti-CXCL13 antibody, an anti-CXCR5 antibody, or a combination thereof. In another embodiment, the method comprises the step of immunizing the subject with an effective amount of CXCL13 and/or CXCR5 immunogen(s) as protein, peptide or encoded gene to induce antibodies that inhibit the biological activity of CXCL13 and/or CXCR5.
A preferred antibody of the present invention is one which binds to human CXCL13 and preferably blocks (partially or completely) the ability of CXCL13 to bind to a receptor, including, but not limited to, CXCR5. Another preferred antibody of the present invention is one which binds to human CXCR5 and preferably blocks (partially or completely) the ability of a cell, such as a tumor or carcinoma cell, expressing the CXCR5 chemokine receptor at its cell surface to bind to a ligand, including, but not limited to, CXCL13. Yet another preferred antibody of the present invention is one which binds to human CXCR5 and preferably blocks (partially or completely) the ability of soluble CXCR5 chemokine receptor to bind to a ligand, including, but not limited to, CXCL13.
In one embodiment, the anti-CXCL13 antibody and/or anti-CXCR5 antibody is a monoclonal antibody. In another embodiment, the anti-CXCL13 antibody and/or anti-CXCR5 antibody is a humanized antibody. In another embodiment, the anti-CXCL13 antibody and/or anti-CXCR6 antibody is a humanized antibody fragment.
In particular embodiments of the present invention, treatment of a subject with an anti-CXCL13 and/or anti-CXCR5 antibody is in conjunction with the treatment of the subject beforehand, at the same time, or afterward with a therapeutically effective amount of at least one other antibody that is specific for another antigen. In one embodiment, the another antigen is another chemokine or chemokine receptor, such as CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL14, CXCL15, CXCL16, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5a, CXCR5b, CXCR6, CXCR7, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL24, CCL25, CCL25-1, CCL25-2, CCL27, CCL28, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCL1, XCL2, XCR1, CX3CR1, or CX3CL1.
In another embodiment, the another antigen is a chemokine or chemokine receptor associated with a carcinoma and selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL25, CXCL12, CXCL16, CCR2, CCR7, CCR8, CCR9, CXCR4, CXCR6, CXCR7, CX3CL1 and CX3CR1.
In another embodiment, the another antigen is a chemokine or chemokine receptor associated with a melanoma and selected from the group consisting of CCL25, CCL27, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL12, CXCL16, CX3CL1, CCR2, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR6, CXCR7 and CX3CR1.
In another embodiment, the another antigen is a chemokine or chemokine receptor associated with a lymphoma and selected from the group consisting of CXCL12, CXCR4, CXCR7, CCR2.
In another embodiment, the another antigen is a chemokine or chemokine receptor associated with a myeloma or leukemia.
In another embodiment, the another antigen is selected from the polypeptides recited in Table 1 and/or Table 2, and fragments of any of said polypeptides.
Other exemplary antigens include molecules such as renin; a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; a-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIII, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-□ and -β; enkephalinase; a serum albumin such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or NT-6), or a nerve growth factor such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); members of the ErbB receptor family such as the EGF receptor; transforming growth factor (TGF) such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD19, CD20 and CD34; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-α, -β, and -γ; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 and/or IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; αv/β3 integrin including either a or b subunits thereof, such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; prostate specific antigen (PSA); a tumor associated antigen such as carcinoembryonic antigen (CEA), CK2, CA125, TA90, HER2, HER3 or HER4 receptor; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mp1 receptor; CTLA-4; protein C; any one of the proteins from the classical, lectin or alternative complement pathways; and fragments of any of the above-listed polypeptides.
The antibody may be administered to the subject with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. In certain embodiments, the antibody is administered directly to a tumor or cancer tissue, including administration directly to the tumor bed during invasive procedures. The antibody may also be placed on a solid support such as a sponge or gauze for administration against the target chemokine to the affected tissues.
Antibodies of the invention can be administered in the usually accepted pharmaceutically acceptable carriers. Acceptable carriers include, but are not limited to, saline, buffered saline, and glucose in saline. Solid supports, liposomes, nanoparticles, microparticles, nanospheres or microspheres may also be used as carriers for administration of the antibodies.
The appropriate dosage (“therapeutically effective amount”) of the antibody will depend, for example, on the condition to be treated, the severity and course of the condition, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, the type of antibody used, and the discretion of the attending physician. The antibody is suitably administered to the patent at one time or over a series of treatments and may be administered to the patient at any time from diagnosis onwards. The antibody may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the condition in question.
As a general proposition, the therapeutically effective amount of the antibody administered will be in the range of about 1 ng/kg body weight/day to about 100 mg/kg body weight/day whether by one or more administrations. In a particular embodiments, the range of antibody administered is from about 1 ng/kg body weight/day to about 1 μg/kg body weight/day, 1 ng/kg body weight/day to about 100 ng/kg body weight/day, 1 ng/kg body weight/day to about 10 ng/kg body weight/day, 10 ng/kg body weight/day to about 1 μg/kg body weight/day, 10 ng/kg body weight/day to about 100 ng/kg body weight/day, 100 ng/kg body weight/day to about 1 μg/kg body weight/day, 100 ng/kg body weight/day to about 10 μg/kg body weight/day, 1 μg/kg body weight/day to about 10 μg/kg body weight/day, 1 μg/kg body weight/day to about 100 μg/kg body weight/day, 10 μg/kg body weight/day to about 100 μg/kg body weight/day, 10 μg/kg body weight/day to about 1 mg/kg body weight/day, 100 μg/kg body weight/day to about 10 mg/kg body weight/day, 1 mg/kg body weight/day to about 100 mg/kg body weight/day and 10 mg/kg body weight/day to about 100 mg/kg body weight/day.
In another embodiment, the antibody is administered at a dosage range of 1 ng-10 ng per injection, 10 ng to 100 ng per injection, 100 ng to 1 μg per injection, 1 μg to 10 μg per injection, 10 μg to 100 μg per injection, 100 μg to 1 mg per injection, 1 mg to 10 mg per injection, 10 mg to 100 mg per injection, and 100 mg to 1000 mg per injection. The antibody may be injected daily, or every 2, 3, 4, 5, 6 and 7 days, or every 1, 2, 3 or 4 weeks.
In another particular embodiment, the dose range of antibody administered is from about 1 ng/kg to about 100 mg/kg In still another particular embodiment, the range of antibody administered is from about 1 ng/kg to about 10 ng/kg, about 10 ng/kg to about 100 ng/kg, about 100 ng/kg to about 1 μg/kg, about 1 μg/kg to about 10 μg/kg, about 10 μg/kg to about 100 μg/kg, about 100 μg/kg to about 1 mg/kg, about 1 mg/kg to about 10 mg/kg, about 10 mg/kg to about 100 mg/kg, about 0.5 mg/kg to about 30 mg/kg, and about 1 mg/kg to about 15 mg/kg.
In other particular embodiments, the amount of antibody administered is, or is about, 0.0006, 0.001, 0.003, 0.006, 0.01, 0.03, 0.06, 0.1, 0.3, 0.6, 1, 3, 6, 10, 30, 60, 100, 300, 600 and 1000 mg/day. As expected, the dosage will be dependant on the condition, size, age and condition of the patient.
The antibody may be administered, as appropriate or indicated, a single dose as a bolus or by continuous infusion, or as multiple doses by bolus or by continuous infusion. Multiple doses may be administered, for example, multiple times per day, once daily, every 2, 3, 4, 5, 6 or 7 days, weekly, every 2, 3, 4, 5 or 6 weeks or monthly. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques.
In particular embodiments of the present invention, therapeutically effective amount of anti-CXCL13 and/or anti-CXCR5 antibody may be administered to a subject in need thereof as a sole therapeutic agent. In a particular embodiment, the therapeutically effective amount of anti-CXCL13 and/or anti-CXCR5 antibody to kill or promote apoptosis of the tumor or carcinoma cells. In another particular embodiment, the therapeutically effective amount of anti-CXCL13 and/or anti-CXCR5 antibody inhibits or prevents the establishment of a tumor or carcinoma. In a further particular embodiment, the therapeutically effective amount of anti-CXCL13 and/or anti-CXCR5 antibody inhibits or prevents the migration or metastasis of tumor or carcinoma cells from an existing tumor or carcinoma. In yet another particular embodiment, the therapeutically effective amount of anti-CXCL13 and/or anti-CXCR5 antibody inhibits or prevents the invasion of tumor or carcinoma cells into non-cancerous tissues.
In particular embodiments of the present invention, therapeutically effective amount of anti-CXCL13 and/or anti-CXCR5 antibody may be administered to a subject in need thereof in conjunction with one or more additional therapeutically effective antibodies. Said one or more additional therapeutically effective antibodies may be directed to additional determinants on CXCL13 and/or CXCR5, other chemokines, other chemokine receptors, other soluble or cell surface ligands or receptors including, but not limited to, tumor or carcinoma specific antigens, viral, bacterial or parasite antigens, products of cancer cells or remnants of apoptosis. The anti-CXCL13 and/or anti-CXCR5 antibody may be administered before, concurrently with, and/or after the one or more additional therapeutically effective antibodies.
In a particular embodiment, the therapeutically effective amount of anti-CXCL13 and/or anti-CXCR5 antibody augments the effectiveness of the one or more additional therapeutically effective antibodies in killing tumor or carcinoma cells. In a more particular embodiment, the therapeutically effective amount of anti-CXCL13 and/or anti-CXCR5 antibody reduces the amount of the one or more additional therapeutically effective antibodies required for killing tumor or carcinoma cells. In a further particular embodiment, the therapeutically effective amount of anti-CXCL13 and/or anti-CXCR5 antibody inhibits or prevents the migration or metastasis of tumor or carcinoma cells from an established tumor or carcinoma, enhancing the local effectiveness of the one or more additional therapeutically effective antibodies in killing tumor or carcinoma cells. In yet another particular embodiment, the therapeutically effective amount of anti-CXCL13 and/or anti-CXCR5 antibody inhibits or prevents the invasion of tumor or carcinoma cells into non-cancerous tissues, enhancing the local effectiveness of the one or more additional therapeutically effective antibodies in killing tumor or carcinoma cells.
In another embodiment, the anti-CXCL13 antibody and/or anti-CXCR5 antibody is an antibody conjugated to a cytotoxic agent. In another embodiment, the anti-CXCL13 antibody and/or anti-CXCR5 antibody is administered with another anti-cancer agent, such as chemotherapy agent.
Another aspect of the present invention relates to a method of inhibiting the interaction of the chemokine CXCL13 with a receptor therefore, comprising contacting the cell with an effective amount of an antibody or functional fragment thereof which binds to a mammalian CXCL13 or a portion of CXCL13.
Another aspect of the present invention relates to a method of inhibiting the interaction of a cell bearing CXCR5 with a ligand thereof, comprising contacting the cell with an effective amount of an antibody or functional fragment thereof which binds to a mammalian CXCR5 or a portion of CXCR5.
In another embodiment, the method of treating cancer comprises administering to an subject in need of such treatment, an effective amount of an expression vector that expresses an anti-CXCL13 antibody, an anti-CXCR5 antibody, or a combination thereof in a cancer or malignant cell. In another embodiment, the method of treating cancer comprises the step of immunizing the subject with an effective amount of CXCL13 and/or CXCR5 immunogen(s) to induce the host to produce antibodies that inhibit the biological activity of CXCL13 and/or CXCR5.
The expression vectors can be any vector that is capable of nucleotide deliver nucleotides encoding an anti-CXCL13 antibody and/or an anti-CXCR5 antibody into a target cell and express the anti-CXCL13 antibody and/or anti-CXCR5 antibody in the target cell. In another embodiment, the expression vector is capable of delivering nucleotides encoding CXCL13 and/or CXCR6 into a target cell to induce the host to produce anti-CXCL13 and/or CXCR5 antibodies. Examples of expression vectors include viral vectors and non-viral vectors.
Viral vectors include, but are not limited to, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, and other large capacity viral vectors, such as herpes virus and vaccinia virus. Also included are any viral families which share the properties of these viruses which make them suitable for use as expression vectors.
Retroviral Vectors
A retrovirus is an animal virus belonging to the virus family of Retroviridae, including any types, subfamilies, genus, or tropisms. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Pat. Nos. 4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the teachings of which are incorporated herein by reference.
Adenoviral Vectors
Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites. Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus.
A viral vector can be one based on an adenovirus which has had one or more viral genes removed and these virions are generated in a complement cell line, such as the human 293 cell line. In one embodiment, the E1 gene is removed from the adenoviral vector. In another embodiment, both the E1 and E3 genes are removed from the adenoviral vector. In another embodiment, both the E1 and E4 genes are removed from the adenoviral vector. In another embodiment, the adenovirus vector is a gutless adenovirus vector.
Adeno-Associated Viral Vectors
Another type of viral vector is based on an adeno-associated virus (AAV). This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, Calif., which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and/or a marker gene, such as the gene encoding the green fluorescent protein, GFP.
In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus.
Typically the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site-specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. U.S. Pat. No. 6,261,834 is herein incorporated by reference for material related to the AAV vector.
Large Payload Viral Vectors
Molecular genetic experiments with large human herpes viruses have provided a means whereby large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpes viruses (Sun et al., Nature genetics 8: 33-41, 1994; Cotter and Robertson, Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have the potential to deliver fragments of human heterologous DNA>150 kb to specific cells. EBV recombinants can maintain large pieces of DNA in the infected B-cells as episomal DNA. Individual clones carried human genomic inserts up to 330 kb appeared genetically stable. The maintenance of these episomes requires a specific EBV nuclear protein, EBNA1, constitutively expressed during infection with EBV. Additionally, these vectors can be used for transfection, where large amounts of protein can be generated transiently in vitro. Herpesvirus amplicon systems are also being used to package pieces of DNA>220 kb and to infect cells that can stably maintain DNA as episomes. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.
Non-Viral vectors include plasmid expression vectors. Plasmid vectors typically include a circular double-stranded DNA loop into which additional DNA segments can be inserted.
In both viral and non-viral expression vectors, the polynucleotide encoding the antibody or antibodies is typically arranged in proximity and orientation to an appropriate transcription control sequence (promoter, and optionally, one or more enhancers) to direct mRNA synthesis. That is, the polynucleotide sequence of interest is operably linked to an appropriate transcription control sequence. Examples of such promoters include: viral promoters such as the immediate early promoter of CMV, LTR or SV40 promoter, polyhedron promoter of baculovirus, E. coli lac or trp promoter, phage T7 and lambda PL promoter, and other promoters known to control expression of genes in eukaryotic cells or their viruses. The promoters may be a tissue specific promoter.
The expression vector typically also contains a ribosome binding site for translation initiation, and a transcription terminator. The vector optionally includes appropriate sequences for amplifying expression. In addition, the expression vectors optionally comprise one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli. 
The expression vector can also include additional expression elements, for example, to improve the efficiency of translation. These signals can include, e.g., an ATG initiation codon and adjacent sequences. In some cases, for example, a translation initiation codon and associated sequence elements are inserted into the appropriate expression vector simultaneously with the polynucleotide sequence of interest (e.g., a native start codon). In such cases, additional translational control signals are not required. However, in cases where only a polypeptide coding sequence, or a portion thereof, is inserted, exogenous translational control signals, including an ATG initiation codon is provided. The initiation codon is placed in the correct reading frame to ensure translation of the polynucleotide sequence of interest. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. If desired, the efficiency of expression can be further increased by the inclusion of enhancers appropriate to the cell system in use (Scharf et al. (1994) Results Probl Cell Differ 20:125-62; Bitter et al. (1987) Methods in Enzymol 153:516-544).
In one embodiment, the expression vector contains an inducible or regulatable expression system. Examples of regulatable expression systems are briefly described below:
Ecdysone System.
The ecdysone system is based on the molting induction system found in Drosophila, but modified for inducible expression in mammalian cells. The system uses an analog of the drosophila steroid hormone ecdysone, muristerone A, to activate expression of the gene of interest via a heterodimeric nuclear receptor. Expression levels have been reported to exceed 200-fold over basal levels with no effect on mammalian cell physiology.
Progesterone System.
The progesterone receptor is normally stimulated to bind to a specific DNA sequence and to activate transcription through an interaction with its hormone ligand. Conversely, the progesterone antagonist mifepristone (RU486) is able to block hormone-induced nuclear transport and subsequent DNA binding. A mutant form of the progesterone receptor that can be stimulated to bind through an interaction with RU486 has been generated. To generate a specific, regulatable transcription factor, the RU486-binding domain of the progesterone receptor has been fused to the DNA-binding domain of the yeast transcription factor GAL4 and the transactivation domain of the HSV protein VP16. The chimeric factor is inactive in the absence of RU486. The addition of hormone, however, induces a conformational change in the chimeric protein, and this change allows binding to a GAL4-binding site and the activation of transcription from promoters containing the GAL4-binding site.
Rapamycin System.
Immunosuppressive agents, such as FK506 and rapamycin, act by binding to specific cellular proteins and facilitating their dimerization. For example, the binding of rapamycin to FK506-binding protein (FKBP) results in its heterodimerization with another rapamycin binding protein FRAP, which can be reversed by removal of the drug. The ability to bring two proteins together by addition of a drug potentiates the regulation of a number of biological processes, including transcription. A chimeric DNA-binding domain has been fused to the FKBP, which enables binding of the fusion protein to a specific DNA-binding sequence. A transcriptional activation domain also has been fused to FRAP. When these two fusion proteins are co-expressed in the same cell, a fully functional transcription factor can be formed by heterodimerization mediated by addition of rapamycin. The dimerized chimeric transcription factor can then bind to a synthetic promoter sequence containing copies of the synthetic DNA-binding sequence. This system has been successfully integrated into adenoviral and AAV vectors. Long term regulatable gene expression has been achieved in both mice and baboons.
Methods for Treating or Preventing Cancer Using Agents that Inhibits the Expression or Activity of CXCL13 or CXCR5
Another aspect of the present invention relates to methods for treating or preventing cancer by using agents that inhibits the expression or activity of CXCL13 or CXCR5. In another embodiment, the method comprises administering to a subject in need of such treatment, an effective amount of an expression vector that expresses an agent that (1) inhibits the expression of CXCL13 and/or CXCR5, or (2) inhibits the interaction between CXCL13 and CXCR5, or (3) inhibits a biological activity of CXCL13 and/or CXCR5. In one embodiment, the biological activity of CXCL13 and CXCR5 includes the interaction between CXCL13 and CXCR5.
In another embodiment, the subject is diagnosed with a cancer that results in elevated CXCL13 and/or CXCR5 expression in the cancer cells. Examples of such cancer include, but are not limited to, melanoma, lymphoma, myeloma, leukemia and carcinoma, such as ovarian cancer, vaginal cancer, cervical cancer, uterine cancer, prostate cancer, anal cancer, rectal cancer, colon cancer, stomach cancer, pancreatic cancer, insulinoma, glucagonoma, adenocarcinoma, adenosquamous carcinoma, neuroendocrine tumor, breast cancer, lung cancer, esophageal cancer, oral cancer, brain cancer, medulloblastoma, neuroectodermal tumor, glioma, pituitary cancer, and bone cancer.
In another embodiment, the method further comprises determining the level of CXCL13 and/or CXCR5 expression in a tissue from the subject, and administering the agent to the subject only if an increased level of CXCL13 and/or CXCR5 is detected in the tissue.
In one embodiment, the expression vector is a viral vector. In another embodiment, the expression vector is a non-vector vector. In another embodiment, the expression vector is capable of delivering nucleotides encoding CXCL13 and/or CXCR5 into a target cell to induce the host to produce anti-CXCL13 and/or CXCR5 antibodies.
In another embodiment, the agent is an anti-CXCL13 antibody, an anti-CXCR5 antibody, or a combination thereof.
In yet another embodiment, the agent is a functional nucleic acid. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction. The functional nucleic acid molecules can act as inhibitors of a specific activity possessed by a target molecule. Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA and polypeptides. Thus, functional nucleic acids can interact with mRNA or the genomic DNA of CXCL13 or CXCR5 to inhibit expression or interact with CXCL13 or CXCR5 protein to inhibit activity. Often functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place. Examples of functional nucleic acid molecules include siRNA, antisense molecules, aptamers, ribozymes, triplex forming molecules, and external guide sequences.
siRNA is involved in RNA interference (RNAi) which involves a two-step mechanism: an initiation step and an effector step. In the first step, input double-stranded (ds) RNA (siRNA) is processed into small fragments, such as 21-23-nucleotide ‘guide sequences’. RNA amplification occurs in whole animals. Typically then, the guide RNAs can be incorporated into a protein RNA complex which is capable of degrading RNA, the nuclease complex, which has been called the RNA-induced silencing complex (RISC). This RISC complex acts in the second effector step to destroy mRNAs that are recognized by the guide RNAs through base-pairing interactions. RNAi involves the introduction by any means of double stranded RNA into the cell which triggers events that cause the degradation of a target RNA. RNAi is a form of post-transcriptional gene silencing. In addition to the siRNAs disclosed herein, disclosed are RNA hairpins that can act in RNAi. For description of making and using RNAi molecules see, e.g., Hammond et al., Nature Rev Gen 2: 110-119 (2001); Sharp, Genes Dev 15: 485-490 (2001), Waterhouse et al., Proc. Natl. Acad. Sci. USA 95(23): 13959-13964 (1998) all of which are incorporated herein by reference in their entireties and at least form material related to delivery and making of RNAi molecules.
RNAi has been shown to work in many types of cells, including mammalian cells. For work in mammalian cells it is preferred that the RNA molecules which will be used as targeting sequences within the RISC complex are shorter. For example, less than or equal to 50 or 40 or 30 or 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides in length. These RNA molecules can also have overhangs on the 3′ or 5′ ends relative to the target RNA which is to be cleaved. These overhangs can be at least or less than or equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nucleotides long.
Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC. It is preferred that antisense molecules bind the target molecule with a dissociation constant (kd) less than or equal to 10-6, 10-8, 10-10, or 10-12. A representative sample of methods and techniques which aid in the design and use of antisense molecules can be found in the following non-limiting list of U.S. Pat. Nos. 5,135,917, 5,994,320, 6,046,319, and 6,057,437.
Aptamers are molecules that interact with a target molecule, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind a chemokines and block its function (see, e.g., Marro et al., Biochem Biophys Res Commun. 2006 Oct. 13; 349:270-6). Aptamers can bind very tightly with kds from the target molecule of less than 10-12 M. It is preferred that the aptamers bind the target molecule with a kd less than 10−6, 10−8, 1010, or 10−12. Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule (U.S. Pat. No. 5,543,293). It is preferred that the aptamer have a kd with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the kd with a background binding molecule. Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in the following non-limiting list of U.S. Pat. Nos. 5,476,766, 5,861,254, 6,030,776, and 6,051,698.
Ribozymes are nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. Ribozymes are thus catalytic nucleic acid. It is preferred that the ribozymes catalyze intermolecular reactions. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes, (see, e.g., U.S. Pat. Nos. 5,334,711 and 5,861,288, WO 9858058 and WO 9718312) hairpin ribozymes (see, e.g., U.S. Pat. Nos. 5,631,115 and 6,022,962), and tetrahymena ribozymes (see, e.g., U.S. Pat. Nos. 5,595,873 and 5,652,107). There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo (see, e.g., U.S. Pat. Nos. 5,580,967 and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions. This property makes ribozymes particularly good candidates for target specific cleavage of nucleic acids because recognition of the target substrate is based on the target substrates sequence. Representative examples of how to make and use ribozymes to catalyze a variety of different reactions can be found in U.S. Pat. Nos. 5,646,042, 5,869,253, 5,989,906, and 6,017,756.
Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which three strands of DNA are forming a complex dependant on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a kd less than 10−6, 10−8, 10−10, or 10−12. Representative examples of how to make and use triplex forming molecules to bind a variety of different target molecules can be found in U.S. Pat. Nos. 5,176,996, 5,683,874, 5,874,566, and 5,962,426.
External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNase P, which cleaves the target molecule. EGSs can be designed to specifically target a RNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate (see, e.g., WO 92/03566 by Yale, and Forster and Altman, Science 238:407-409 (1990)).
Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukaryotic cells. (Yuan et al., Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 by Yale; WO 95/24489 by Yale; Yuan and Altman, EMBO J 14:159-168 (1995), and Carrara et al., Proc. Natl. Acad. Sci. USA 92:2627-2631 (1995)). Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules be found in the following non-limiting list of U.S. Pat. Nos. 5,168,053, 5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162.
Methods for Prevention or Inhibition of Migration or Metastasis of Cancer Cells with Elevated Expression of CXCL13 and/or CXCR5
Another aspect of the present invention relates to a method for prevention or inhibition of the migration or metastasis of cancer cells with elevated expression of CXCL13 and/or CXCR5 in a subject.
In one embodiment, the method comprises the step of administering to the subject a therapeutically effective amount of an anti-CXCL13 antibody, or an anti-CXCR5 antibody, or a combination thereof.
In another embodiment, the method comprises the step of administering to the subject an expression vector that expresses an anti-CXCL13 antibody, or an anti-CXCR5 antibody, or a combination thereof in said subject.
In another embodiment, the method comprises administering to the subject an expression vector that expresses an agent capable of inhibiting the expression of CXCL13 or CXCR5, or a biological activity of CXCL13 or CXCR5, or the interaction between CXCL13 and CXCR5. In one embodiment, the expression vector is capable of delivering nucleotides encoding CXCL13 and/or CXCR5 into a target cell to induce the host to produce anti-CXCL13 and/or CXCR5 antibodies.
Expression of CXCL13 and/or CXCR5 in cancer cells can be determined using methods well known in the art, such as immunostaining or quantitative PCR. Cancer cells that are known to overexpress CXCL13 and/or CXCR5 include, but are not limited to, melanoma cells and carcinoma cells. Examples of carcinoma include, but are not limited to, ovarian cancer, vaginal cancer, cervical cancer, uterine cancer, prostate cancer, anal cancer, rectal cancer, colon cancer, stomach cancer, pancreatic cancer, insulinoma, adenocarcinoma, adenosquamous carcinoma, neuroendocrine tumor, breast cancer, lung cancer, esophageal cancer, oral cancer, brain cancer, medulloblastoma, neuroectodermal tumor, glioma, pituitary cancer, and bone cancer.
In one embodiment, the cancer cells are brain cancer cells. In another embodiment, the cancer cells are bone cancer cells. In another embodiment, the cancer cells are pituitary cancer cells. In yet another embodiment, the cancer cells are ovarian cancer cells.
Method for Enhancing the Effect of Chemotherapy
Another aspect of the present invention relates to a method for enhancing the effect of chemotherapy. In one embodiment, the method comprises administering to a subject who is under chemotherapy for a cancer, an effective amount of an anti-CXCL13 antibody, or an anti-CXCR5 antibody, or a combination thereof.
In another embodiment, the method comprises administering to a subject who is under chemotherapy for a cancer, an effective amount of an expression vector that expresses anti-CXCL13 antibody, or an anti-CXCR5 antibody, or a combination thereof.
In another embodiment, the method comprises administering to a subject who is under chemotherapy for a cancer an expression vector that expresses an agent capable of inhibiting the expression of CXCL13 or CXCR5, or a biological activity of CXCL13 or CXCR5, or the interaction between CXCL13 and CXCR5. In another embodiment, the expression vector is capable of delivering nucleotides encoding CXCL13 and/or CXCR5 into a target cell to induce the host to produce anti-CXCL13 and/or CXCR5 antibodies.
In one embodiment, the subject is under chemotherapy for melanoma, lymphoma, myeloma, leukemia or carcinoma. In another embodiment, the subject is under chemotherapy for brain cancer. In another embodiment, the subject is under chemotherapy for bone cancer. In another embodiment, the subject is under chemotherapy for pituitary cancer. In yet another embodiment, the subject is under chemotherapy for ovarian cancer.
Compositions and Kits for Treating of Preventing Cancer
Another aspect of the present invention relates to compositions and kits for treating or preventing cancer. In one embodiment, the composition comprises (1) an anti-CXCL13 antibody, an anti-CXCR5 antibody, or a combination thereof, and (2) a pharmaceutically acceptable carrier. In another embodiment, the composition comprises (1) an expression vector carrying the coding sequence for an anti-CXCL13 antibody, an anti-CXCR5 antibody, or a combination thereof, and (2) a pharmaceutically acceptable carrier. In another embodiment, the composition comprises (1) an expression vector carrying the coding sequence for an agent that inhibits the expression of CXCL13 or CXCR5, or a biological activity of CXCL13 or CXCR5, or the interaction between CXCL13 and CXCR5, and (2) a pharmaceutically acceptable carrier.
The composition of the present invention may contain a single type of antibody, such as an anti-CXCL13 or anti-CXCR5 antibody alone, or both types of antibodies. The composition may also contain therapeutically effective amounts of antibodies specific for one or more additional antigens as described above as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect one another. For example, where the carcinoma being treated is ovarian cancer, it may be desirable to prepare a therapeutic formulation comprising anti-CXCL13 and/or -CXCR5 with one or more further anti-cancer determinant antibodies, such as an anti-CEA, anti-CA125 and/or anti-TA90 in a single formulation. In some embodiments of the present invention, a therapeutic antibody may be combined with an chemotherapy agent or a cytotoxic agent. In other embodiments of the present invention, a therapeutic antibody may be combined with an anti-inflammatory agent or a thrombolytic agent. Such agents are suitably present in combination in amounts that are effective for the purpose intended.
As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, lubricants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary agents can also be incorporated into the compositions. In certain embodiments, the pharmaceutically acceptable carrier comprises serum albumin.
The pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intrathecal, intra-arterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical) and transmucosal administration. In certain embodiments, the pharmaceutical composition is administered directly into a tumor tissue.
Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine; propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or ethyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the injectable composition should be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the requited particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a neuregulin) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Stertes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the pharmaceutical compositions are formulated into ointments, salves, gels, or creams as generally known in the art.
In certain embodiments, the pharmaceutical composition is formulated for sustained or controlled release of the active ingredient. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from e.g. Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein includes physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within orange of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. In certain embodiments, single dosage contains 0.01 ug to 50 mg of a chimeric neuregulin. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
The present invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures and Tables, are incorporated herein by reference.
Example 1
In Vitro Analysis of DOCK2 Expression in Cancer Cell Lines and CXCL13-CXCR5 Mediation of Cancer Cell Invasion
Total cell lysates (60 μg) from RWPE-1, LNCaP, and PC3 cells were resolved by SDS-PAGE and subjected to immunoblotting using antibodies against DOCK2 (FIG. 1A). GAPDH served as loading control. In FIG. 1B, DOCK2 silencing conditions were optimized by transfecting PC3 cells with 2 μM of DOCK2 siRNA duplex following manufacturer's protocol (Santa Cruz), and incubating cells for 0, 24, 48, and 72 hours. The efficacy of DOCK2 silencing was determined by Western blot analysis.
While CXCL13:CXCR5 interaction is known to mediate DOCK2-dependent chemotaxis of neutrophils, it was also found that CXCL13:CXCR5 interaction is also capable of promoting cancer cell metastasis and invasion independent of DOCK2. In FIG. 2, LNCaP and PC3 cells were tested for their ability to invade MatrigelTM Matrix and migrate through an 8.0 μm porous membrane in the presence of CXCL13 (100 ng/ml), anti-human CXCR5 antibody (1 μg/ml), DOCK2 or control siRNA. Cells which invaded to the lower surface of the membrane were stained with crystal violet and counted by microscopy at 40× magnification. Percent cell invasion was calculated following manufacturer's instructions (BD Biosciences). Error bars represent standard error of means of 3 independent experiments. Asterisks (*) indicate significant differences (p<0.05) relative to CXCL13-treated cells (control).
CXCL13 also regulates activation of Akt and ERK1/2, as shown in FIG. 3. FACE assays were performed to measure active and total Akt or ERK1/2 in LNCaP and PC3 cell lines. Cells were treated with anti-human CXCR5 antibody, DOCK2 siRNA, Control siRNA, or JNK inhibitor in the presence of CXCL13 (100 ng/ml) for 0, 5 or 10 minutes. Experiments were performed in triplicate and results show the ratio of active (phosphorylated) to total Akt or ERK1/2. Error bars represent ±standard error of means of 3 independent experiments. Asterisks (*) indicate significant (p<0.05) decrease in phosphorylation.
FIG. 4 shows that CXCL13 induces JNK activation through DOCK2 in PC3 cells. RWPE-1, LNCaP, and PC3 cells were treated with DOCK2 siRNA and corresponding control in the presence or absence of CXCL13 (100 ng/ml). Lysates were collected 5 minutes following CXCL13 stimulation and samples were resolved on SDS-PAGE. Membranes were blotted for phospho-JNK (46 kDa). GAPDH serves as loading control.
In FIG. 5, it is shown that CXCL13 regulates prostate cancer (PCa) cell proliferation through JNK and DOCK2. RWPE-1, LNCaP, and PC3 cells were grown in reduced serum conditions (2% FBS) in the presence or absence of 100 ng/ml CXCL13, 1 μg/ml anti-CXCR5 antibody, DOCK2 siRNA, and/or 10 μM JNK inhibitor. MTT assay was done over 3 days to assess cell proliferation. Error bars represent ±standard error of means of 3 independent experiments. Asterisks (*) indicate significant (p<0.05) changes relative to CXCL13 treated cells.
FIG. 6 shows JNK inhibition and DOCK2 knockdown lead to reduction of PCa cell proliferation that is not due to apoptosis. RWPE-1, LNCaP, and PC3 cells were grown in reduced serum conditions (2% FBS) in the presence or absence of 100 ng/ml CXCL13, 1 μg/ml anti-CXCR5 antibody, DOCK2 siRNA, 10 μM JNK inhibitor, or 1 μM Wortmannin. Caspase activity was measured using the CASPASE-GLO 3/7 Assay (Promega, Madison, Wis.) according to the manufacturer's directions. Asterisks (*) indicate significant (p<0.05) changes relative to no additions.
FIG. 7 shows CXCL13 modulation of signaling cascades in PCa cell lines. CXCL13 through its cognate receptor CXCR5 elicits Akt and ERK1/2 activation. In LNCaP cells CXCL13 also regulates JNK activation, presumably via Gαq/11 coupled to CXCR5, which mediates activation of phospholipase C (PLC) and protein kinase C (PKC). In PC3 cells, however, JNK activation is mediated through DOCK2.
FIGS. 8A-C demonstrate the expression of G-protein α subunit isoforms in prostate cancer cell lines. Equal amounts of protein (50 μg) from RWPE-1, LNCaP, C4-2B, and PC3 cells were resolved by SDS-Page. Expression of (A) Gαi1,2,3, Gαs; (B) Gα12, Gα13 and (C) Gαq/11 and Gα16 were determined by immunoblot. GAPDH served as a loading control.
FIGS. 9A-B demonstrates the expression of G-protein β and γ subunit isoforms in prostate cancer cell lines. Equal amounts of protein (50 μg) from RWPE-1, LNCaP, C4-2B, and PC3 cells were resolved by SDS-Page. Expression of Gβ1,2,3,4,5 and Gγ1,2,3,4,5,7,9,10,13 were determined by immunoblot. GAPDH served as a loading control.
FIGS. 10 A-C show expression of CXCR5 and associated G proteins in prostate cancer cell lines treated with or without CXCL13. (A) C) CXCR5 protein levels were analyzed by Western blot of RWPE-1, LNCaP, C4-2B, and PC3 cell lysates (50 Ng). GAPDH served as loading control. (B) Cell lines were treated with or without CXCL13 and lysed. CXCR5 was immuno-precipitated (IP) to pull down associated proteins from total cell lysates. The IP cell lysates were resolved by SDS-PAGE and the expression of (B) Gαi1, Gαi2, Gα13, Gαs, Gαq/11, Gα12, Gα13, (C) Gβ1, Gβ2, Gβ3, Gβ4, and (D) Gγ5, Gγ7, Gγ9, Gγ10 were examined by immunoblot.
The validation of Gq/11 and Gαi2 protein association with CXCR5 by immunoprecipitation is shown in FIGS. 11A-B. Cell lines were treated with or without CXCL13 and lysed (A) Gαq/11 and (B) Gαi2 were immunoprecipitated (IP) from total cell lysates. The IP cell lysates were resolved by SDS-PAGE and CXCR5 expression was examined by immunoblot.
Identification of CXCR4 and CXCR5 coupled to Gα13 following CXCL13 stimulation is shown in FIGS. 12A-C. Cell lines were treated with or without CXCL13 and lysed. Antibody against Gα13 was used to immunoprecipitate (IP) it from total cell lysates. The IP cell lysates were resolved by SDS PAGE and immunoblotted for (FIG. 12A) CXCR5 and (FIG. 12B) CXCR4. (FIG. 12C) Western blot analysis of CXCR4 expression was also performed for CXCR5 IP lysates before and after CXCL13 treatment. GAPDH served as a loading control.
FIG. 13 depicts a hypothetical model of CXCR5 interactions in prostate cancer cells. CXCR5 associates with CXCR4 and couples with Gαq/11/Gβ3/Gγ9 heterotrimers in androgen-dependent LNCaP cell lines or Gαi2/Gβ3/Gγ9 heterotrimers in hormone refractory C4-2B and PC3 cell lines in the absence of its specific ligand, CXCL13. Upon CXCL13 stimulation, G-proteins dissociate from CXCR5 to activate effector molecules. In addition, CXCL13-activated CXCR5 causes, associates or sequesters Gα13 protein favoring signals that would promote PCa cell motility.
Table 1 shows the different networks that are affected by anti-CXCL13 and/or anti-CXCR5 treatment of prostate cancer cells, and the functions in prostate cancer cells that each of those networks is involved in. Score indicates the number of molecules known to participate in each respective network. Focus molecules (indicated by underline) are those molecules of particular interest or importance in each network.







TABLE 1







Highest scoring networks involved in CXCL13-treated


metastatic prostate cancer











Network
Molecules in Network

# of Focus
Top


ID
(Focus Molecules in Bold)
Score
Molecules
Functions





1
AKT1, AKT2, RTK, AMPK,
31
19
Cancer, Cell



ATM/ATR, BRCA1, CDC2,


Cycle



CDC25A, CDC25B/C,






CDC25C, CDK2, CDKN1B,






CHEK1, CHEK2, Cyclin A,






Cyclin B, Cyclin D, Cyclin E,






E2f, Fcer1, Foxo, Ige,






Laminin, LIMK1, MAP2K2,






MAP2K3, MEF2, Mek, Pkg,






PRKAA1, RAF1, Rb, RB1,






Scf, STMN1





2
Actin, α Actinin, β Arrestin,
26
20
Cellular



Calpain, CAV1, CFL1,


Movement,



Cofilin, Collagen(s), CTTN, 


Cell



Dynamin, Erm, EZR, F


Morphology



Actin, FAK-Src,






FCGR1A/2A/3A, G3BP1,






Integrin αVβ3, KRT18,






MAP2K1/2, NF2, NTRK2,






Pak, phosphatase, PTEN,






PTK2, PXN, Rac,






Ras homolog, Rock, SRC,






Talin, VASP





3
AKT1, ALP, Calmodulin,
21
14
Cancer,



CaMKII, Caspase, CDKN1A,


Reproductive



Ck2, Creb, CREB1,


System



CTNNB1, Cytochrome c,


Disease



ERBB2, ESR1,






FSH, GLRX2, HDAC8, 






Histone h3, Histone h4,






Hsp70, HSP84-2,






HSP90AB1, ICAM1, JUN,






Nfat, PDPK1, Pp2b,






Proteasome, RNA polymer-






ase II, Rxr, Smad, SYN1,






TFIIH, Tubulin, YWHAZ









Table 2 shows the proteins that have been found to be regulated by CXCL13 and CXCR5 in prostate cancer cells. The molecules are arranged according to the particular biological functions they are associated with in the cells and the functions or diseases for which their increased expression in the cells can be used as a marker.







TABLE 2







Proteins regulated by CXCL13 and


their relevant biological functions in PC3 cells









Biological




functions




and

P-


diseases
Molecules
value





Growth
AKT1, AKT2, BAD, BCL2, BCL2L1, CAV1,
1.11E−09


of tumor
CDC2, CDK2, ELK1, JUN, MAPK3, MAPK8,



cell lines
NF2, PTK2, RAF1, SRC, STMN1



Proliferation
AKT1, AKT2, BAD, CAV1, GJA1, ITGB3,
6.66E−08


of tumor
JUN, JUNB, LIMK1, MAPK3, MAPK8,



cell lines
PDPK1, SRC



Anti-
AKT1, AKT2, BAD, BCL2, BCL2L1, CAV1,
4.26E−07


Apoptosis
CDC2, ITGB3, JUN, MAPK3, MAPK8,




PDPK1, PTK2, SRC, STMN1, VAV1



Prostate
AKT1, AKT2, CDC2, CDK2, ITGB3, JUN,
9.16E−07


carcinoma
RAF2, SRC



Metastasis
AKT1, ITGB1, NF2, PTK2, RELA, SRC
9.29E−07


Cell cycle
BCL2, CAV1, CDC25C, CDK2, MAPK8,
1.85E−05


progression
RAF1, VAV1



Survival
AKT1, AKT2, BCL2, BCL2L1, CAV1, CDK2,
2.05E−05


of tumor
CDKN1A, CDKN1B, CHEK1, CHEK2,



cell lines
CREB1, EGFR, ERBB2, FRAP1, JAK1, MET,




NFKB1, NFKB2, NTRK2, PDGFRB,




PRKAA1, PTK2, RELA, RELB, SRC, STAT3









FIG. 14 depicts how CXCL13 regulates key molecules involved in cell cycle. Phospho-specific antibody microarrays were separately hybridized with CXCL13-treated or untreated PC3 cell lysates. Ratios of phosphorylated to unphosphorylated molecules were calculated and the datasets uploaded into the Ingenuity Pathways Analysis application. Networks were algorithmically generated based on molecules' connectivity. Results were normalized to GAPDH levels. Colors represent fold changes in phosphorylation. Gray indicates no change in phosphorylation status, green indicates decreased phosphorylation, pink indicates baseline phosphorylation, and red indicates increased phosphorylation relative to baseline.
FIG. 15 depicts how CXCL13 regulates key molecules involved in cell migration. Phospho-specific antibody microarrays were separately hybridized with CXCL13-treated or untreated PC3 cell lysates. Ratios of phosphorylated to unphosphorylated molecules were calculated and the datasets uploaded into the Ingenuity Pathways Analysis application. Networks were algorithmically generated based on molecules' connectivity. Results were normalized to GAPDH levels. Colors represent fold changes in phosphorylation. Gray indicates no change in phosphorylation status, green indicates decreased phosphorylation, pink indicates baseline phosphorylation, and red indicates increased phosphorylation relative to baseline.
FIG. 16 depicts how CXCL13 regulates key molecules involved in cell survival and growth. Phospho-specific antibody microarrays were separately hybridized with CXCL13-treated or untreated PC3 cell lysates. Ratios of phosphorylated to unphosphorylated molecules were calculated and the datasets uploaded into the Ingenuity Pathways Analysis application. Networks were algorithmically generated based on molecules' connectivity. Results were normalized to GAPDH levels. Colors represent fold changes in phosphorylation. Gray indicates no change in phosphorylation status, green indicates decreased phosphorylation, pink indicates baseline phosphorylation, and red indicates increased phosphorylation relative to baseline.
The top ten signaling pathways regulated by CXCL13 based on their significance (p-value) calculated using the right-tailed Fisher's Exact test using the entire dataset are shown in FIG. 17.
It was found that CXCL13 mediates differential phosphorylation of proteins (colored molecules) belonging to the P13K/Akt and SAPK/JNK signaling pathways, as shown in FIG. 18. The two canonical pathways were merged and overlaid with the analyzed microarray data from CXCL13-treated or untreated PC3 cells. Gray indicates no change in phosphorylation status, green indicates decreased phosphorylation, pink indicates baseline phosphorylation, and red indicates increased phosphorylation relative to baseline.
FIG. 19 summarizes the signaling pathways modulated by CXCL13:CXCR5 interactions. CXCL13 binding to CXCR5 results in the activation of PI3K/Akt, Raf/MEK/ERK, Integrin/33/Src/FAK, and DOCK2/Rac/JNK pathways involved in cell survival, invasion, and growth respectively.
A confirmation of major CXCL13-CXCR5 cell signaling cascades is shown in FIG. 20. LNCaP (blue circles) or PC3 (magenta circles) cells received no additions (open circles) or 100 ng/ml of CXCL13 (closed circles) for 5 or 10 minutes. FACETM assays (Active Motif, Carlsbad, Calif.) were used to detect both active and inactive (total) P13K, ERK, FAK, Src kinase and NFkb proteins, 5 or 10 minutes after stimulation. Ratios of active (phosphorylated) to total proteins are presented ±SEM from 3 separate experiments performed in triplicate.
CXCL13-CXCR5 signaling events required for AKT activation are shown in FIG. 21. FACE assays were performed to measure active and total AKT levels in LNCaP and PC3 cell lines. Cells were treated with (or without) CXCL13 for 5 or 10 minutes, along with or without CXCR5 blockade, pertussis toxin, U-73122, wortmannin, PI-103, TGX221, and AS605240, DOCK2 siRNA, SU6656, and PF-573228. Experiments were performed in triplicate and results show the ratio of p-AKT to total AKT.
FIGS. 22A-B show CXCL13-mediated CXCR5 ligation and translocation to nuclei. LNCaP (FIG. 22A) and PC3 (FIG. 22B) cancer cell lines were stained with FITC (green)-conjugated anti-CXCR5 antibody, Alexa455 (orange)-conjugated anti-CXCL13 antibody and 7AAD (red) as a nuclear stain 30 and 60 minutes after treatment with 0 or 100 ng/ml of CXCL13. Histograms indicate the degree of signal correlation between CXCR5 and CXCL13 or these pairs with nuclei. The gate for positive population thresholds was determined by referencing dark field and 7AAD similarity scores with the Amnis Imagestream INSPIRETM and IDEASTM acquisition, and analysis software and system. The percentage of translocated cells are given above the ‘Translocated’ region bars.
Example 2
Anti-CXCL13 Antibody Treatment Inhibits Metastasis and Tumor Growth in Bone
Anti-CXCL13 antibody treatment is shown in FIGS. 23A-B to inhibit prostate cancer progression and bone metastasis. Two groups of ten B6.Cg-Foxn-Nu/J male mice (ten-week old) were challenged with 106 luciferase-positive PC3 cells in 50 μl of saline by intracardiac injection. Prostate tumors were allowed to develop over 30 days; afterwards groups received either 0.5 μg of control (FIG. 23A) or anti-CXCL13 (FIG. 23B) antibodies every three days for an additional 30 days. This representative image shows the changes in tumor burden and bone metastasis that was analyzed by in vivo imaging using a Caliper/Xenogen IVIS100 imaging system (Caliper, San Diego, Calif.).
FIGS. 24A-B show that CXCL13 blockade inhibits prostate tumor growth in bone. Male Nu/Nu mice were intra-tibially injected with 106 luciferase-positive PC3 (PC3-luc) cells and tumors were allowed to develop for one week. Subsequently, the mice were intraperitoneally injected with 475 μg/kg isotype control or anti-CXCL13 antibody suspended in 100 μl of sterile saline every 72 hours for one week. Experimental groups were imaged every week for four weeks using the Caliper/Xenogen In Vivo imaging system 100 and analyzed using the Caliper LIVING IMAGE® (Caliper, San Diego, Calif.) software. FIG. 24A displays representative images of PC3-luc tumor growth in bone. FIG. 24B shows luminescence (photons/sec/cm2)±SEM of PC3-luc tumors in bone 7, 14, 21, and 28 days post challenge. Asterisk (*) indicates significant (p<0.01) differences between isotype control and anti-CXCL13 antibody-treated groups.
CXCL13 blockade also abrogates osteolytic prostate tumor growth in bone, as shown in FIGS. 25A-B. Male Nu/Nu mice were intra-tibially injected with 106 luciferase-positive PC3 (PC3-luc) cells and tumors were allowed to develop for one week. Subsequently, mice were intraperitoneally injected with 475 μg/kg isotype control or anti-CXCL13 antibody suspended in 100 of sterile saline every 72 hours for one week. Experimental groups were imaged 28 days post challenge using a Siemens microCT Scan System. Low (FIG. 25A) and high FIG. 25B) resolution images from five mice in each group processed using OsiriX imaging software are shown.
FIGS. 26A-B demonstrate that CXCL13 blockade inhibits loss of bone mineral density (BMD) induced by prostate cancer bone metastasis. Male Nu/Nu mice were intra-tibially injected with 106 of luciferase positive PC3 (PC3-luc) cell lines and tumors were allowed to develop for one week. Subsequently, the mice were intraperitoneally injected with 475 ug/kg isotype control or anti-CXCL13 antibody suspended in 100 ul of sterile saline every 72 hours for one week. Experimental groups were imaged 28 days post challenge using a Siemens microCT Scan System. FIG. 26A displays representative in mineral density images. FIG. 26B shows the moral diaphysis BMD (mg/cm3) scans for each subject, which were quantified using MicroView software version 2.1.1 (General Electric Medical). Asterisk (*) indicates statistical significance (p<0.0001) between isotype control or anti-CXCL13 antibody-treated group.
Example 3
Detection of CXCL13 and CXCR5 Expression in Various Tumors
FIG. 27 shows CXCL13 levels in serum of normal healthy controls and lung cancer subjects. ELISA assays, capable of detecting >5 pg/mL of CXCL13, were performed to quantify CXCL13 levels se from normal healthy donors (n=9) or patients diagnosed with squamous cell carcinoma (SSC; n=17) or adenocarcinoma (Adeno Cu; n=14). Solid circles indicate individual serum CXCL13 levels and lines show median concentrations for each group. Asterisks (*) show significant differences (p<0.01) between normal healthy donor (i.e., control) or lung cancer patient serum samples.
CXCR5 expression by non-neoplastic lung and lung cancer tissue is shown in FIG. 28. Lung tissue from non-neoplastic (NN; n=8), squamous cell carcinoma (SCC; n=24), and adenocarcinoma (AdenoCa; n=54) were stained with isotype control or anti-CXCR5 antibodies. Brown (DAB) color show CXCR5 staining. An Aperio ScanScope CS system with a 40× objective captured digital images of each slide. Representative cases are indicated and immuno-intensities of CXCR5 were quantified using image analysis Aperio ImageScope v.6.25 software. Asterisks (*) show significant differences (p<0.01) between non-neoplastic and lung cancer tissue.
FIG. 29 illustrates CXCR5 expression by non-neoplastic mammary and breast cancer tissue. Breast tissue from non-neoplastic (NN; n=8) and adenocarcinoma (AdenoCa; n=16) were stained with isotype control or anti-CXCR5 antibodies. Brown (DAB) color show CXCR5 staining. An Aperio ScanScope CS system with a 40× objective captured digital images of each slide. Representative cases are indicated and immuno-intensities of CXCR5 were quantified using image analysis Aperio ImageScope v.6.25 software. Asterisks/*) show significant differences (p<0.01) between non-neoplastic and cancerous tissue.
CXCR5 and CXCL13 expression are also increased in colon cancer tissue relative to nonneoplastic controls, as shown in FIG. 30. Colon tissue from non-neoplastic (n=8) and adenocarcinoma (n=16) were stained with isotype control, anti-CXCR5, or anti-CXCL13 antibody. Brown (DAB) and magenta stain indicates CXCR5 and CXCL13 positivity, respectively. An Aperio ScanScope CS system with a 40× objective captured digital images. Representative cases are shown along with relative colon cancer to non-neoplastic control tissue immuno-intensities ratios of CXCR5 and CXCL13 that were quantified using Aperio ImageScope v.6.25 software. Asterisks (*) show significant differences (p<0.01) between non-neoplastic and cancerous tissue.
It was also found that CXCR5 and CXCL13 expression by ovarian cancer tissue relative to nonneoplastic controls is significantly higher, as shown in FIG. 31. Ovarian tissue from non-neoplastic (n=8) and adenocarcinoma (n=16) were stained with isotype control, anti-CXCR5, or anti-CXCL13 antibody. Brown (DAB) and magenta stain indicates CXCR5 and CXCL13 positivity, respectively. Aperio ScanScope CS system with a 40× objective captured digital images. Representative cases are shown along with relative ovarian cancer to non-neoplastic control tissue immuno-intensities ratios of CXCR5 and CXCL13 that were quantified using Aperio ImageScope v.6.25 software. Asterisks (*) show significant differences (p<0.01) between non-neoplastic and cancerous tissue.
Example 4
Detecting Chemokine Expression Levels with Real Time-PCR Analysis Primer Design
Messenger RNA sequences for 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, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL24, CCL25, CCL25-1, CCL25-2, CCL27, CCL28, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCL1, XCL2, XCR1, CX3CR1, or CX3CL1 were obtained from the NIH-NCBI gene bank database. Primers were designed using the BeaconJ 2.0 computer program. Thermodynamic analysis of the primers was conducted using computer programs: Primer PremierJ and MIT Primer 3. The resulting primer sets were compared against the entire hu an genome to confirm specificity.
Real Time PCR Analysis
Cancer cell lines (ATCC, Rockville, Md.) were cultured in RMPI-1640 containing 10% fetal calf serum supplemented with non-essential amino acids, L-glutamate, and sodium pyruvate (complete media). Primary tumor and normal-paired matched tissues were obtained from clinical isolates (Clinomics Biosciences, Frederick, Md. and UAB Tissue Procurement, Birmingham, Ala.). Messenger RNA (mRNA) was isolated from 106 cells using TriReagent (Molecular Research Center, Cincinnati, Ohio) according to manufacturer's protocols. Potential genomic DNA contamination was removed from these samples by treatment with 10 U/Fl of RNase free DNase (Invitrogen, San Diego, Calif.) for 15 minutes at 37° C. RNA was then precipitated and resuspended in RNA Secure (Ambion, Austin, Tex.). The cDNA was generated by reverse transcribing approximately 2 μg of total RNA using Taqman7 reverse transcription reagents (Applied Biosystems, Foster City, Calif.) according to manufacturer's protocols. Subsequently, cDNAs were amplified with specific human cDNA primers, to 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, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL24, CCL25, CCL25-1, CCL25-2, CCL27, CCL28, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR11, XCL1, XCL2, XCR1, CX3CR1, or CX3CL1 using SYBR7 Green PCR master mix reagents (Applied Biosystems) according to manufacturer's protocol. The level of copies of mRNA of these targets were evaluated by real-time PCR analysis using the BioRad Icycler and software (Hercules, Calif.).
The RT-PCR products obtained using 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-, CCL7-, CCL8-, CCL9-, CCL10-, CCL11-, CCL12-, CCL13-, CCL14-, CCL15-, CCL16-, CCL17-, CCL18-, CCL19-, CCL20-, CCL21-, CCL22-, CCL24-, CCL25-, CCL25-1-, CCL25-2-, CCL27-, CCL28-, CCR1-, CCR2-, CCR3-, CCR4-, CCR5-, CCR6-, CCR7-, CCR8-, CCR9-, CCR10-, CCR11-XCL1-, XCL2-, XCR1-, CX3CR1-, or CX3CL1-specific primer sets did not cross react with other gene targets due to exclusion of primers that annealed to host sequences (NIH-NCBI Genebank). The primers produced different size amplicon products relative the polymorphisms that resulted in CXCR5a versus CXCR5b and CCL25, CCL25-1, versus CCL25-2. To this end, RT-PCR analysis of adenoma, carcinoma, leukemia, lymphoma, melanoma, and/or myeloma cell lines and tumor tissue revealed that chemokines and chemokine receptors were differentially expressed by cancer cells.
Example 5
Anti-Chemokine and Anti-Chemokine Receptor Antibodies Inhibit Tumor Cell Growth In Vitro and In Vivo
Anti-Sera Preparation
The 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, CX3CR1, and CX3CL1 (SEQ ID NOS:1-21) were synthesized (Sigma Genosys, The Woodlands, Tex.) and conjugated to hen egg lysozyme (Pierce, Rockford, Ill.) to generate the antigen for subsequent immunizations for anti-sera preparation or monoclonal antibody generation. The endotoxin levels of chemokine peptide conjugates were quantified by the chromogenic Limulus amebocyte lysate assay (Cape Cod, Inc., Falmouth, Miss.) and shown to be <5 EU/mg. 100 μg of the antigen was used as the immunogen together with complete Freund's adjuvant Ribi Adjuvant system (RAS) for the first immunization in a final volume of 1.0 ml. This mixture was administered in 100 ml aliquots on two sites of the back of the rabbit subcutaneously and 400 ml intramuscularly in each hind leg muscle. Three to four weeks later, rabbits received 100 μg of the antigen in addition to incomplete Freund's adjuvant for 3 subsequent immunizations. Anti-sera were collected when anti-CXCR1, -CXCR2, -CXCL1, -CXCL2, -CXCL3, -CXCL5, -CXCL6-CXCL7, -CXCL8, -CXCL12, -CXCR5a, -CXCR5b, -CXCL13, -CXCR6, -CXCL16, -CCL16, -CCL25, -CCL25-1, -CCL25-2, -CX3CR1, and -CX3CL1 antibody titers reached 1:1,000,000. Subsequently, normal or anti-sera were heat-inactivated and diluted 1:50 in PBS.
Monoclonal Antibody Preparation
The 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, CX3CR1, and CX3CL1 were synthesized (Sigma Genosys) and conjugated to hen egg lysozyme (Pierce) to generate the “antigen” for subsequent immunizations for anti-sera preparation or monoclonal antibody generation. The endotoxin levels of chemokine peptide conjugates were quantified by the chromogenic Limulus amebocyte lysate assay (Cape Cod, Inc., Falmouth, Miss.) and shown to be <5 EU/mg. 100 μg of the antigen was used as the immunogen together with complete Freund's adjuvant Ribi Adjuvant system (RAS) for the first immunization in a final volume of 200 μl. This mixture was subcutaneously administered in 100 μl aliquots at two sites of the back of a rat, mouse, or immunoglobulin-humanized mouse. Two weeks later, animals received 100 μg of the antigen in addition to incomplete Freund's adjuvant for 3 subsequent immunizations. Serum were collected and when anti-CXCR1, -CXCR2, -CXCL1, -CXCL2, -CXCL3, -CXCL5, -CXCL6-CXCL7, -CXCL8, -CXCL12, -CXCR5a, -CXCR5b, -CXCL13, -CXCR6, -CXCL16, -CCL16, -CCL25, -CCL25-1, -CCL25-2, -CX3CR1, or -CX3CL1 antibody titers reached 1:2,000,000, hosts were sacrificed and splenocytes were isolated for hybridoma generation. Briefly, B cells from the spleen or lymph nodes of immunized hosts were fused with immortal myeloma cell lines (e.g., YB2/0). Hybridomas were next isolated after selective culturing conditions (i.e., HAT-supplemented media) and limiting dilution methods of hybridoma cloning. Cells that produce antibodies with the desired specificity were selected using ELISA. Hybridomas from normal rats or mice were humanized with molecular biological techniques in common use. After cloning a high affinity and prolific hybridoma, antibodies we e isolated from ascites or culture supernatants and adjusted to a titer of 1:2,000,000 and diluted 1:50 in PBS.
Anti-Sera or Monoclonal Antibody Treatment
Immunodeficient nude NIH-III mice (8 to 12 weeks old, Charles River Laboratory, Wilmington, Mass.), which lack T, B, and NK cells, received 1×106 cancer cells, subcutaneously, for the establishment of a tumor. The established solid tumor was then removed from the host for immediate implantation or stored in liquid nitrogen for later implantation. Freshly isolated or liquid nitrogen frozen tumor tissue (1 g) were surgically implanted in the intestinal adipose tissue for the generation of tumor. Once the xenografted tumor growth reached 5 mm in size, the NIH-III mice received 200 μl intraperitoneal injections of either anti-sera or monoclonal antibodies every three days and the tumor was monitored for progression or regression of growth.
Data Analysis
SigmaStat 2000 (Chicago, Ill.) software was used to analyze and confirm the statistical significance of data. The data were subsequently analyzed by the Student's t-test, using a two-factor, unpaired test. In this analysis, treated samples were compared to untreated controls. The significance level was set at p<0.05.
In Vitro Growth Studies
The adenoma, carcinoma, leukemia, lymphoma, melanoma, and/or myeloma cell lines were grown in complete media 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 CX3CL1. The growth of cancer cell lines expressing CXCR1 and/or CXCR2 were inhibited by antibodies to CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, or CXCL8. Similarly, the growth of cancer cell lines expressing CXCR4 were inhibited by antibodies to CXCR4 or CXCL12. The growth of cancer cell lines expressing CXCR5a or CXCR5b were inhibited by antibodies to CXCR5a, CXCR5b, or CXCL13. The proliferation of cancer cell lines expressing CXCR6 were inhibited by antibodies to CXCR6 or CXCL16. The growth of cancer cell lines expressing CCR9 were inhibited by antibodies to CCR9, CCL25, CCL25-1, or CCL25-2. The propagation of cancer cell lines expressing CX3CR1 were inhibited by antibodies to CX3CR1 or CXC3L1. Of interest, antibodies against the soluble ligands, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCL12, CXCL13, CXCL16, CCL16, CCL25, CCL25-1, CCL25-2, or CX3CL1, were more effective at growth inhibition that those directed against the membrane receptors.
In Vitro Angiogenesis Studies
Microvascular endothelial cells (Cell Systems, Kirkland, Wash.) were grown according to supplier's protocols and allowed to form microvascular venules in an in vitro assay for angiogenesis (BD-Biocoat, Hercules, Calif.), 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 CX3CL1. The angiogenesis was inhibited by antibodies against CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCR4, CXCL12, CXCR6 or CXCL16.
In Vivo Growth Studies
Cancer cell lines or primary tumor tissue were adoptively transferred into NIH-III mice and allowed to form the xenograft tumor of interest. 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 CX3CL1 differentially affected the progression and regression of tumor size. In certain cases, antibodies directed towards CXCR1, CXCR2, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8, CXCR4, CXCL12, CXCR6 or CXCL16 effectively lead to both regression and impeding progression of tumor growth. Antibodies directed against CXCR4, CXCL12, CXCR5a, CXCR5b, CXCL13, CCL16, CCR9, CCL25, CCL25-1, CCL25-2, CX3CR1, or CX3CL1 were effective at inhibiting the progression of tumor size.
The protein sequences of the chemokines used herein are recorded in NIH-NCBI GenBank as: (1) CXCR1 (ACCESSION# NP 000625), SEQ ID NO:1, (2) CXCR2 (ACCESSION# NP 001548), SEQ ID NO:2, (3) CXCL1 (ACCESSION# NP 001502), SEQ ID NO:3, (4) CXCL2 (ACCESSION# NP 002080), SEQ ID NO:4, (5) CXCL3 (ACCESSION# NP 002081), SEQ ID NO:5, (6) CXCL5 (ACCESSION# NP 002985), SEQ ID NO:6, (7) CXCL6 (ACCESSION# NP 002984), SEQ ID NO:7, (8) CXCL7 (ACCESSION# NP 002695). SEQ ID NO:8, (9) CXCL8 (IL-8, ACCESSION# NP 000575), SEQ ID NO:9, (10) CXCR4 (ACCESSION# NP 003458), SEQ ID NO:10, (11) CXCL12 (ACCESSION# NP 000600), SEQ ID NO:11, (12) CXCR5A (ACCESSION# NP 116743), SEQ ID NO:12, (13) CXCR5B (ACCESSION# NP 001707), SEQ ID NO:13, (14) CXCL13 (ACCESSION# NP 006410), SEQ ID NO:14, (15) CXCR6 (ACCESSION# NP 006555), SEQ ID NO:15, (16) CXCL16 (ACCESSION# NP 071342), SEQ ID NO:16, (17) CCL16 (ACCESSION# NP 004581), SEQ ID NO:17, (18) CCL25 (ACCESSION# NP-005615.2), SEQ ID NO:18, (19) CCL25-1 (ACCESSION# NP 005615), SEQ ID NO:19, (20) CCL25-2 (ACCESSION# NP 683686), SEQ ID NO:20, (21) CX3CR1 (ACCESSION# NP 001328), SEQ ID NO:21, and (22) CX3CL1 (ACCESSION # NP 002987), SEQ ID NO:22.
The cDNA sequences are known and are available in NIH-NCBI GenBank under the following accession numbers: (23) CXCR1 (ACCESSION# NM 000634), SEQ ID NO:23, (24) CXCR2(ACCESSION# NM 001557), SEQ ID NO:24, (25) CXCL1 (ACCESSION# NM 001511), SEQ ID NO:25, (26) CXCL2 (ACCESSION# NM 002089), SEQ ID NO:26, (27) CXCL3 (ACCESSION# NM 002090), SEQ ID NO:27, (28) CXCL5 (ACCESSION# NM 002994), SEQ ID NO:28, (29) CXCL6 (ACCESSION# NM 002993), SEQ ID NO:29, (30) CXCL7 (ACCESSION# NM 002704), SEQ ID NO:30, (31) CXCL8 (IL-8, ACCESSION# NM 000584), SEQ ID NO:31, (32) CXCR4 (ACCESSION# NM 003467), SEQ ID NO:32, (33) CXCL12 (ACCESSION# NM 000609), SEQ ID NO:33, (34) CXCR5A (ACCESSION# NM 032966), SEQ ID NO:34, (35) CXCR5B (ACCESSION# NM 001716), SEQ ID NO:35, (36) CXCL13 (ACCESSION# NM 006419), SEQ ID NO:36, (37) CXCR6 (ACCESSION# NM 006564), SEQ ID NO:37, (38) CXCL16 (ACCESSION# NM 022059), SEQ ID NO:38, (39) CCL16 (ACCESSION# NM 004590), SEQ ID NO:39, (40) CCL25 (ACCESSION# NM 005624.3), SEQ ID NO:40, (41) CCL25-1 (ACCESSION# NM 005624), SEQ ID NO:41, (42) CCL25-2 (ACCESSION# NM 148888), SEQ ID NO:42, (43) CX3CR1 (ACCESSION# NM 001337), SEQ ID NO:43, and (44) CX3CL1 (ACCESSION# NM 002996), SEQ ID NO:44.
As shown in the table below, the particular chemokines which are most which any tumor expresses may vary. The methods of the present invention may be customized for a particular patient, depending on the chemokines over-expressed by the patient's own tumor. It is possible to identify the particular chemokines which are over-expressed in the tumor using methods of the invention and administer antibodies against that over-expressed chemokine. The tailoring of treatment for the cancer patient is novel, and is a particularly valuable aspect of the invention.
TABLE 3 indicates the differing amounts of particular chemokines over-expressed in particular tumors that were studied.









TABLE 3







Chemokine



Chemokine
Receptor







Carcinoma
CCL1, CCL2, CCL4, CCL17, CCL19,
CCR2, CCR7,



CCL21, CCL22, CCL25
CCR8, CCR9



CXCL12, CXCL13, CXCL16
CXCR4, CXCR5,



CX3CL1
CXCR6 CX3CR1


Leukemia
CCL1, CCL4, CCL17, CCL19, CCL21,
CCR7, CCR8,



CCL22, CCL25
CCR9



CXCL12
CXCR4, CXCR7


Lymphoma
CXCL12, CXCL13
CXCR4, CXCR5


Melanoma
CCL25, CCL27
CCR9, CCR10



CXCL1, CXCL2, CXCL3, CXCL5,
CXCR1, CXCR2,



CXCL6, CXCL7, CXCL8, CXCL12,
CXCR4, CXCR5,



CXCL13, CXCL16
CXCR6, CXCR7



CX3CL1
CX3CR1


Sarcoma
CCL1, CCL3, CCL4, CCL5, CCL7,
CCR3, CCR5,



CCL8, CCL11, CCL13, CCL17,
CCR8



CCL22, CCL24




CXCL12
CXCR4, CXCR7



CX3CL1
CX3CR1









Example 6
CXCR5-CXCL13 Induced Anti-Apoptotic and/or Survival Signal Involved in PCa Chemo Resistance
LNCaP (hormone responsive, wild type p53 expression), PC3 (hormone refractory, p53 null), and DU145 (hormone refractory, p53 mutated) cell lines are grown with or without CXCL16 and with or without doxorubicin (1 μM/2 μM/4 μM), etoposide (20 μM/40 μM), estramustine (4 μM/10 μM), or docetaxel (10 nM/20 nM/40 nM) for 4, 8, 12, and 24 hours. Expression and activation of cell survival, pro- and anti-apoptotic signals (Akt, Src, Ca KII, FAK, FKHR, FOXO, CREB, NF-κB, Myc, Fos, Jun Apaf1, Bax, Bcl2, BclXL, BaK, Bad, Bik, Bim, TP53, Caspase-3, -6, -8, -9, survivin, vitronectin, β-Catenin) and molecules responsible for drug resistance or metabolism (Twist-1, Snail-1, Glutathione-S-transferase-π (GST-π), p53, topoisomerase I, IIα, IIβ, and ABC drug transporters) are accessed by real-time PCR and Western blot. Briefly, after treatment of cells, changes in the gene expression is tested using real-time PCR. Activation of signaling molecules is also be tested by phosphorylation specific antibody (i.e., Western blot analysis). To further confirm the role of the activated signaling molecules, following CXCL16 treatment, expression or activity of the candidate molecules is inhibited using chemical inhibitors or siRNAs and target genes are analyzed by real-time PCR and Western blot analysis. Subsequently, the response of treated cells to chemotherapeutic drugs is evaluated by Vybrant apoptosis assay (Molecular probes) kit.
RNA Isolation and Real-Time PCR
Total RNA is isolated by TRIZOLTM (Invitrogen) method and quantified by UV spectrophotometry. Quality of RNA is analyzed by electrophoresis. The cDNA synthesis is completed using the ISCRIPTTM cDNA synthesis kit (BioRad) as described by the manufacturer. Real-time PCR is performed using IQTM SYBR green supermix (BioRad) as described by manufacturer and specific primers designed against FAK, FKHR, FOXO, Apaf1, Bax, Bcl2, BclXL, BaK, Bad, Bid, XIAP, Bik, Bim, TP53, cytochrome C, Caspase-3, -6, -8, -9, survivin, lamin, CamKII, vitronectin, β-Catenin, cadherins, Twist-1, Snail-1, CREB, NF-κB, Myc, Fos, Jun, β-actin and GAPDH. The results are calculated by delta Ct to quantify fold changes in mRNAs compared to untreated groups.
Western Blotting
Cells are harvested and resuspended in lysis buffer to extract total protein. Lysis buffer contains 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 5 mM EDTA supplemented with protease inhibitors, 1 mM phenylmethylsulphonylfluoride, 1 mM benzamidine, 10 μg/mL soybean trypsin inhibitor, 50 μg/mL leupeptin, 1 μg/mL pepstatin and 20 μg/mL aprotinin. Cell lysates are stored on ice for 30 min, centrifuged (14000×g) for 20 min at 4° C., and supernatant is used for Western blot analysis of genes demonstrating significant modulation in mRNA level. Similarly, phosphor-specific antibodies are used to test changes in the level of phosphorylation of Akt1/2/3, mTOR, FAK, FKHR, FOXO, and GSK-3β. Moreover, activation of caspases and PARP, following cleavage are evaluated using specific antibodies. The results obtained after chemiluminescent detection of protein bands by ECL plus reagent (Pharmecia) on X-ray film is normalized to β-actin and/or GAPDH using Image J image analysis software (NIH).
Detection of Cytochrome C Release
Cells are collected and washed in PBS, and resuspended in extraction buffer containing 220 mM mannitol, 68 mM sucrose, 50 mM PIPES-KOH, pH 7.4, 50 mM KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT, and protease inhibitors. After 30 min incubation on ice, cells are homogenized using Glass-Teflon homogenizer and homogenates will be spun at 14,000 g for 15 mM. Cytosolic extracts are used for Western blot analysis using anti-cytochrome C monoclonal antibody (PharMingen).
siRNA Transfection, Chemical Inhibitor, and Apoptosis Detection
Prostate cancer cell lines are transfected with gene specific and nonspecific control siRNAs (Dharmacon) using LipofectAMINE 2000 (Invitrogen). Optimum gene knock-down time and siRNA concentration are confirmed by Western blot analysis and further evaluated for cell survival following drug treatment with or without CXCL16, control antibody, and/or anti-CXCR6 antibody. The detection of changes in live, apoptotic, and necrotic cells is evaluated as follows: cell survival is tested by Vybrant apoptosis as described by the manufacturer (Molecular probe), using FACScan flow cytometer and CELLQUESTTM software (BD Phammingen). Change in down-stream gene expression after gene knockdown is tested using real-time PCR and western blotting.
Cells treated with CXCL13 show enhanced expression of cell survival and drug transporter proteins which show differences in their expression pattern in hormone responsive and non responsive cells. Anti-CXCL13 Abs effectively reverse the effect of CXCL13 in PCa cells. Doxorubicin, estramustine, etoposide and docetaxel induce apoptosis in PCa cells without CXCL13 treatment (or CXCR5 blockade).
Example 7
CXCR5-CXCL13 Induced Changes in ABC Drug Transporters
LNCaP, PC3, and DU145 cells are grown with or without CXCL13, control antibody, and/or anti-CXCR5 antibodies along with or without doxorubicin, estramustine, etoposide or docetaxel for 4, 8, 12 or 16 hours as described earlier. After treatment, changes in the ABC transporter and Twist-1 mRNA expression are quantified by real-time PCR, as described above, using specific primers directed for ABC and Twist-1 cDNA. The genes demonstrating significant alterations in mRNA expression are further tested by Western blot analysis. Nuclear extracts from treated cells are evaluated by chromatin immuno-precipitation (ChIP) assay to determine whether the transcriptional factors induced by CXCL13 bind the promoter region of ABC transporters and Twist-1.
Chromatin Immuno-Precipitation (ChIP)
The results from Example 4 provide information about the genes that are regulated as well as those that may modulate transcription factors activated by CXCR5-CXCL13 interaction. Based on these results, target transcription factors and genes are selected. Specific PCR primers are designed against the promoter region of these genes containing the binding sites of transcription factors. PCR primer are used to amplify the DNA being precipitated along with transcription factors. Cells are harvested by trypsinization in the presence of 20 mM butyrate. 50,000 cells are re-suspended in 500 μl PBS/butyrate. Proteins and DNA are cross-linked with 1% formaldehyde for 8 min at room temperature and cross-linking is stopped with 125 mM glycine for 5 min. Cells are centrifuged at 470 g in a swing-out rotor with soft deceleration settings for 10 min at 4° C. and washed twice in 0.5 ml ice-cold PBS/butyrate by vortexing followed by centrifugation. Cells are lysed by addition of lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS, protease inhibitor cocktail (Sigma-Aldrich), 1 mM PMSF, 20 mM butyrate, vortexing and subsequent centrifugation. This procedure is known to produce chromatin fragments of 500 bp. The sonicated lysate is diluted 8-fold in RIPA buffer containing a protease inhibitor cocktail, 1 mM PMSF, and 20 mM butyrate (RIPA ChIP buffer). RIPA ChIP buffer (330 μl) is added to the pellet and mixed by vortexing. Immunoprecipitation and washes of the ChIP material is accomplished by the use of antibody-directed against specific transcription factors. Chromatin is aliquoted into tubes containing antibody-bead complexes. Input sample is placed in a tube for phenol-chloroform isoamyl alcohol isolation. The immunoprecipitated material is washed three times and transferred into a new tube while in TE. DNA elution in 1% SDS, cross-link reversal and proteinase K digestion is carried out in a single step for 2 hrs at 68° C. DNA is extracted with phenol-chloroform isoamylalcohol, and ethanol-precipitation in presence of acrylamide carrier (Sigma-Aldrich) and dissolved in TE. Immunoprecipitated DNA from 3-4 independent ChIPs is analyzed by real time PCR. Real-time PCR data is expressed as percent (±SD) precipitated (antibody-bound) DNA relative to input DNA, in three independent replicate ChIP assays.
Phosphorylation and activation of transcription factors such as CREB, Fos, Jun, and NFkB via CXCR5-CXCL13 signaling subsequently leads to increases in expression of ABC transporters and Twist-1. Decreases in gene expression are observed if negative regulatory elements are present in the same promoter. Since hormone-dependent and refractory PCa cells have differences in the expression of these intracellular signaling molecules, they show variations in genes to be modulated by hormone dependent and refractory conditions. The modulation in gene expression shows differences with drug treatment in presence of CXCL13 and in absence of CXC L13 treatment.
Example 8
In Vivo Evaluation of CXCL13:CXCR5-Directed Therapy
Male nude mice are subcutaneously challenged by luciferase expressing androgen responsive (LNCaP-Luc) and non-responsive (PC3-Luc) cells. Tumor development is measured non-invasively using in vivo imaging system. After establishment of a measurable tumor, mice are divided into treatment (A, B, C, D and E) and control groups (F, G, H, I, J and K). Group “A” receives CXCL13 or CXCR5 neutralizing antibodies (12.5 mg/kg/day) every alternate day and controls (group F) receive isotype control antibodies (12.5 mg/kg/day). Group “B,” “C,” “D” and “E” receive CXCL13 or CXCR5 neutralizing antibodies (12.5 mg/kg/day) with intraperitoneal injection of doxorubicin (5 mg/kg/day on days 1 to 3 followed by administration on days 15 to 17), intravenous injection of etoposide (10 mg/kg/day; on day 1, 5, 9, 14, 19 and 24), intravenous injection of estramustine (4 g/kg/day on day 1-5 and day 26-31), or intraperitoneal injection of docetaxel (8 mg/kg/day twice a week for 4 weeks), respectively. Controls for these treatment groups (“G,” “H,” “I” and “J,” respectively) receive theses drugs using similar concentration and injection protocol with isotype control antibodies (12.5 mg/kg/day). Group “K” receives PBS and serves as placebo. Tumor progression and regression in treatment and controls are evaluated by non-invasive in vivo imaging. The tumor from treated groups and untreated control groups is excised and evaluated for the changes in the cell survival and drug resistance proteins by immunohistochemistry.
Statistics (Significance) and Sample Size
Sample size (or power) calculations are relevant to the design of preliminary studies and determining the requirements for proposed experiments. To interpret our results, significance tests and statistical analysis are also critical. The traditional α-value, i.e., p=0.01, is used to evaluate the statistical significance of this study. The proposed experiment will require a minimum of 10 mice per group. The data is expressed as the mean±SEM and compared using a two-tailed paired (or unpaired) students t-test for normally distributed samples or an unpaired Mann Whitney U test as a non-parametric test for samples not normally distributed. The results are analyzed using SYSTAT (Systat Software Inc.) statistical program. Single-factor and two-factor variance ANOVA analyses are used to evaluate groups and subgroups, respectively. Hence, results are considered statistically significant if p values are <0.05.
Animals
Six to eight week old male nude mice are subcutaneously injected with PCa cells. Briefly, 5×106 Luciferase expressing PC3 cells are resuspended in 100 μl of sterile PBS and injected into the flanks of nude mice under isoflurane anesthesia. Luciferase expressing LNCaP cells (5×106 cell) are mixed with 50% Matrigel (Becton Dickinson) and injected in the flanks of nude mice under isoflurane anesthesia.
Analysis of In Vivo Tumor Growth
Tumor bearing nude mice receive 150 mg/kg D-Luciferin (Xenogen) by intra-peritoneal injection Using 25×5/8 inch gauge needle 15 minutes before imaging. The mice are imaged using the IVIS100 in vivo imaging system and results expressed in photons/sec/cm2/sr. Tumor volume is measured by use of calipers and calculated by the formula (Larger diameter)×(smaller diameter)2×0.5.
Cell Survival, Apoptotic and Drug Resistant Gene Expression Analysis
Tumors from all groups are excised three days after completion of treatment protocols. Tumors are fixed in 4% PFA and embedded in paraffin. Paraffin sections (thickness 7 μm) are mounted on glass slides, deparaffinized and re-hydrated (Xylene for 5 min; absolute, 95% and 70% ethanol for 1 min each). The rehydrated sections are used for peroxidase based immunohistochemical staining for drug transporters, PI3K, Akt, FAK, FKHR, FOXO, Apaf1, Bax, Bcl2, BclXL, BaK, Bad, Bid, XIAP, Bik, Bim, TP53, Cytochrome C, Caspase-3, -6, -8, -9, survivin, lamin, CamKII, vitronectin, β-Catenin, cadherins, Twist-1, CREB, NF-κB, Myc, Fos, Jun, CXCR5 and CXCL13. After staining, slides are scanned and analyzed by the Aperio scanscope (Aperio) system.
CXCL13 neutralization leads to decreased cell survival in response to drugs, thus reduction of tumor volume. However, the response also varies among the tumors formed by hormone sensitive (LNCaP) and hormone refractory (PC3 cells). Further, chemotherapeutic drugs have lower efficacy in the tumors with a functional CXCR5-CXCL13 axis, which may enhance 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 that will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention, which is defined by the following claims. The claims are intended to cover the components and steps in any sequence that is effective to meet the objectives there intended, unless the context specifically indicates the contrary. All the references cited in the specification are herein incorporated by reference in their entirely.

## Claims

1. A method for treating cancer in a subject, comprising:
administering to said subject a therapeutically effective amount of an anti-CXCL13 antibody, or an anti-CXCR5 antibody, or a combination thereof,
wherein said cancer is prostate cancer, and wherein said anti-CXCL13 antibody, or said anti-CXCR5 antibody, or said combination of anti-CXCL13 antibody and said anti-CXCR5 antibody is given in a dosage range of 1 ng/kg body weight/day to about 100 mg/kg body weight/day.

2. The method of claim 1, wherein said anti-CXCL13 antibody, or anti-CXCR5 antibody, or a combination thereof is administered directly into a cancerous tissue.

3. The method of claim 1, wherein said anti-CXCL13 antibody, or anti-CXCR5 antibody, or a combination thereof is administered in conjunction with a chemotherapeutic agent.

4. The method of claim 1, wherein said anti-CXCL13 antibody, or said anti-CXCR5 antibody, or a combination thereof is administered in conjunction with one or more antibodies that bind specifically to one or more chemokines or chemokine receptors selected from the group consisting of CCL1, CCL2, CCL4, CCL17, CCL19, CCL21, CCL22, CCL25, CXCL12, CXCL16, CCR2, CCR7, CCR8, CCR9, CXCR4, CXCR6, CX3CL1 and CX3CR1.

5. The method of claim 1, further comprising:
determining the level of CXCL13 and/or CXCR5 expression in a tissue from said subject, and, if an increased level of CXCL13 and/or CXCR5 is detected, administering to said subject a therapeutically effective amount of said anti-CXCL13 antibody, said anti-CXCR5 antibody, or a combination thereof.

6. The method of claim 1, wherein said anti-CXCL13 antibody, or said anti-CXCR5 antibody, or said combination of anti-CXCL13 antibody and said anti-CXCR5 antibody is given in a dosage range of 1 ng/kg body weight/day to about 100 ng/kg body weight/day.

7. The method of claim 1, wherein said anti-CXCL13 antibody, or said anti-CXCR5 antibody, or said combination of anti-CXCL13 antibody and said anti-CXCR5 antibody is given in a dosage range of 10 ng/kg body weight/day to about 1 μg/kg body weight/day.

8. The method of claim 1, wherein said anti-CXCL13 antibody, or said anti-CXCR5 antibody, or said combination of anti-CXCL13 antibody and said anti-CXCR5 antibody is given in a dosage range of 100 ng/kg body weight/day to about 10 μg/kg body weight/day.

9. The method of claim 1, wherein said anti-CXCL13 antibody, or said anti-CXCR5 antibody, or said combination of anti-CXCL13 antibody and said anti-CXCR5 antibody is given in a dosage range of 1 μg/kg body weight/day to about 100 μg/kg body weight/day.

10. The method of claim 1, wherein said anti-CXCL13 antibody, or said anti-CXCR5 antibody, or said combination of anti-CXCL13 antibody and said anti-CXCR5 antibody is given in a dosage range of 10 μg/kg body weight/day to about 1 mg/kg body weight/day.

11. The method of claim 1, wherein said anti-CXCL13 antibody, or said anti-CXCR5 antibody, or said combination of anti-CXCL13 antibody and said anti-CXCR5 antibody is given in a dosage range of 100 μg/kg body weight/day to about 10 mg/kg body weight/day.

12. A method for prevention or inhibition of the migration or metastasis of cancer cells with elevated expression of CXCL13 and/or CXCR5 in a subject, comprising:
administering to the subject a therapeutically effective amount of an anti-CXCL13 antibody, or an anti-CXCR5 antibody, or a combination thereof,
wherein said anti-CXCL13 antibody, or said anti-CXCR5 antibody, or said combination of anti-CXCL13 antibody and said anti-CXCR5 antibody is given in a dosage range of 1 ng/kg body weight/day to about 100 mg/kg body weight/day;
wherein said cancer cells are prostate cancer cells.

13. A method for enhancing the effect of chemotherapy, comprising:
administering to a subject who is under chemotherapy for a cancer, an effective amount of an anti-CXCL13 antibody, or an anti-CXCR5 antibody, or a combination thereof,
wherein said cancer is prostate cancer.