WO2011123903A1

WO2011123903A1 - Chemokine receptor heteromultimers, compounds that bind thereto and uses thereof

Info

Publication number: WO2011123903A1
Application number: PCT/AU2011/000412
Authority: WO
Inventors: Marina Kochetkova; Shaun Mccoll; Michael John Kleinig; Andrew Mark Coley
Original assignee: Adelaide Research & Innovation Pty Ltd
Priority date: 2010-04-08
Filing date: 2011-04-08
Publication date: 2011-10-13

Abstract

The present disclosure provides a CXCR4-CCR7 heteromultimer, molecules that bind thereto and/or modulate function thereof and uses therefor.

Description

CHEMOKINE RECEPTOR HETEROMULTIMERS, COMPOUNDS THAT BIND THERETO AND USES THEREOF

INCORPORATION BY REFERENCE

The present application claims priority from Australian Provisional Application

No. 2010901487 entitled "Chemokine receptors in metastatic cancer", filed on 8 April 2010, the entire contents of which are incorporated by reference.

FIELD

The present disclosure relates to chemokine receptor heteromultimers, compounds that bind thereto, and uses thereof, e.g., in diagnosis, prognosis, therapy or prophylaxis of a condition.

BACKGROUND

Chemokines are small chemoattractant cytokines known to play a role in many important physiological processes, including directing immune cell migration and/or immune cell survival and proliferation, immune cell differentiation, cancer cell survival and cancer metastasis. Upon secretion, chemokines accumulate in localized areas by binding to cell surface carbohydrate -containing structures and extracellular matrix components, and recruit receptor-bearing cells. Chemokines and their receptors are also involved in many developmental processes including central nervous system development, cardiogenesis and lymphopoiesis. In addition to their normal physiological roles, aberrant expression and/or regulation of chemokines and their receptors is associated with a many diseases including inflammatory diseases, atherosclerosis, cancer and human immunodeficiency virus infection. The chemokine network consists of approximately 50 human ligands and 20 receptors.

Chemokine receptors are 7 transmembrane G protein-coupled receptors (GPCRs). When a chemokine agonist binds on the extracellular side of its receptor, it induces a conformational change of the receptor that is transmitted to heterotrimeric G proteins bound on the intracellular face. Upon activation of the heterotrimeric G proteins, the Ga subunit exchanges GDP for GTP and dissociates from the receptor and from the βγ subunits, and both G protein complexes go on to activate other downstream signaling events. Classically, chemokine receptors and other GPCRs have been thought to function as monomers and have been studied as isolated systems to identify particular pathways activated by a given ligand/receptor. However, although GPCRs may activate G proteins as monomers, they are also known to homo- and heteromultimerize.

Multimerization of GPCRs, including chemokine receptors may prove a degree of specificity to signaling by these molecules, given the potential redundancy that occurs by chemokines binding to multiples monomeric receptors and monomeric receptors binding to several chemokines. Since chemokine receptors play a role in several processes important in human disease, heteromultimeric forms of these receptors represent new diagnostic/therapeutic targets for many diseases. SUMMARY

In work leading up to the present invention, the inventors demonstrated that CXCR4 and CCR7 form a heteromultimer in certain cells, such as cancer cells and, in the case of cancer cells, the formation of the heteromultimer is associated with its metastatic potential. Without limiting the present disclosure to any specific mode of action, the inventors believe that the CXCR4-CCR7 heteromultimer allows efficient G- protein coupling in response to ligand binding, which may not occur for the individual monomers or homomultimers of CXCR4 or CCR7 in some cell types. In this regard, ligand binding to the CXCR4-CCR7 heteromultimer, but not the individual monomers or homomultimers, may facilitate G protein dependent and/or G protein independent signaling events including, for example, changes in Ca²⁺ mobilization (e.g. increase in Ca²⁺ flux), changes in cAMP levels (e.g. inhibition of cAMP synthesis) or activation of the MAP kinase family members, other kinases, phosphatases or phospholipases.

The studies performed by the inventors in cancer cells are a model system for other cell types. For example, the studies on the role the CXCR4-CCR7 heteromultimer plays in metastasis is useful as a model system for determining the effect of the heteromultimer in chemtotaxis, e.g., of immune system cells or stem cells. The inventors also determined that by inhibiting function of the CXCR4-CCR7 heteromultimer, they can induce anoikis.

The inventors also reasoned that the CXCR4-CCR7 heteromultimer provided the basis for identifying or isolating certain cells and/or for killing certain cells, e.g., by inducing immune effector function or through the use of a toxic compound.

The inventors also determined that they could modulate the function of CXCR4 by modulating CCR7 expression and/or activity and vice versa. The inventors also demonstrated that they could inhibit binding of an anti-CCR7 antibody to its target antigen using an anti-CXCR4 antibody. The findings by the inventors provide the basis for various reagents comprising or that bind to or modulate a CXCR4-CCR7 heteromultimer and methods of use thereof.

In one example, the present disclosure provides an isolated or recombinant CXCR4-CCR7 heteromultimer. The heteromultimer can comprise at least one CXCR4 monomer with contact with at least one CCR7 monomer.

For example, the heteromultimer is a heterodimer comprising one CXCR4 monomer in contact with one CCR7 monomer.

In another example, the heteromultimer comprises a plurality of CXCR4 monomers and a plurality of CCR7 monomers.

In a further example, the heteromultimer is a hetero-oligomer comprising at least one CXCR4 homomultimer in contact with at least one CCR7 homomultimer or a plurality of CXCR4 monomers associated with a plurality of CCR7 monomers.

Heteromultimers encompassed by the present invention are not limited to those only comprising CXCR4, and CCR7, i.e., they can additionally comprise a protein other than CXCR4 and CCR7. For example, the heteromultimer can comprise G proteins and/or an arrestin and/or another chemokine receptor and/or a G protein coupled receptor other than a chemokine receptor and/or another receptor.

In one example, the CXCR4 and/or CCR7 is a fusion protein. For example, the CXCR4 and/or CCR7 is fused to a detectable tag, such as a detectable tag is selected from the group consisting of a fluorescent label, an enzyme and an epitope tag.

In one example, the present disclosure provides a fusion protein comprising a CXCR4 and a CCR7 in contact with one another. In one example, the CXCR4 and CCR7 are separated by a linker. For example, the N-termini or C-termini of the CXCR4 and CCR7 are linked.

The present disclosure also provides an expression construct comprising a nucleic acid encoding CXCR4 operably linked to a promoter and a nucleic acid encoding CCR7 operably linked to a promoter.

In one example, the expression construct comprises the following operably linked components in 5' to 3' order:

(i) a promoter

(ii) a nucleic acid encoding a first polypeptide;

(iii) an internal ribosome entry site; and

(iv) a nucleic acid encoding a second polypeptide,

wherein the first polypeptide is CXCR4 and the second polypeptide is CCR7, or vice versa. The present disclosure also provides a composition comprising:

(i) a first expression construct comprising a nucleic acid encoding CXCR4 operably linked to a promoter; and

(ii) a second expression construct comprising a nucleic acid encoding CCR7 operably linked to a promoter.

The present disclosure also provides a recombinant cell comprising the heteromultimer of the present disclosure, e.g., the heteromultimer is on the surface of the cell.

In one example, the CXCR4 and/or CCR7 is a recombinant protein.

In one example, the cell comprises an expression construct described herein or comprising:

In one example, the cell is a mammalian cell. For example, the cell is selected from the group consisting of a cancer cell, an immune cell, a mesodermal cell, an endothelial cell, an epithelial cell, an ectodermal cell and a stem cell. For example, the cell is an immune cell, a tonsil cell, a thymus cell, an endothelial cell, an endothelial progenitor cell, a mesangial cell, a hepatic cell, a lung cell, a bone marrow CD34⁺ cell, a cord blood CD34⁺ cell, a lymph node cell, a neuronal cell, a CHO cell, a human embryonic kidney (HEK cell) or a neoplastic cell. Exemplary immune cells include an immune cell selected from the group consisting of B lymphoblasts, B cells, dendritic cells, plasmacytoid dendritic cells, regulatory T (Treg) cells and T cells.

In one exemplary form of the disclosure the cell is a cancer cell or a metastatic cancer cell. For example, the cell is from an adenocarcinoma, a squamous cell carcinoma, a digestive/gastrointestinal cancer, an endocrine cancer, an eye cancer, a musculoskeletal cancer, a breast cancer, a neurologic cancer, a genitourinary cancer, a germ cell cancer, a head and neck cancer, a hematologic/blood cancer, a respiratory cancer, a skin cancer, an AIDS-related malignancy or a gynelogic cancer.

For example, the cell is a cancer cell or metastatic cancer cell selected from the group consisting of a breast cancer cell, a lymphoma cell, a leukemia cell, a head and neck cancer cell, a thyroid cancer cell, a gastric cancer cell, an endometrial cancer cell, a lung cancer cell, a cervical cancer cell, a melanoma cell, a non-melanoma skin cancer cell, a pancreatic cancer cell, a prostate cancer cell, an esophageal cancer cell, a nasopharyngeal cancer cell, a colorectal cancer cell, an osteocarcinoma cancer cell, a kidney cancer cell, an ovarian cancer cell, a myeloma cell, a neuroblastoma cell, a myosarcoma cell and a testicular cancer cell. In one example, the cell is a breast cancer cell or a metastatic breast cancer cell.

The present disclosure also provides an isolated population of cells enriched for cells expressing a CXCR4-CCR7 heteromultimer. In one example, the cells are mammalian cells, such as cancer cells, immune cells, mesodermal cells, endothelial cells, epithelial cells, ectodermal cells and stem cells.

For example, the cells are immune cells, tonsil cells, thymus cells, endothelial cells, endothelial progenitor cells, mesangial cells, hepatic cells, lung cells, bone marrow CD34⁺ cells, cord blood CD34⁺ cells, or lymph node cells.

Exemplary immune cells are selected from the group consisting of B lymphoblasts, B cells, dendritic cells, plasmacytoid dendritic cells, regulatory T (Treg) cells, monocytes, macrophages, granulocytes and T cells.

In one example, the cells are cancer cells or metastatic cancer cells. For example, the cells are from an adenocarcinoma, a squamous cell carcinoma, a digestive/gastro intestinal cancer, an endocrine cancer, an eye cancer, a musculoskeletal cancer, a breast cancer, a neurologic cancer, a genitourinary cancer, a germ cell cancer, a head and neck cancer, a hematologic/blood cancer, a respiratory cancer, a skin cancer, an AIDS-related malignancy or a gynelogic cancer.

For example, the cells are cancer cells or metastatic cancer cells selected from the group consisting of breast cancer cells, lymphoma cells, leukemia cells, head and neck cancer cells, thyroid cancer cells, gastric cancer cells, endometrial cancer cells, lung cancer cells, cervical cancer cells, melanoma cells, non-melanoma skin cancer cells, pancreatic cancer cells, prostate cancer cells, esophageal cancer cells, nasopharyngeal cancer cells, colorectal cancer cells, osteocarcinoma cancer cells, kidney cancer cells, ovarian cancer cells, myeloma cells, neuroblastoma cells, myosarcoma cells and testicular cancer cells. In one example, the cells are breast cancer cells or metastatic breast cancer cells.

The present disclosure also provides a binding molecule that specifically binds to a CXCR4-CCR7 heteromultimer.

In one example, the binding molecule does not detectably bind to a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer. Methods for detecting binding to a heteromultimer, a monomer and/or a homomultimer will be apparent to the skilled artisan and/or described herein.

In one example, the molecule specifically binds to one or more of the following:

(i) the heterodimeric interface of CXCR4 and CCR7 heteromultimer; (ii) an epitope formed in a CXCR4-CCR7 heteromultimer which is not present in a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer; or

(iii) an epitope exposed in the CXCR4-CCR7 heteromultimer which is not sufficiently exposed in a CXCR4 monomer and/or a CXCR4 homomultimer and/or a

CCR7 monomer and/or a CCR7 homomultimer to permit binding of the molecule.

The present disclosure also provides a binding molecule that prevents formation of CXCR4-CCR7 heteromultimer and/or disrupts a CXCR4-CCR7 heteromultimer.

In one example, a binding molecule described herein reduces or prevents activity of a CXCR4-CCR7 heteromultimer.

In another example, a binding molecule described herein enhances or induces activity of a CXCR4-CCR7 heteromultimer.

In a further example, a binding molecule described herein does not detectably reduce or prevent activity of a CXCR4-CCR7 heteromultimer.

Methods for determining the effect of a binding molecule on a heteromultimer or on CXCR4 or on CCR7 will be apparent to the skilled artisan and/or described herein.

The present disclosure also provides a binding molecule that specifically binds to CXCR4 and modulates CCR7 activity or that specifically binds to CCR7 and modulates CXCR4 activity. In one example, the binding molecule antagonizes the activity.

In one example, the binding molecule is a small molecule, an aptamer, a protein, a ligand or an antibody or antigen binding fragment thereof.

In one example, the binding molecule is an antibody or an antigen binding fragment thereof. Exemplary antibodies include monoclonal antibodies, chimeric antibodies, humanized antibodies and human antibodies.

Exemplary antigen binding fragments contemplated by the present disclosure include:

(i) a domain antibody (dAb);

(ii) a Fv;

(iii) a scFv or stabilized form thereof (e.g., a disulfide stabilized scFv);

(iv) a dimeric scFv or stabilized form thereof);

(iv) a diabody, triabody, tetrabody or higher order multimer;

(v) Fab fragment;

(vi) a Fab' fragment;

(vii) a F(ab') fragment; (viii) a F(ab')2 fragment;

(ix) any one of (i)-(viii) fused to a Fc region of an antibody;

(x) any one of (i)-(viii) fused to an antibody or antigen binding fragment thereof that binds to an immune effector cell.

In one example, the binding molecule kills a cell to which it binds. In the case of an antibody or antigen binding fragment thereof, the present disclosure contemplates those capable of inducing effector function to thereby kill a cell to which it binds and/or that is conjugated to a toxic compound that kills a cell to which is binds.

The present disclosure also provides a binding molecule described herein having a compound conjugated thereto. For example, the compound is selected from the group consisting of a radioisotope, a detectable tag, a therapeutic compound, a colloid, a toxin, a nucleic acid, a peptide, a protein, a compound that increases the half life of the protein in a subject and mixtures of two or more thereof.

The present disclosure also provides a composition comprising the heteromultimer described herein or the expression construct described herein or the composition comprising expression constructs described herein or the cell described herein, the population of cells described herein or the binding molecule of described herein and, optionally, a pharmaceutically acceptable carrier..

In one example, the composition is a pharmaceutical composition and comprises the cell described herein, the population of cells described herein or the binding molecule of described herein and a pharmaceutically acceptable carrier.

The present disclosure also provides a method for detecting a CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer, the method comprising contacting a sample with the binding molecule described herein such that the molecule binds to the heteromultimer, if present, and detecting the bound heteromultimer.

The present disclosure also provides a method for detecting a CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer, the method comprising:

(i) contacting a sample with a first binding molecule that specifically binds to CXCR4 such that the molecule binds to the heteromultimer, if present;

(ii) contacting the sample the a second binding molecule that specifically binds to CCR7 such that the molecule binds to the heteromultimer, if present; and

(iii) detecting heteromultimer bound the first and second binding molecules.

In one example, the first binding molecule is immobilized on a solid support and the second binding molecule is conjugated to a detectable tag, or vice versa. The present disclosure also provides a method for isolating a cell expressing a CXCR4-CCR7 heteromultimer or a population of cells enriched for CXCR4-CCR7 heteromultimer expressing cells, the method comprising performing a method described herein to detect a cell or population enriched for cells and isolating a cell(s) detected by the method.

In one example, the method additionally comprises culturing the isolated cells.

In one example, the method additionally comprises formulating the cells and/or an extract thereof into a pharmaceutical composition, e.g., to thereby produce a vaccine.

In one example, the cell is or the population comprises a cancer cell, an immune cell, a mesodermal cell, an endothelial cell, an epithelial cell, an ectodermal cell and a stem cell. For example, the cell is or the population comprises an immune cell, a tonsil cell, a thymus cell, an endothelial cell, an endothelial progenitor cell, a mesangial cell, an hepatic cell, a lung cell, a bone marrow CD34⁺ cell, a cord blood CD34⁺ cell, a lymph node cell, a neuronal cell, a CHO cell or a human embryonic kidney (HEK cell).

In one example, the cell is or the population comprises an immune cell selected from the group consisting of B lymphoblasts, B cells, dendritic cells, plasmacytoid dendritic cells, regulatory T (Treg) cells, monocytes, macrophages, granulocytes and T cells.

In another example, the cell is or the population comprises a cancer cell or a metastatic cancer cell. For example, the cell is or the population is from an adenocarcinoma, a squamous cell carcinoma, a digestive/gastrointestinal cancer, an endocrine cancer, an eye cancer, a musculoskeletal cancer, a breast cancer, a neurologic cancer, a genitourinary cancer, a germ cell cancer, a head and neck cancer, a hematologic/blood cancer, a respiratory cancer, a skin cancer, an AIDS-related malignancy or a gynelogic cancer.

For example, the cell or population comprises a cancer cell or metastatic cancer cell selected from the group consisting of a breast cancer cell, a lymphoma cell, a leukemia cell, a head and neck cancer cell, a thyroid cancer cell, a gastric cancer cell, an endometrial cancer cell, a lung cancer cell, a cervical cancer cell, a melanoma cell, a non-melanoma skin cancer cell, a pancreatic cancer cell, a prostate cancer cell, an esophageal cancer cell, a nasopharyngeal cancer cell, a colorectal cancer cell, an osteocarcinoma cancer cell, a kidney cancer cell, an ovarian cancer cell, a myeloma cell, a neuroblastoma cell, a myosarcoma cell and a testicular cancer cell. In one example, the cell is a breast cancer cell or a metastatic breast cancer cell. In one example the method additionally comprises formulating the cells with a pharmaceutically acceptable carrier to thereby produce a pharmaceutical composition.

The present disclosure also provides a cell or population of cells isolated by a method described herein.

The present disclosure also provides a method for diagnosing and/or prognosing a condition in a subject, the method comprising performing a method described herein to detect a CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer or the level of the heteromultimer or the cell in a sample from a subject, wherein detection of CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4- CCR7 heteromultimer or the level of the heteromultimer or the cell or failure to detect CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer is diagnostic or prognostic of the condition.

In one example, the disclosure also provides a method for diagnosing and/or prognosing a condition in a subject, the method comprising performing a method described herein to detect CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer in a sample from a subject, wherein detection of CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer or failure to detect CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4- CCR7 heteromultimer is diagnostic or prognostic of the condition.

In one example, the method additionally comprises:

(i) determining or estimating the amount of the CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer in the sample;

(ii) comparing the amount of the CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer in a standard derived from samples from one or more normal and/or healthy subject(s);

wherein an increased or decreased amount of the CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer at (i) compared to the amount of the CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer in the standard.

The present disclosure additionally provides a method for monitoring the efficacy of treatment of a condition, the method comprising performing a method described herein to detect a CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer or the level of the heteromultimer or the cell in a sample from a subject receiving treatment for the condition, wherein detection of CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer or the level of the heteromultimer or the cell or failure to detect CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer is indicative of whether or not the subject is responding to treatment .

In one example, the method comprises:

(i) determining or estimating the amount of a CXCR4-CCR7 heteromultimer or cells expressing the CXCR4-CCR7 heteromultimer in a sample from a subject undergoing treatment for a condition by performing a method described herein;

(ii) comparing the amount of CXCR4-CCR7 heteromultimer or cells expressing the CXCR4-CCR7 heteromultimer in a standard,

wherein:

(a) the standard is derived from one or more normal and/or healthy subject(s) and a similar amount of CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4- CCR7 heteromultimer at (i) compared to the standard indicates that the subject is responding to treatment for the condition;

(b) the standard is derived from one or more normal and/or healthy subject(s) and an increased or decreased amount of CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer at (i) compared to the standard indicates that the subject is not responding to treatment for the condition;

(c) the standard is derived from the subject undergoing treatment at an earlier point in time and a similar amount of CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer at (i) compared to the standard indicates that the subject is not responding to treatment for the condition; or

(d) the standard is derived from the subject undergoing treatment at an earlier point in time and an increased or decreased amount of CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer at (i) compared to the standard indicates that the subject is responding to treatment for the condition.

The present disclosure also contemplates imaging methods, e.g., for detecting a

CXCR4-CCR7 heteromultimer expressing cell in vivo. In one example, the disclosure provides a method for localizing and/or detecting and/or diagnosing and/or prognosing condition associated with a cell expressing the CXCR4-CCR7 heteromultimer, the method comprising detecting in vivo the binding molecule described herein bound to the CXCR4-CCR7 heteromultimer expressing cell, if present, wherein the molecule is conjugated to a detectable tag.

In one example, the method additionally comprises administering the binding molecule to the subject.

In on example of a diagnostic/prognostic/imaging method described herein, the condition is selected from the group consisting of cancer, an inflammatory condition, an autoimmune condition, an obstructive condition, an infectious condition, an excitotoxic condition, an immunodeficiency condition, a metabolic condition, pain, a circulatory condition and a degenerative condition.

In one example, the condition is associated with or caused by a cell selected from the group consisting of an immune cell, a tonsil cell, a thymus cell, an endothelial cell, an endothelial progenitor cell, a mesangial cell, an hepatic cell, a lung cell, a bone marrow CD34⁺ cell, a cord blood CD34⁺ cell, or a lymph node cell. For example, the condition is selected from the group consisting of an inflammatory condition, an autoimmune condition, graft versus host disease, graft rejection, liver disease, liver cirrhosis, kidney disease, renal failure, neutropenia, viral infection or hepatitis.

In one exemplary form of the disclosure, the condition is cancer or a metastasis thereof. For example, the cancer is selected from the group consisting of an adenocarcinoma, a squamous cell carcinoma, a digestive/gastrointestinal cancer, an endocrine cancer, an eye cancer, a musculoskeletal cancer, a breast cancer, a neurologic cancer, a genitourinary cancer, a germ cell cancer, a head and neck cancer, a hematologic/blood cancer, a respiratory cancer, a skin cancer, an AIDS-related malignancy or a gynelogic cancer.

For example, the cancer is selected from the group consisting of a breast cancer, a lymphoma, a leukemia, a head and neck cancer, a thyroid cancer, a gastric cancer, an endometrial cancer, a lung cancer, a cervical cancer, a melanoma, a non-melanoma skin cancer, a pancreatic cancer, a prostate cancer, an esophageal cancer, a nasopharyngeal cancer, a colorectal cancer, an osteocarcinoma cancer, a kidney cancer, an ovarian cancer, a myeloma, a neuroblastoma, a myosarcoma and a testicular cancer. In one example, the cancer is breast cancer or a metastatic breast cancer.

The present disclosure also provides a method for ascertaining the metastatic potential of a cancer cell, the method comprising detecting the presence or amount of a CXCR4-CCR7 heteromultimer in/on the cell or in a sample that contains or has contained the cell, wherein the presence or amount of the CXCR4-CCR7 heteromultimer in/on the cell or in a sample that contains or has contained the cell is indicative of the metastatic potential of the cell.

The present method also contemplates detecting the heteromultimer or an antigenic component thereof or a cell membrane comprising the heteromultimer in a sample separate from an intact living cell, e.g., as a result of cell death or lysis or in cell culture medium.

In one example, the method additionally comprises obtaining the cell from a cancer. In one example, the cancer cell is from breast cancer, lymphoma, leukemia, head and neck cancer, thyroid cancer, gastric cancer, endometrial cancer, lung cancer, cervical cancer, melanoma, non-melanoma skin cancer, pancreatic cancer, prostate cancer, nasopharyngeal cancer, colorectal cancer, osteocarcinoma cancer, kidney cancer and ovarian cancer. For example, the cancer cell is from breast cancer.

In one example, the presence of a CXCR4-CCR7 heteromultimer in/on the cell is detected by performing a method described herein.

The present disclosure also provides a method for identifying a subject suffering from a metastasis or at risk of developing a metastasis, the method comprising ascertaining the metastatic potential of a cancer cell in a sample from the subject by performing a method described herein. The result of such a method identifies a subject suffering from a metastasis or at risk of developing a metastasis.

The present disclosure additionally provides obtaining the results of a diagnostic/prognostic/imaging method as described herein and administering a therapeutic agent. For example, the therapeutic agent is selected from the group consisting of:

(i) an agent that reduces or prevents the activity or expression of a CXCR4-CCR7 heteromultimer

(ii) an agent that kills a cell expressing a CXCR4-CCR7 heteromultimer;

(iii) an agent reduces or prevents the activity or expression of CXCR4;

(iv) an agent that kills a cell expressing CXCR4;

(v) an agent reduces or prevents the activity or expression of CCR7; and

(iv) an agent that kills a cell expressing CCR7.

In one example, the agent is not specific to the heteromultimer. For example, a chemotherapeutic agent can kill a cell expressing the heteromultimer.

In one example, the agent is the binding molecule as described herein or a binding molecule that binds to CXCR4 or CCR7.

The present disclosure also provides a method of treating or preventing a condition, the method comprising administering the cell or population of cells as described herein or the binding molecule as described herein or the composition as described herein to a subject in need thereof.

The present disclosure also provides the cell or population of cells as described herein or the binding molecule as described herein or the composition as described herein for use in the treatment or prevention of a condition or for use in medicine. The present disclosure also provides for the use of the cell or population of cells as described herein or the binding molecule as described herein or the composition as described herein in the manufacture of a medicament.

In one example, the condition is selected from the group consisting of cancer, an inflammatory condition, an autoimmune condition, an obstructive condition, an infectious condition, an excitotoxic condition, an immunodeficiency condition, a metabolic condition, pain, a circulatory condition and a degenerative condition.

For example, the condition is associated with or caused by a cell selected from the group consisting of an immune cell, a tonsil cell, a thymus cell, an endothelial cell, an endothelial progenitor cell, a mesangial cell, an hepatic cell, a lung cell, a bone marrow CD34⁺ cell, a cord blood CD34⁺ cell, or a lymph node cell.

For example, the condition is selected from the group consisting of an inflammatory condition, an autoimmune condition, graft versus host disease, graft rejection, liver disease, liver cirrhosis, kidney disease, renal failure, neutropenia, a viral infection or hepatitis.

In one exemplary form of the disclosure the condition is cancer or a metastasis thereof. For example, the cancer is selected from the group consisting of an adenocarcinoma, a squamous cell carcinoma, a digestive/gastrointestinal cancer, an endocrine cancer, an eye cancer, a musculoskeletal cancer, a breast cancer, a neurologic cancer, a genitourinary cancer, a germ cell cancer, a head and neck cancer, a hematologic/blood cancer, a respiratory cancer, a skin cancer, an AIDS-related malignancy or a gynelogic cancer.

In one example, the method or use comprises administering (or making use of) the binding molecule as described herein, wherein the molecule reduces or prevents the activity of a CXCR4-CCR7 heteromultimer and/or kills a cell expressing a CXCR4- CCR7 heteromultimer.

The present disclosure also provides a method for treating or preventing a cancer or metastasis thereof in a subject wherein the cancer expresses a CXCR4-CCR7 heteromultimer, the method comprising administering to the subject the binding molecule as described herein, wherein the molecule reduces or prevents the activity of a CXCR4-CCR7 heteromultimer and/or kills a cell expressing a CXCR4-CCR7 heteromultimer.

The present disclosure also provides the binding molecule as described herein for use in treating or preventing a cancer or metastasis thereof in a subject, wherein the molecule reduces or prevents the activity of a CXCR4-CCR7 heteromultimer and/or kills a cell expressing a CXCR4-CCR7 heteromultimer.

The present disclosure also provides for the use of the binding molecule as described herein, which reduces or prevents the activity of a CXCR4-CCR7 heteromultimer and/or kills a cell expressing a CXCR4-CCR7 heteromultimer in the manufacture of a medicament for treating or preventing a cancer or metastasis thereof in a subject

In one example, the cancer is a metastatic cancer.

In one example, the cancer is selected from the group consisting of an adenocarcinoma, a squamous cell carcinoma, a digestive/gastrointestinal cancer, an endocrine cancer, an eye cancer, a musculoskeletal cancer, a breast cancer, a neurologic cancer, a genitourinary cancer, a germ cell cancer, a head and neck cancer, a hematologic/blood cancer, a respiratory cancer, a skin cancer, an AIDS-related malignancy or a gynelogic cancer.

For example, the cancer is selected from the group consisting of a breast cancer, a lymphoma, a leukemia, a head and neck cancer, a thyroid cancer, a gastric cancer, an endometrial cancer, a lung cancer, a cervical cancer, a melanoma, a non-melanoma skin cancer, a pancreatic cancer, a prostate cancer, an esophageal cancer, a nasopharyngeal cancer, a colorectal cancer, an osteocarcinoma cancer, a kidney cancer, an ovarian cancer, a myeloma, a neuroblastoma, a myosarcoma and a testicular cancer.

In one exemplary form of the disclosure the cancer is breast cancer.

In one example, the method or use additionally comprises administering (or making use of) an anticancer drug and/or treating the subject with radiation therapy.

The present disclosure also provides a method for reducing an immune response in a subject, the method comprising administering to the subject the binding molecule as described herein, wherein the molecule reduces or prevents the activity of a CXCR4- CCR7 heteromultimer and/or that kills a cell expressing a CXCR4-CCR7 heteromultimer.

The present disclosure also provides the binding molecule as described herein for use in reducing an immune response in a subject, wherein the molecule reduces or prevents the activity of a CXCR4-CCR7 heteromultimer and/or that kills a cell expressing a CXCR4-CCR7 heteromultimer.

The present disclosure also provides for the use of the binding molecule as described herein, which reduces or prevents the activity of a CXCR4-CCR7 heteromultimer and/or that kills a cell expressing a CXCR4-CCR7 heteromultimer in the manufacture of a medicament for reducing an immune response in a subject.

In one example, the subject suffers from an inflammatory condition and/or an autoimmune condition.

The present disclosure also provides a method for reducing an immune response in a subject, the method comprising identifying a subject mounting an immune response characterized by the presence of immune cells expressing a CXCR4-CCR7 heteromultimer and administering to the subject an agent that reduces or prevents the activity or expression of a CXCR4-CCR7 heteromultimer and/or that kills a cell expressing a CXCR4-CCR7 heteromultimer or another therapy to reduce an immune response.

The present disclosure also provides an agent that reduces or prevents the activity or expression of a CXCR4-CCR7 heteromultimer and/or that kills a cell expressing a CXCR4-CCR7 heteromultimer for use in reducing an immune response in a subject, wherein the subject is mounting an immune response characterized by the presence of immune cells expressing a CXCR4-CCR7 heteromultimer.

The present disclosure also provides for the use of an agent that reduces or prevents the activity or expression of a CXCR4-CCR7 heteromultimer and/or that kills a cell expressing a CXCR4-CCR7 heteromultimer in the manufacture of a medicament for reducing an immune response in a subject, wherein the subject is mounting an immune response characterized by the presence of immune cells expressing a CXCR4- CCR7 heteromultimer.

In one example, the method or use additionally comprises performing a method described herein to identify the subject.

The present disclosure also provides a method for inducing an immune response in a subject comprising administering a binding molecule described herein that induces or enhances activity of the heteromultimer and/or administering a composition comprising a ligand of CXCR4 (e.g., stromal derived factor-1) and a ligand of CCR7 (e.g., CCL19 and/or CCL21) to the subject.

The present disclosure also provides the binding molecule that induces or enhances activity of the heteromultimer and/or a composition comprising a ligand of CXCR4 (e.g., stromal derived factor- 1) and a ligand of CCR7 (e.g., CCL19 and/or CCL21) for use in inducing an immune response in a subject.

The present disclosure also provides for use of the binding molecule that induces or enhances activity of the heteromultimer and/or a composition comprising a ligand of CXCR4 (e.g., stromal derived factor- 1) and a ligand of CCR7 (e.g., CCL19 and/or CCL21) for the manufacture of a medicament for inducing an immune response in a subject.

In one example, the method or use additionally comprises administering (or use of) an antigen.

The present disclosure also provides a method for inducing or enhancing mobilization of stem cells in a subject, the method comprising administering to the subject a binding molecule described herein, wherein the molecule reduces or prevents the activity of a CXCR4-CCR7 heteromultimer.

The present disclosure also provides a binding molecule as described herein for use in inducing or enhancing mobilization of stem cells in a subject, wherein the molecule reduces or prevents the activity of a CXCR4-CCR7 heteromultimer.

The present disclosure also provides for use of a binding molecule as described herein for the manufacture of a medicament for inducing or enhancing mobilization of stem cells in a subject, wherein the molecule reduces or prevents the activity of a CXCR4-CCR7 heteromultimer.

The present disclosure also provides a method for inducing migration of a stem cell to a location in a subject, the method comprising administering a binding molecule that induces or enhances activity of the heteromultimer and/or administering a composition comprising a ligand of CXCR4 (e.g., stromal derived factor-1) and a ligand of CCR7 (e.g., CCL19 and/or CCL21) to the location to the subject.

The present disclosure also provides a binding molecule that induces or enhances activity of the heteromultimer and/or a composition comprising a ligand of CXCR4 (e.g., stromal derived factor-1) and a ligand of CCR7 (e.g., CCL19 and/or CCL21) for use in inducing migration of a stem cell to a location in a subject.

The present disclosure also provides for the use of the binding molecule that induces or enhances activity of the heteromultimer and/or a composition comprising a ligand of CXCR4 (e.g., stromal derived factor-1) and a ligand of CCR7 (e.g., CCL19 and/or CCL21) for the manufacture of a medicament for inducing migration of a stem cell to a location in a subject. In one example, the stem cell is a hematopoietic stem cell or an endothelial stem cell. For example, the stem cell is a hematopoietic stem cell which is mobilized from bone marrow.

The present disclosure also provides a method of reducing or preventing chemotaxis of a cell, the method comprising contacting the cell with the binding molecule as described herein, wherein the molecule reduces or prevents the activity of a CXCR4-CCR7 heteromultimer.

The present disclosure also provides the binding molecule as described herein for use in reducing or preventing chemotaxis of a cell, wherein the molecule reduces or prevents the activity of a CXCR4-CCR7 heteromultimer.

The present disclosure also provides for use of the binding molecule as described herein for the manufacture of a medicament for reducing or preventing chemotaxis of a cell, wherein the molecule reduces or prevents the activity of a CXCR4-CCR7 heteromultimer

The present disclosure also provides a method of inducing chemotaxis of a cell, comprising contacting the cell with binding molecule that induces or enhances activity of the heteromultimer and/or a composition comprising a ligand of CXCR4 (e.g., stromal derived factor- 1) and a ligand of CCR7 (e.g., CCL19 and/or CCL21).

The present disclosure also provides a binding molecule that induces or enhances activity of the heteromultimer and/or a composition comprising a ligand of CXCR4 (e.g., stromal derived factor- 1) and a ligand of CCR7 (e.g., CCL19 and/or CCL21) for use in inducing chemotaxis of a cell.

The present disclosure also provides for the use of a binding molecule that induces or enhances activity of the heteromultimer and/or a composition comprising a ligand of CXCR4 (e.g., stromal derived factor- 1) and a ligand of CCR7 (e.g., CCL19 and/or CCL21) for the manufacture of a medicament for inducing chemotaxis of a cell.

The present disclosure also provides a method of modulating CXCR4 activity in a cell, the method comprising identifying a cell expressing a CXCR4-CCR7 heteromultimer and contacting the cell with an agent that modulates the activity or expression of CCR7. In one example, the agent is a ligand of CCR7, e.g., CCL19 or CCL21. Additional agents are described herein.

The present disclosure also provides a method of modulating CCR7 activity in a cell, the method comprising identifying a cell expressing a CXCR4-CCR7 heteromultimer and contacting the cell with an agent that modulates the activity or expression of CXCR4. In one example, the agent is a ligand of CXCR4, e.g., SDF-1. Other agents are described herein. The present disclosure also provides a method for identifying an agent for treating or preventing a condition in a subject, the method comprising identifying an agent that inhibits the formation or activity of a CXCR4-CCR7 heteromultimer in a cell.

In one example, the method comprises contacting a CXCR4-CCR7 heteromultimer with an agent and detecting the amount or activity of the heteromultimer, wherein a reduction in the amount or activity of the heteromultimer is indicative of an agent that inhibits the formation or activity of a CXCR4-CCR7 heteromultimer in a cell.

The present disclosure also provides a method for identifying a binding molecule, the method comprising identifying binding molecule that specifically binds to a CXCR4-CCR7 heteromultimer.

In one example, the method comprises identifying a binding molecule that binds to binds to a CXCR4-CCR7 heteromultimer and that does not detectably bind to CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer.

In one example, the method additionally comprises identifying a binding molecule that kills a cell to which it binds. For example, the binding molecule induces an effector function or delivers a toxic compound to a cell or induces apoptosis of a cell to which it binds.

The present disclosure also provides a method for identifying a binding molecule that specifically binds to CXCR4 or CCR7, the method comprising:

(i) identifying a binding molecule that binds to a CXCR4 monomer and/or a CXCR4 homomultimer or a CCR7 monomer and/or a CCR7 homomultimer; and (ii) contacting the molecule identified at (i) to a CXCR4-CCR7 heteromultimer and identifying a molecule that does not detectably bind to the heteromultimer.

In one example, the present disclosure provides a method for identifying a binding molecule that specifically modulates the activity of CXCR4 or CCR7, the method comprising:

(i) identifying a binding molecule that modulates the activity of a CXCR4 monomer and/or a CXCR4 homomultimer or a CCR7 monomer and/or a CCR7 homomultimer; and

(ii) contacting the molecule identified at (i) to a CXCR4-CCR7 heteromultimer or cell expressing the CXCR4-CCR7 heteromultimer and identifying a molecule that does not detectably modulate the activity of the CXCR4-CCR7 heteromultimer. In on example, the binding molecule is an antibody or antigen binding fragment thereof. For example, the antibody is produced by immunizing non-human mammal with a CXCR4-CCR7 heteromultimer or cell expressing the CXCR4-CCR7 heteromultimer or contacting a library of antibodies or antigen binding fragments thereof with a CXCR4-CCR7 heteromultimer or cell expressing the CXCR4-CCR7 heteromultimer.

In one example, a method as described herein additionally comprises providing the agent or binding molecule. For example, the method additionally comprises formulating the agent or binding molecule into a pharmaceutical composition.

The present disclosure also provides a method for producing an antibody that specifically binds to a CXCR4-CCR7 heteromultimer, the method comprising:

(i) immunizing a non-human mammal with a CXCR4-CCR7 heteromultimer or a cell expressing a CXCR4-CCR7 heteromultimer;

(ii) isolating cells producing antibodies that bind to the CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer;

(iii) contacting antibodies produced by the cells isolated at (ii) with a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer or a cell expressing same;

(iv) isolating a cell producing antibodies that do not detectably bind a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer or a cell expressing same; and

(v) producing the antibody.

In one example, the non-human mammal is a mouse.

In one example, the method additionally comprises isolating nucleic acid encoding the antibody or variable regions thereof, inserting the nucleic acid into an expression construct, introducing the expression construct into a cell and expressing the antibody.

In one example, the method comprises isolating nucleic acid encoding the variable regions of the antibody, inserting the nucleic acid into an expression construct in operable connection with human constant regions and expressing the antibody.

In one example, the method comprises humanizing the antibody and producing the humanized antibody.

In one example, the method additionally comprises isolating the antibody.

The present disclosure provides a method for producing an antigen binding fragment of an antibody that specifically binds to a CXCR4-CCR7 heteromultimer, the method comprising: (i) contacting particles having displayed thereon one or more antibody variable regions with a CXCR4-CCR7 heteromultimer or a cell expressing a CXCR4-CCR7 heteromultimer and isolating particles that bind to the heteromultimer or cell expressing the heteromultimer; and

(ii) contacting the particles isolated at (i) with a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer or a cell expressing same and isolating particles that do not detectably bind thereto,

thereby producing an antigen binding fragment of an antibody that specifically binds to a CXCR4-CCR7 heteromultimer.

Alternatively, the present disclosure provides a method for producing an antigen binding fragment of an antibody that specifically binds to a CXCR4-CCR7 heteromultimer, the method comprising:

(i) contacting particles having displayed thereon one or more antibody variable regions with a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer or a cell expressing same and isolating particles that do not detectably bind thereto; and

(i) contacting particles isolated at (i) with a CXCR4-CCR7 heteromultimer or a cell expressing a CXCR4-CCR7 heteromultimer and isolating particles that bind to the heteromultimer or cell expressing the heteromultimer,

In one example, the method additionally comprises isolating nucleic acid encoding the one or more antibody variable regions in the antigen binding fragment of an antibody that specifically binds to a CXCR4-CCR7 heteromultimer, inserting the nucleic acid into an expression construct, introducing the expression construct into a cell and expressing the antigen binding fragment of an antibody.

In one example, the method additionally comprises inserting the nucleic acid into an expression construct in operable connection with human constant regions to produce a nucleic acid encoding an antibody and expressing the antibody.

In one example, the method additionally comprises isolating the antibody.

In one example, the antibody variable regions are human antibody variable regions.

In one example, the particles are phage. For example, the phage have displayed on their surface a domain antibody or a scFv or a Fab or a single chain Fab. Exemplary display methods, including phage display methods are known in the art. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows microscopic images of shR A mediated knock-down of CCR7 and the associated abrogation of MDA-231 lung colonization in a SCID mouse metastatic model. Shown are excised lungs from mice injected with indicated cell mixes and analysed by fluorescence microscopy.

Figure 2 shows fluorescent images of CXCR4 and CCR7 staining in MDA-231 cells including an overlay. Shown are confocal images of the same cell mid section with membrane-localized CCR7 (left panel) and CXCR4 (middle panel) detected by monoclonal antibodies following by incubation with Cy3 and Alexa-468 conjugated secondary antibodies. Co-localizing pixels highlighted in white (right panel).

Figure 3 shows pictures of FRET imaging showing a direct interaction between CXCR4 and CCR7. MDA-231 cells were stained with mouse anti-CXCR4 and rat anti- CCR7 antibodies and Cy3 and Cy5 conjugated secondary antibodies as indicated. FRET was calculated as an increase in Cy3 fluorescence after Cy5 bleaching using Leica Application Suite Advance Fluorescence software and shown in the right panel.

Figure 4 shows the results of Western blotting demonstrating that CXCR4 and CCR7 co-precipitate from metastatic cancer cells. Lysates were prepared from MDA- 361 cells which were untreated or stimulated as indicated in the Figure. Immunocomplexes were precipitated by incubation with monoclonal anti-CXCR4 and CCR7 and ϋβ proteins were detected in the same gel by Western Blot with respective antibodies. The negative control is an isotype matched murine IgG and is shown in lane 1.

Figure 5 shows co-precipitation of CXCR4 and CCR7 in invasive (MDA-231 and MDA-361) but not in nonmetastatic (MDA-134 and MDA-453) cells. Lysates were prepared from indicated cell lines, immunoprecipitated with anti-CXCR4 monoclonal antibodies and immunocomplexes complexes were resolved and blotted with anti- CCR7 and anti-CXCR4.

Figure 6 shows a graph of the relative cell death of control cells, CXCR4 shRNA cells and CCR7 shRNA cells treated with CXCL12 or CCL21. Untreated, CXCL12 treated or CCL21 treated wild type or shRNA expressing MDA-231 cells as indicated were prevented from attachment for 24hrs following by TU EL staining and FACS analysis. Proportion of apoptotic cells was estimated as percent TUNEL +ve cells relative to the whole cell population from triplicate samples.

Figure 7 panel (A) shows a graph of intracellular calcium concentrations in wild type, CXCR4 shRNA or CCR7 shRNA expressing cells treated with CXCL12 at the time point indicated by the arrow. Changes in levels of intracellular calcium concentration were recorded as [Ca ]i (nM). Panel (B) shows a graph of intracellular calcium concentrations in CXCR4 shRNA expressing cells transfected with a CXCR4 expression plasmid. Cells were loaded with the Fura-2AM and treated with CXCL12 or CCL21 at the time point indicated by the arrow. Changes in levels of intracellular calcium concentration were recorded as [Ca⁺⁺]i (nM).

Figure 8A is a graphical representation showing a dose-dependent decrease in allophycocyanin (APC) fluorescence that is representative of the anti-CCR7 antibodies binding to the cell surface, after the addition of the anti-CXCR4 antibodies in MDA- 361 but not in ZR-75-1 cells. The fluorescent profile of cells stained with the isotype control antibody is also presented (filled grey histogram).

Figure 8B is a graphical representation showing the extent of the reduction in anti-CCR7 antibody binding to the cell surface estimated as the percent signal in APC mean fluorescence intensity (MFI) values relative to the non-inhibited situation, after the addition of the increasing amounts of the anti-CXCR4 antibodies.

KEY TO SEQUENCE LISTING

SEQ ID NO 1 amino acid sequence of Homo sapiens CXCR4

SEQ ID NO 2 amino acid sequence of Homo sapiens CCR7

SEQ ID NO 3 amino acid sequence of Homo sapiens SDF-1 alpha

SEQ ID NO 4 amino acid sequence of Homo sapiens SDF-1 beta

SEQ ID NO 5 amino acid sequence of Homo sapiens CCL19

SEQ ID NO 6 amino acid sequence of Homo sapiens CCL21

DETAILED DESCRIPTION

General

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al, (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al, (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

Selected Definitions

The term "CXCR4" as used herein encompasses human CXCR4 as described under Gene ID 7852 of the NCBI Entrez Gene database, or orthologs thereof including those recited in Gene ID 7852. For the purposes of nomenclature only, an exemplary sequence of CXCR4 is set forth in SEQ ID NO: 1.

The term "CCR \syn. CD 197) as used herein encompasses human CCR7 as described under Gene ID 1236 of the NCBI Entrez Gene database, or orthologs thereof including those recited in Gene ID 1236. For the purposes of nomenclature only, an exemplary sequence of CCR7 is set forth in SEQ ID NO: 2.

The term "SDF-1" (syn. CXCL12) as used herein encompasses human SDF-1 as described under Gene ID 6387of the NCBI Entrez Gene database, or orthologs thereof including those recited in Gene ID 6387. This term encompasses SDF-1 a and SDF-1 β. For the purposes of nomenclature only, an exemplary sequence of SDF-1 a is set forth in SEQ ID NO: 3. For the purposes of nomenclature only, an exemplary sequence of SDF-1 β is set forth in SEQ ID NO: 4.

The term "CCL19" as used herein encompasses human CCL19 as described under Gene ID 6363 of the NCBI Entrez Gene database, or orthologs thereof including those recited in Gene ID 6363. For the purposes of nomenclature only, an exemplary sequence of CCR7 is set forth in SEQ ID NO: 5.

The term "CCL21" as used herein encompasses human CCL21 as described under Gene ID 6366 of the NCBI Entrez Gene database, or orthologs thereof including those recited in Gene ID 6366. For the purposes of nomenclature only, an exemplary sequence of CCR7 is set forth in SEQ ID NO: 6.

The term "CXCR4-CCR7 heteromultimer", as used herein, is intended to mean a protein complex comprising at least one CXCR4 monomer in contact with at least one CCR7 monomer. For example, the heteromultimer may comprise a heteroligomer such as a heterodimer, a heterotrimer, a heteroquadromer, a heteropentamer, a heterohexamer, a heteroheptamer or a hetero-octamer comprising at least one CXCR4 monomer in contact with at least one CCR7 monomer. In some examples, the heteromultimer may comprise a heterotrimer or a heterodimer. In some examples, the heteromultimer comprises one or more CXCR4 homomultimers and/or one or more CCR7 homomultimers. As would be appreciated, a CXCR4-CCR7 heteromultimer as referred to herein may further comprise monomers or components in addition to at least one CXCR4 monomer and at least one CCR7 monomer. In some examples, the CXCR4 and/or CCR7 can be associated with other proteins, provided that at least one CXCR4 is in contact with at least one CCR7.

As used herein, the term "homomultimer" shall be understood to mean a protein complex comprising only one G protein coupled receptor (GPCR), for example chemokine receptor, such as, only CXCR4 or CCR7. A homomultimer can comprise non-GPCR proteins, such as a G protein or a ligand.

As used herein, the term "monomer" shall be taken to mean a GPCR, e.g., a chemokine receptor that is not in direct contact with another GPCR, e.g., chemokine receptor.

By "isolated" is meant that a protein, heteromultimer or cell is substantially removed from its naturally-occurring environment, e.g., is in a heterologous environment and/or that it is substantially free of contaminating agents, e.g., at least about 70% or 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99% free of contaminating agents.

As used herein, the term "enriched" or "enrich" in the context of a cell population shall be taken to mean that the number or percentage of cells expressing a CXCR4-CCR7 heteromultimer is greater than the number or percentage in a naturally occurring cell population. For example, a population enriched in cells expressing a CXCR4-CCR7 heteromultimer is made up of at least about 0.02% of said cells, or at least about 0.05% of said cells or at least about 0.1% of said cells or at least about 0.2% of said cells or at least about 0.5% of said cells or at least about 0.5% of said cells or at least about 0.8% of said cells or at least about 1% of said cells or at least about 2% of said cells or at least about 3% of said cells or at least about 4% of said cells or at least about 5% of said cells or at least about 10% of said cells or at least about 15% of said cells or at least about 20% of said cells or at least about 25% of said cells or at least about 30% of said cells or at least about 40% of said cells or at least about 50% of said cells or at least about 60% of said cells or at least about 70% of said cells or at least about 80% of said cells or at least about 85% of said cells or at least about 90% of said cells or at least about 95% of said cells or at least about 97% of said cells or at least about 98% of said cells or at least about 99% of said cells.

As used herein, the term "binding molecule" shall be understood to mean any compound capable of binding to a protein or a heteromultimer. Preferably, the binding molecule is capable of specifically binding to a protein or a heteromultimer. Exemplary binding molecules are aptamers, small molecule, proteins, peptides, ligands, antibodies and antigen binding fragments of antibodies. On exemplary binding molecule described herein is an antibody or an antigen binding fragment thereof.

As used herein, the term "antibody" refers to an immunoglobulin molecule capable of binding to a target protein or heteromultimer and/or an epitope thereof and/or an immunogenic fragment thereof and/or a modified form thereof (e.g., glycosylated, etc.) through at least one antigen binding site, located in the variable region of the immunoglobulin molecule. This term encompasses not only intact polyclonal or monoclonal antibodies, but also humanized antibodies, human antibodies, chimeric antibodies. This term also encompasses antibody-like molecules, such as, heavy chain antibodies from camelids or shark IgNAR.

As used herein, the term "antigen binding fragment" shall be taken to mean any fragment of an antibody that retains at least one variable region of an antibody and the ability to bind to the target protein or heteromultimer preferably specifically or selectively. This term includes a domain antibody, a Fab fragment, a Fab' fragment, a F(ab') fragment, a single chain antibody (SCA or SCAB) amongst others. An "Fab fragment" consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain. A Fab fragment can also be produced recombinantly. An "Fab' fragment" of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner. An Fab' fragment can also be produced recombinantly. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction or by recombinant production. An "Fv fragment" is a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains. A "single chain antibody" or scFv is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible polypeptide linker. Additional antigen binding domains of antibodies are described herein and/or known in the art.

As used herein, the term "specifically binds" shall be taken to mean that a binding molecule reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a protein (e.g., a CXCR4-CCR7 heteromultimer) than it does with another protein or epitope (e.g., with a CXCR4 and/or CCR7 monomer and/or homomultimer). For example, a binding molecule that specifically binds to a CXCR4-CCR7 heteromultimer binds with greater affinity, avidity, more readily, and/or with greater duration than it binds CXCR4 and/or CCR7 monomer and/or homomultimer. In this regard, the degree of greater affinity, avidity, more readily, and/or with greater duration will depend on the application of the binding molecule. For example, for detection/diagnostic/prognostic purposes the degree of specificity should be sufficiently high to permit quantification (where required). For therapeutic/prophylactic applications, the degree of specificity should be sufficient to provide a therapeutic/prophylactic effect without serious adverse effects resulting from cross-reactivity of the binding molecule. Preferably, the binding compound binds to a CXCR4-CCR7 heteromultimer and does not detectably bind to CXCR4 and/or CCR7 monomer and/or homomultimer, e.g., as determined by a Western blot and/or FACS and/or ELISA and/or antibody panning. In one example, wherein the binding molecule is an antibody, the antibody binds to a CXCR4-CCR7 heteromultimer and does not bind to CXCR4 and/or CCR7 monomer and/or homomultimer to a significantly greater level than an isotype control antibody, e.g., as determined by a Western blot or FACS analysis. It is also to be understood by reading this definition that "specific binding" does not necessarily require exclusive binding or non-detectable binding of another molecule, this is encompassed by the term "selective binding". Generally, but not necessarily, reference to binding means specific binding.

As used herein, "does not detectably bind" shall be understood to mean that a binding molecule, e.g., an antibody, does not bind to an antigen at a level significantly greater than background, e.g., binds to the antigen at a level less than 10%, or 8% or 6% or 5% above background. In the case of an antibody, the antibody binds to the antigen at a level less than 10% or 8% or 6% or 5% greater than an isotype control antibody. In one example, the binding is detected by Western blotting and/or FACS and/or ELISA and/or antibody panning (e.g., with antibody variable regions on the surface of a particle, such as a phage) and/or Biacore analysis.

As used herein, the term "heterodimeric interface" shall be taken to mean a region of a CXCR4-CCR7 surrounding sites at which CXCR4 and CCR7 contact one another and/or are linked to one another and/or are sufficiently close to permit binding of a binding molecule such that it specifically binds to the heteromultimer while having reduced or undetectable binding to a CXCR4 monomer or CXCR4 homomultimer or a CCR7 or CCR7 homomultimer. In this regard, this term does not limit to only those residues in CXCR4 and CCR7 that actually contact one another. Rather, the residues need only be within the region of the proteins involved in heterodimerization and exposed as to permit binding and to permit specific binding to a CXCR4-CCR7 heteromultimer. In one example, the "heterodimeric interface" is an epitope that is produced by dimerization of the protein, e.g., comprises amino acids of CXCR4 and CCR7.

As used herein, the term "epitope" (syn. "antigenic determinant") shall be understood to mean a region of a CXCR4-CCR7 heteromultimer to which a binding molecule binds. This term is not necessarily limited to the specific residues or structure to which the molecule makes contacts. For example, this term includes the region spanning amino acids contacted by the molecule and/or 5-10 or 2-5 or 1-3 amino acids outside of this region. In some examples, the epitope is a linear series amino acids. However, an epitope can also comprise a series of discontinuous amino acids that are positioned close to one another when the CXCR4-CCR7 heteromultimer is folded, i.e., a "conformational epitope". The skilled artisan will also be aware that the term "epitope" is not limited to peptides or polypeptides. For example, the term "epitope" includes chemically active surface groupings of molecules such as sugar side chains, phosphoryl side chains, or sulfonyl side chains, and, in certain embodiments, may have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope or peptide or polypeptide comprising same can be administered to an animal to generate antibodies against the epitope.

An epitope is "sufficiently exposed to permit binding" if, in the context of a CXCR4-CCR7 heteromultimer, will be understood to mean that when the CXCR4- CCR7 heteromultimer is in its native context (e.g., in a cell membrane) a binding molecule is able to bind to the heteromultimer without prior denaturation. For example, the epitope is exposed to solute (or surface exposed) in such a manner that the binding molecule can bind thereto. Such an epitope may not be exposed so as to permit binding in the context of a CXCR4 monomer or CXCR4 homomultimer or a CCR7 or CCR7 homomultimer.

In the context of the present disclosure, "effector functions" refer to those biological activities mediated by cells or proteins that bind to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody that result in killing of a cell. Examples of effector functions induced by antibodies include: complement dependent cytotoxicity; antibody-dependent-cell-mediated cytotoxicity (ADCC); antibody-dependent-cell-phagocytosis (ADCP); and B-cell activation. In the context of the present disclosure, the term "effector function induced by an antibody" or like term is used interchangeable with "effector function of an antibody" or like term and each provides literal support for the other.

As used herein, the term "disrupts a CXCR4-CCR7 heteromultimer" means that a binding molecule is capable of disassociate a previously formed CXCR4-CCR7 heteromultimer such that the CXCR4 and CCR7 are no longer in contact with one another. This is distinct from preventing formation of a heteromultimer, in which case the binding molecule binds to one or both of CXCR4 and/or CCR7 and prevents them from associating one another. As used herein, the term "activity of a CXCR4-CCR7 heteromultimer" shall be taken to mean any activity conferred by the heteromultimer upon a cell, including signal transduction. This term encompasses CXCR4 activity conferred by the heteromultimer and/or CCR7 activity conferred by the heteromultimer and/or alternative G protein coupling conferred by the heteromultimer. The term "alternate G protein coupling", as used herein, is intended to mean an arrangement of G proteins that couple with the CXCR4-CCR7 heteromultimer in response to activation that is different to the arrangement of G proteins that couple with CXCR4 or CCR7 monomers or homomultimers in response to activation. G proteins and various G protein couplings may include, for example, those described in Hamm, The Journal of Biological Chemistry 273(2): 669-672, 1998. Methods for determining G protein dependent and/or G protein independent signaling events including, for example, changes in Ca²⁺ mobilization (e.g. increase in Ca²⁺ flux), changes in cAMP levels (e.g. inhibition of cAMP synthesis) or activation of the MAP kinase family members, other kinases, phosphatases or phospholipases are known in the art and/or described herein.

As used herein, the term "CXCR4 activity" shall be taken to mean any activity conferred by CXCR4 upon a cell, including signal transduction. For example, CXCR4 can induce cell survival and/or chemotaxis and/or cell maturation and/or cell differentiation. CXCR4 signal transductions can be G protein dependent or G protein independent. For example, G protein dependent signaling can involve the G; family of a G proteins, the Gq family as well as βγ subunits. Signaling through G; can regulate PI3K or PKC. βγ signaling can regulate PI3K and/or Rho and/or ΡΙΧβ and/or RAS- mediated signaling. Additional exemplary CXCR4 activities include, chemotaxis (e.g., of monocytes, T cells, T_H17 cells, CD34⁺ bone marrow cells, pro-B cells, pre-B cells, NK cells, endothelial cells, EPCs and/or plasmacytoid dendritic cells(pDCs)), promotion of angiogenesis, neuronal development, lung development, T cell activation, memory T cell development, protection of dendritic cells from apoptosis, hematopoiesis, pDC development, inflammation, hepatitis -mediated liver damage, liver cirrhosis/fibrosis, development of or progression of numerous cancers and metastasis of numerous cancers.

As used herein, the term "CCR7 activity" shall be taken to mean any activity conferred by CCR7 upon a cell, including signal transduction. For example, CCR7 can induce cell survival and/or chemotaxis and/or endocytosis and/or cell maturation. CCR7 signal transductions can be G protein dependent or G protein independent. For example, G protein dependent signaling can involve the G; or G₁₂ families of a G proteins as well as βγ subunits. Signaling through G; can regulate PI3K, p38, Erkl/2 Cdc42/Roc. Gi2 can regulate Rho-mediated signaling. Additional exemplary CCR7 activities include, chemotaxis (e.g., of dendritic cells, T cells (both CD4⁺ and CD8⁺), B cells, cord blood and bone marrow CD34⁺ cells, and mesangial cells), proliferation (e.g., of mesangial cells, T cells and cord blood and bone marrow CD34⁺ cells), dendritic cell endocytosis, dendritic cell differentiation, T cell differentiation, cell survival (e.g., of dendritic cells, mesangial cells and T cells), inflammation, hepatitis- mediated liver damage, liver cirrhosis/fibrosis, development of or progression of numerous cancers and metastasis of numerous cancers.

The term "metastatic potential" as used herein is intended to mean the ability or propensity of a cancer cell to migrate from a primary tumor site to other parts of the body. A cancer cell with a high metastatic potential is a cancer cell with a relatively high ability or propensity to move to other parts of the body. In contrast, a cancer cell with a low metastatic potential is a cancer cell which lacks the ability or has low propensity of movement to other parts of the body.

For the foregoing, it will be apparent to the skilled person that a "metastasis" is the occurrence of a cell from one tissue in another part of the body, for example, the occurrence of a cancer cell outside the site of the primary tumor. For example, leukemia or lymphoma cells in the blood stream are metastatic, since the primary sites of these cancers are the bone marrow or lymph nodes, respectively. Exemplary other forms of cancers that become metastatic include breast cancer cell, a lymphoma cell, a leukemia cell, a head and neck cancer cell, a thyroid cancer cell, a gastric cancer cell, an endometrial cancer cell, a lung cancer cell, a cervical cancer cell, a melanoma cell, a non-melanoma skin cancer cell, a pancreatic cancer cell, a prostate cancer cell, an esophageal cancer cell, a nasopharyngeal cancer cell, a colorectal cancer cell, an osteocarcinoma cancer cell, a kidney cancer cell and an ovarian cancer cell. The term "metastasis" also encompasses micrometastasis.

As used herein, the terms "treating", "treat" or "treatment" include administering a therapeutically effective amount of a compound described herein sufficient to reduce or eliminate at least one symptom of a specified disease or condition. The term 'treat" or "treating" or "treatment" as used herein in relation to a cancer in a subject is intended to mean that the binding molecule or agent reduces or inhibits the rate or extent of growth of the cancer or inhibits the metastatic potential of the cancer. In some examples, treating a cancer may reduce the size and/or growth rate of the cancer.

As used herein, the terms "preventing", "prevent" or "prevention" include administering a therapeutically effective amount of an inhibitor(s) and/or agent(s) described herein sufficient to stop or hinder the development of at least one symptom of a specified disease or condition. The term "preventing", "prevent" or "prevention" as used herein in relation to a cancer in a subject is intended to mean that the agent substantially prevents the formation of a cancer or prevents the transformation of a non- metastatic cancer to a metastatic cancer that would otherwise occur had the subject not been treated with the agent.

As used herein, a "condition" is a disruption of or interference with normal function, and is not to be limited to any specific condition, and will include diseases or disorders. In an example, the condition is a CXCR4-CCR7 heteromultimer-mediated condition. A "CXCR4-CCR7 heteromultimer-mediated condition" is any condition that is caused by or associated with a cell expressing a CXCR4-CCR7 heteromultimer. The skilled artisan will be readily able to determine such conditions based on the disclosure herein and/or by performing an assay to detect a cell expressing a CXCR4- CCR7 heteromultimer as described herein. In this regard, in some examples the condition is selected from the group consisting of cancer, an inflammatory condition, an autoimmune condition, an obstructive condition, an infectious condition, an excitotoxic condition, an immunodeficiency condition, a metabolic condition, pain, a circulatory condition and a degenerative condition. In some example, the condition is caused by a cell selected from the group consisting of an immune cell, a tonsil cell, a thymus cell, an endothelial cell, an endothelial progenitor cell, a mesangial cell, an hepatic cell, a lung cell, a bone marrow CD34⁺ cell, a cord blood CD34⁺ cell, or a lymph node cell. In one example, the condition is selected from the group consisting of an inflammatory condition, an autoimmune condition, graft versus host disease, graft rejection, liver disease, liver cirrhosis, kidney disease, renal failure, neutropenia, a viral infection or hepatitis. A description of some additional conditions to be treated according to the present disclosure in included herein. Alternatively, or additionally, a condition is associated with a cancer cell or a metastatic cancer cell, for example the condition is cancer or metastasis.

As used herein, the term "mobilization of stem cells" shall be understood to mean causing a stem cell to enter the blood stream from another tissue in the body. For example, a hematopoietic stem cell can be "mobilized" from bone marrow into the blood stream.

As used herein, the term "normal individual" shall be taken to mean that the subject is selected on the basis that they do not have an increased or reduced number of cells expressing a CXCR4-CCR7 heteromultimer. For example, the number of cells expressing a CXCR4-CCR7 heteromultimer in a given tissue is determined in a population of subjects and a subject that falls within the range detected or the mean (+/- standard error or standard deviation or 2x standard deviation) is considered a normal individual.

A "healthy subject" is one that has not previously been diagnosed as suffering from the condition being diagnosed/prognosed and/or is not at risk of developing the condition being diagnosed/prognosed.

A "standard derived from samples from one or more normal and/or healthy subject(s)" shall be taken to mean data established by measuring the amount of a CXCR4-CCR7 heteromultimer or cells expressing the heteromultimer in one or more (or a population of) normal and/or healthy individuals. This term encompasses determining such data at the time of performing a diagnostic/prognostic assay or data determined at a previous point in time. The latter situation assists diagnostic/prognostic assays of the present since it means that it is not necessary to perform an assay with one or more control samples each time a diagnostic/prognostic assay is performed.

The term "expression construct" is to be taken in its broadest context and includes a nucleic acid comprising one or more promoter sequences operably linked with first and second nucleic acids as described herein.

The term "expression vector" refers to a nucleic acid comprising an expression construct that is additionally capable of maintaining and or replicating nucleic acid in an expressible format. For example, an expression vector may comprise a plasmid, bacteriophage, phagemid, cosmid, virus sub-genomic or genomic fragment. Selection of appropriate vectors is within the knowledge of those having skill in the art.

As used herein, the term "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid, e.g., in response to a developmental and/or external stimulus, or in a tissue specific manner. In the present context, the term "promoter" is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative which confers, activates or enhances the expression of a nucleic acid to which it is operably linked. Preferred promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter the spatial expression and/or temporal expression of said nucleic acid. As used herein, the term "operably linked to" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

A "subject" is an animal subject. Suitable subjects include, for example, mammalian subjects such as humans, primates, livestock animals such as horses, cattle, sheep, pigs, goats or the like, companion animals such as dogs or cats, laboratory test animals such as mice, rats, guinea pigs or birds, or animals of veterinary significance, or animals of economic significance. The subject may also include non-mammalian animal subjects such as birds including poultry birds such as chickens; reptilian subjects including companion reptiles such as turtles, tortoises and snakes; fish including wild-caught fish and fish in aquaculture. In one example, the subject is a human or non-human primate, for example, a human.

Heteromultimers and Cells Expressing Heteromultimers

The present disclosure provides an isolated or recombinant CXCR4-CCR7 heteromultimer. A recombinant heteromultimer can be in isolated form or can be in the context of a cell membrane (which can be isolated) or can be within a cell. In this regard, a recombinant heteromultimer can comprise one of CXCR4 or CCR7 which is expressed in recombinant form and the other of CXCR4 or CCR7 which is expressed in its native state in a cell. Alternatively, both CXCR4 and CCR7 can be expressed in recombinant form.

Methods for expressing a protein in recombinant form will be apparent to the skilled artisan. To facilitate the production of a recombinant protein, nucleic acid encoding same is isolated or synthesized. Typically, the nucleic acid encoding the protein is/are isolated using a known method, such as, for example, amplification (e.g., using PCR or splice overlap extension) or isolated from nucleic acid from an organism using one or more restriction enzymes or isolated from a library of nucleic acids. Methods for such isolation will be apparent to the ordinary skilled artisan and/or described in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987), Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).

For example, nucleic acid (e.g.., genomic DNA or RNA that is then reverse transcribed to form cDNA) from a cell or organism capable of expressing CXCR4 and/or CCR7 is isolated using a method known in the art and cloned into a suitable vector. The vector is then introduced into a suitable organism, such as, for example, a bacterial cell. Methods and nucleic acids for producing a CXCR4 polypeptide are described, for example, in US5840856. Methods and nucleic acids for producing a CCR7 polypeptide are described, for example, in W094/12519.

Other methods for the production of a nucleic acid of the invention will be apparent to the skilled artisan and are encompassed by the present invention.

Following production, a nucleic acid is inserted into a suitable expression construct such that it is operably linked to a promoter. For example, the nucleic acid is inserted to an expression vector.

Many vectors for expression in cells are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequences encoding a polypeptide(s), an enhancer element, a promoter, and a transcription termination sequence. The skilled artisan will be aware of suitable sequences for expression of a protein. For example, exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, a factor leader, or acid phosphatase leader) or mammalian secretion signals (e.g., herpes simplex gD signal).

Exemplary promoters active in mammalian cells include cytomegalovirus immediate early promoter (CMV-IE), human elongation factor 1-oc promoter (EF1), small nuclear RNA promoters (Ula and Ulb), oc-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, β-actin promoter; hybrid regulatory element comprising a CMV enhancer/ β- actin promoter or an immunoglobulin promoter or active fragment thereof. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO). Additional suitable cells for expressing a CXCR4-CCR7 heteromultimer are disclosed herein.

Typical promoters suitable for expression in yeast cells such as for example a yeast cell selected from the group comprising Pichia pastoris, Saccharomyces cerevisiae and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUPl promoter, the PH05 promoter, the nmt promoter, the RPR 1 promoter, or the TEF1 promoter.

Typical promoters suitable for expression in insect cells include, but are not limited to, the OPEI2 promoter, the insect actin promoter isolated from Bombyx muri, the Drosophila sp. dsh promoter and the inducible metallothionein promoter. Preferred insect cells for expression of recombinant proteins include an insect cell selected from the group comprising, BT1-TN-5B1-4 cells, and Spodoptera frugiperda cells (e.g., sfl9 cells, sf21 cells). Suitable insects for the expression of the nucleic acid fragments include but are not limited to Drosophila sp. The use of S. frugiperda is also contemplated.

Methods for a nucleic acid or an expression construct into a cell for expression are known to those skilled in the art. The method used for a given cell depends on the known successful techniques. Methods for introducing recombinant DNA into cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine (Gibco, MD, USA) and/or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation and microparticle bombardment such as by using DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA) amongst others.

The host cells used to produce the heteromultimer may be cultured in a variety of media, depending on the cell type used. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPM1-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing other cell types discussed herein are known in the art.

Fusion Proteins

The present disclosure additionally contemplates a heteromultimer in which one or both of CXCR4 and CCR7 is a fusion protein. For example, the protein is fused to a tag or label. In some examples, a tag or label facilitates purification or isolation or detection of the protein. Suitable tags will be apparent to the skilled artisan and include, for example, influenza virus hemagglutinin (HA), Simian Virus 5 (V5), polyhistidine (e.g., hexa-HIS), c-myc or FLAG.

In one example a label is a detectable tag. In one example, a detectable tag is a fluorescent protein. Examples of fluorescent proteins include, but are not limited to, BFP (blue fluorescent protein), CFP (cyan fluorescent protein), GFP (green fluorescent protein), YFP (yellow fluorescent protein), EBFP2 (enhanced blue fluorescent protein 2), Azurite, mTFPl (monomeric teal fluorescent protein 1), mECFP (monomeric enhanced cyan fluorescent protein), Cerulean, Emerald, EGFP (enhanced green fluorescent protein), T-Sapphire, mKO (monomeric Kusabira Orange) and mOrange (monomeric Orange). In another example, a detectable tag is a bioluminescent protein. Examples of bioluminescent proteins include, but are not limited to, a luciferase, a β-galactosidase, a lactamase, a horseradish peroxidase, an alkaline phophatase, a β -glucuronidase or a β - glucosidase.

Examples of luciferases include, but are not limited to, a Renilla luciferase, a

Firefly luciferase, a Coelenterate luciferase, a North American glow worm luciferase, a click beetle luciferase, a railroad worm luciferase, a bacterial luciferase, a Gaussia luciferase, Aequorin, a Arachnocampa luciferase, or a biologically active variant or fragment of any one, or chimera of two or more, thereof. An example of a biologically active variant of Renilla luciferase is RLuc8.

Isolation of Proteins

A heteromultimer produced as described herein can be isolated. Methods for isolating a protein of the invention are known in the art (e.g., and/or described in Scopes, 1994) and/or described herein.

For example, where the protein is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The protein prepared from the cells can be purified using, for example, hydroxyl apatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the protein to be recovered.

The skilled artisan will also be aware that a polypeptide/multimeric protein can be modified to include a tag to facilitate purification or detection, e.g., a poly-histidine tag, e.g., a hexa-histidine tag, or a influenza virus hemagglutinin (HA) tag, or a Simian Virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. For example a hexa-his tag containing protein is purified using methods known in the art, such as, by contacting a sample comprising the protein with nickel-nitrilotriacetic acid (Ni-NTA) that specifically binds a hexa-his tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein. Alternatively, or in addition a ligand or antibody that binds to a tag is used in an affinity purification method.

The present disclosure also encompasses an isolated cell membrane comprising a CXCR4-CCR7 heteromultimer of the present disclosure.

Binding Molecules

As will be apparent from the description herein, the present disclosure provides a binding molecule that binds specifically to a CXCR4-CCR7 heteromultimer.

In some examples, the binding molecule does not detectably bind to a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer.

In some examples, the binding molecule specifically binds to one or more of the following:

(i) the heterodimeric interface of CXCR4 and CCR7 heteromultimer;

(ii) an epitope formed in a CXCR4-CCR7 heteromultimer which is not present in a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer; or

(iii) an epitope exposed in the CXCR4-CCR7 heteromultimer which is not sufficiently exposed in a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer to permit binding of the molecule.

In the case of (ii) or (iii), either or both CXCR4 and/or CCR7 may undergo a conformational change in the heteromultimer thereby producing a conformational epitope not present in a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer (ii) or that exposes an epitope not exposed in a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer (iii).

In some examples, the CXCR4-CCR7 heteromultimer binding molecules may specifically bind to an epitope of the CXCR4-CCR7 heteromultimer. In some examples, the epitope may comprise portions of both the CXCR4 and CCR7 molecules. Such CXCR4-CCR7 heteromultimer binding molecules would typically have a low binding affinity, or substantially no binding affinity, to the CXCR4 and CCR7 monomers or homomultimers.

In one example, a binding molecule as described herein according to any example does not detectably bind to a heteromultimer other than a CXCR4-CCR7 heteromultimer. For example, the binding molecule does not bind to a heteromultimer comprising chemokine receptors other than CXCR4 or CCR7 and/or comprising CXCR4 or CCR7 but not both CXCR4 or CCR7 or a heteromultimer comprising both CXCR4 and CCR7, wherein the CXCR4 and CCR7 are not in contact with one another.

Antibodies

In one example, a binding molecule is an antibody or an antigen binding fragment thereof. Antibodies used as the CXCR4-CCR7 heteromultimer binding molecule may include, for example, monoclonal antibodies, polyclonal antibodies, multivalent antibodies, chimeric antibodies, multispecific antibodies, and antibody fragments that exhibit the desired binding specificity. Antibodies to the CXCR4-CCR7 heteromultimer may be obtained commercially or generated by methods known in the art. For example, antibodies may be prepared using methods generally disclosed by Howard and Kaser (2007) or Harlow and Lane (1988). Examples of the therapeutic use of antibodies and antigen binding fragments thereof are provided by Stockwin and Holmes (2003).

Hybridoma Preparation

Methods for producing antibodies by production of hybridomas are known in the art. Generally, in such methods CXCR4-CCR7 heteromultimer or immunogenic fragment or epitope thereof or a cell expressing and displaying same (i.e., an immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non-human animal, for example, a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig. The immunogen may be administered intranasally, intramuscularly, sub-cutaneous ly, intravenously, intradermally, intraperitoneally, or by other known route.

The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. One or more further immunizations may be given, if required to achieve a desired antibody titer. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal is bled and the serum isolated and stored, and/or the animal is used to generate monoclonal antibodies (mAbs).

Monoclonal antibodies are one exemplary form of antibody contemplated by the present disclosure. The term "monoclonal antibody" or "mAb" refers to a homogeneous antibody population capable of binding to the same antigen(s), for example, to the same epitope within the antigen. This term is not intended to be limited as regards to the source of the antibody or the manner in which it is made.

For the production of mAbs any one of a number of known techniques may be used, such as, for example, the procedure exemplified in US4196265 or Harlow and Lane (1988), supra.

For example, a suitable animal is immunized with an immunogen under conditions sufficient to stimulate antibody producing cells. Rodents such as rabbits, mice and rats are exemplary animals. Mice genetically-engineered to express human immunoglobulin proteins and, for example, do not express murine immunoglobulin proteins, can also be used to generate an antibody of the present disclosure (e.g., as described in WO2002/066630).

Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsies of spleens, tonsils or lymph nodes, or from a peripheral blood sample. The B cells from the immunized animal are then fused with cells of an immortal myeloma cell, generally derived from the same species as the animal that was immunized with the immunogen.

Hybrids are amplified by culture in a selective medium comprising an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary agents are aminopterin, methotrexate and azaserine.

The amplified hybridomas are subjected to a functional selection for antibody specificity and/or titer, such as, for example, by flow cytometry and/or immunohistochemstry and/or immunoassay (e.g. radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, and the like).

Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to produce cell lines secreting MAbs (e.g., as described in Largaespada et al, 1996).

Antibodies are also screened to identify/isolate an antibody capable specifically binding to a CXCR4-CCR7 heteromultimer, e.g., using a method as described herein.

Library Screening

In another example an antibody is identified by screening a library of antibody variable domain containing proteins.

Examples of this libraries contemplated by disclosure include naive libraries, immunized libraries or synthetic libraries. Naive libraries are derived from B- lymphocytes of a suitable host which has not been challenged with any immunogen, nor which is exhibiting symptoms of infection or inflammation. Immunized libraries are made from a mixture of B-cells and plasma cells obtained from a suitably "immunized" host, i.e., a host that has been challenged with an immunogen, such as a CXCR4-CCR7 heteromultimer or a cell expressing the heteromultimer. In one example, the mR A from these cells is translated into cDNA using methods known in the art (e.g., oligo-dT primers and reverse transcriptase). In an alternative example, nucleic acids encoding antibodies from the host cells (mRNA or genomic DNA) are amplified by PCR with suitable primers. Primers for such antibody gene amplifications are known in the art (e.g., US6096551 and/or WO00/70023). In a further example, the mRNA from the host cells is synthesized into cDNA and these cDNAs are then amplified in a PCR reaction with antibody specific primers (e.g., US6319690).

In another example, a database of published antibody sequences of human origin is established where the antibody sequences are aligned to each other. The database is used to define subgroups of antibody sequences which show a high degree of similarity in both the sequence and the canonical fold of CDR loops (as determined by analysis of antibody structures). For each of the subgroups a consensus sequence is deduced which represents the members of this subgroup; the complete collection of consensus sequences represent therefore the complete structural repertoire of human antibodies. These artificial genes are then constructed, e.g., by total gene synthesis or by the use of synthetic genetic subunits. These genetic subunits correspond to structural sub- elements at the polypeptide level. On the DNA level, these genetic subunits are defined by cleavage sites at the start and the end of each of the sub-elements, which are unique in the vector system. All genes which are members of the collection of consensus sequences are constructed such that they contain a similar pattern of corresponding genetic sub-sequences. For example, said polypeptides are or are derived from the HuCAL consensus genes: VKI, VK2, VK3, VK4, νλΐ, Υλ2, Υλ3, V_H1A, V_H1B, V_H2, VH3, VH4, VH5, VH6, CK, Ck, CR\ or any combination of said HuCAL consensus genes. This collection of DNA molecules can then be used to create "synthetic libraries" of antibodies or fragments thereof, e.g., Fv, disulphide-linked Fv, single-chain Fv (scFv), Fab fragments, or Fab' fragments which may be used as sources of proteins that bind specifically to an antigen. US6300064 discloses methods for making synthetic libraries.

In another example, synthetic human antibodies are made by synthesis from defined V-gene elements. Winter (EP0368684) has provided a method for amplifying (e.g., by PCR), cloning, and expressing antibody variable region genes. Starting with these genes he was able to create libraries of functional antibody fragments by randomizing the CDR3 of the heavy and/or the light chain. This process is functionally equivalent to the natural process of VJ and VDJ recombination which occurs during the development of B-cells in the immune system. For example, repertoires of human germ line VH gene segments can be rearranged in vitro by joining to synthetic "D-segments" of five random amino acid residues and a J-segment, to create a synthetic third complementarity determining region (CDR) of eight residues. US5885793 discloses methods of making such antibody libraries such as these.

The proteins according to the disclosure may be soluble secreted proteins or may be presented as a fusion protein on the surface of a cell, or particle (e.g., a phage or other virus, a ribosome or a spore).

Various display library formats are known in the art. For example, the library is an in vitro display library (i.e., the proteins are displayed using in vitro display wherein the expressed domain is linked to the nucleic acid from which it was expressed such that said domain is presented in the absence of a host cell). Accordingly, libraries produced by in vitro display technologies are not limited by transformation or transfection efficiencies. Examples of methods of in vitro display include ribosome display, covalent display and mRNA display.

In another example, the display library is a phage display library wherein the expressed proteins comprising a VH are displayed on the surface of a bacteriophage, as described, for example, in US5821047; US6248516 and/or US6190908. The basic principle described relates to the fusion of a first nucleic acid comprising a sequence encoding a protein comprising a VH to a second nucleic acid comprising a sequence encoding a phage coat protein, such as, for example a phage coat proteins selected from the group, M13 protein-3, M13 protein-7, or M13, protein-8. These sequences are then inserted into an appropriate vector, i.e., one that is able to replicate in bacterial cells. Suitable host cells, such as, for example E. coli, are then transformed with the recombinant vector. Said host cells are also infected with a helper phage particle encoding an unmodified form of the coat protein to which a nucleic acid fragment is operably linked. Transformed, infected host cells are cultured under conditions suitable for forming recombinant phagemid particles comprising more than one copy of the fusion protein on the surface of the particle. This system has been shown to be effective in the generation of virus particles such as, λ phage, T4 phage, Ml 3 phage, T7 phage and baculovirus. Such phage display particles are then screened to identify a displayed domain having a conformation sufficient for binding to a target antigen.

Other viral display libraries include a retroviral display library wherein the expressed peptides or protein domains are displayed on the surface of a retroviral particle, e.g., as described in US6297004 The present disclosure also contemplates bacterial display libraries, e.g., as described in US5516637; or yeast display libraries, e.g., as described in US6423538.

Methods for screening display libraries are known in the art. In one example, a display library of the present disclosure is screened using affinity purification. Affinity purification techniques are known in the art and are described in, for example, Scopes (1994). Methods of affinity purification typically involve contacting the proteins comprising a VH displayed by the library with a target antigen (e.g., a CXCR4-CCR7 heteromultimer or a cell expressing same) and, following washing, eluting those domains that remain bound to the antigen. The antigen is preferably bound to another molecule to allow for ease of purification, such as, for example, a molecule selected from the group consisting of protein G, Sepharose, agarose, biotin, glutathione S- transferase (GST), and FLAG epitope. Accordingly, the target protein or nucleic acid is isolated simply through centrifugation, or through binding to another molecule, e.g. streptavidin, or binding of a specific antibody, e.g. anti-FLAG antibodies, or anti-GST antibodies. Alternatively, the antigen is immobilized on a solid surface. Such affinity purification methods are also referred to as panning and, when the antibody domain is displayed on a phage the method can be referred to as phage panning.

In another example, the display library of the present disclosure is expressed so as to allow identification of a bound peptide using FACS analysis.

Alternatively the library is screened using a biosensor-based assay, such as, for example, Biacore sensor chip technology (Biacore AB, UK). The Biacore sensor chip is a glass surface coated with a thin layer of gold modified with carboxymethylated dextran, to which the target heteromultimer or cell expressing same is covalently attached. The libraries of the present disclosure are then exposed to the Biacore sensor chip comprising the antigen.

An antigen binding fragment identified as binding to the heteromultimer can then be expressed as a fusion with the relevant contact domains to produce a complete antibody.

Antibodies or antigen binding fragments thereof are also screened to identify/isolate an antibody capable specifically binding to a CXCR4-CCR7 heteromultimer, e.g., using a method as described herein.

Chimeric Antibodies

An antibody of the present disclosure may be a synthetic antibody, or may be modified to make it a synthetic antibody. In one example, an antibody described herein is a chimeric antibody. The term "chimeric antibody" refers to 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 (e.g., murine, such as mouse) 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 (e.g., primate, such as human) or belonging to another antibody class or subclass. Typically chimeric antibodies utilize rodent or rabbit variable regions and human constant regions, in order to produce an antibody with predominantly human domains. Methods for producing chimeric antibodies are described in, e.g., US4816567 and/or US5807715.

The term "constant region" (CR) as used herein, refers to the portion of the antibody molecule which confers effector functions. The heavy chain constant region can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu. Further, heavy chains of various subclasses (such as the IgG subclasses of heavy chains) are responsible for different effector functions and thus, by choosing the desired heavy chain constant region, antibodies with desired effector function can be produced. Preferred heavy chain constant regions are gamma 1 (IgGl), gamma 2 (IgG2), gamma 3 (IgG3) and gamma 4 (IgG4). Light chain constant regions can be of the kappa or lambda type, preferably of the kappa type.

As used herein, "variable region" refers to the portions of the light and/or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and, for example, includes amino acid sequences of CDRs; i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, the variable region comprises three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain. The amino acid positions assigned to CDRs and FRs can be defined according to Kabat (1987 and 1991, supra) or other numbering systems in the performance of methods according to the present disclosure, e.g., the hypervariable loop numbering system of Chothia and Lesk (1987 and/or 1989, and/or Al-Lazikani et ah, 1997). For example, according to the numbering system of Kabat, a VH FRS and CDRs positioned as follows residues 1-30 (FR1 ), 31-25 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3) and 103- 1 13 (FR4), numbered according to the Kabat numbering system. For example, according to the numbering system of Kabat, a V_L FRS and CDRs are positioned as follows residues 1-23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3) and 98-107 (FR4).

As used herein, the term "complementarity determining regions" (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat et ah, (1991) and/or those residues from a "hypervariable loop" Chothia and Lesk (1987 and/or 1989, and/or Al- Lazikani et ah, 1997) or other numbering system.

"Framework regions" (hereinafter FR) are those variable domain residues other than the CDR residues. Humanized and Human Antibodies

The antibodies of the present disclosure may be humanized or human.

The term "humanized antibody" shall be understood to refer to a subclass of chimeric antibodies having an antigen binding site or components of a variable region derived from an antibody from a non-human species and the remaining antibody structure of the molecule based upon the structure and/or sequence of a human antibody. The antigen-binding site comprises the CDRs from the non-human antibody grafted onto appropriate FRs in the variable domains of a human antibody and the remaining regions from a human antibody. Antigen binding sites may be wild type or modified by one or more amino acid substitutions. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues.

Methods for humanizing non-human antibodies or parts thereof (e.g., variable regions) are known in the art. Humanization can be performed following the method of US5225539, or US5585089. Other methods for humanizing an antibody are not excluded.

The term "human antibody" as used herein in connection with antibodies refers to antibodies having variable regions (e.g. VH, VL) and, optionally constant regions derived from or corresponding to sequences found in humans, e.g. in the human germline or somatic cells. The "human" antibodies can include amino acid residues not encoded by human sequences, e.g. mutations introduced by random or site directed mutations in vitro (in particular mutations which involve conservative substitutions or mutations in a small number of residues of the antibody, e.g. in 1, 2, 3, 4 or 5 of the residues of the antibody, e.g. in 1, 2, 3, 4 or 5 of the residues making up one or more of the CDRs of the antibody). These "human antibodies" do not actually need to be produced by a human, rather, they can be produced using recombinant means and/or isolated from a transgenic animal (e.g., mouse) comprising nucleic acid encoding human antibody constant and/or variable regions (e.g., as described above). Human antibodies can be produced using various techniques known in the art, including phage display libraries (e.g., as described herein).

Human antibodies which recognize a selected epitope can also be generated using a technique referred to as "guided selection." In this approach a selected non- human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope (e.g., as described in US5565332).

De-immunized Antibodies and Immunoglobulins

The present disclosure also contemplates a de-immunized antibody or protein.

De-immunized antibodies and proteins have one or more epitopes, e.g., B cell epitopes or T cell epitopes removed (i.e., mutated) to thereby reduce the likelihood that a mammal will raise an immune response against the antibody or protein. Methods for producing de-immunized antibodies and proteins are known in the art and described, for example, in WO00/343 17, WO2004/108158 and WO2004/064724.

Methods for introducing suitable mutations and expressing and assaying the resulting protein will be apparent to the skilled artisan based on the description herein.

Heavy Chain Antibodies

Heavy chain antibodies differ structurally from many other forms of antibodies, in so far as they comprise a heavy chain, but do not comprise a light chain. Accordingly, these antibodies are also referred to as "heavy chain only antibodies". Heavy chain immunoglobulins are found in, for example, camelids and cartilaginous fish (also called IgNAR).

The variable regions present in naturally occurring heavy chain antibodies are generally referred to as "VHH domains" in camelid antibodies and V-NAR in IgNAR, in order to distinguish them from the heavy chain variable regions that are present in conventional 4-chain antibodies (which are referred to as "VH domains") and from the light chain variable regions that are present in conventional 4-chain antibodies (which are referred to as "VL domains").

A general description of heavy chain antibodies from camelids and the variable regions thereof and methods for their production and/or isolation and/or use is found inter alia in the following references WO94/04678, WO97/49805 and WO 97/49805.

A general description of heavy chain immunoglobulins from cartilaginous fish and the variable regions thereof and methods for their production and/or isolation and/or use is found inter alia in WO2005/1 18629. Antibody Fragments

Single-Domain Antibodies

In some examples, an antigen binding fragment of an antibody of the disclosure is or comprises a single-domain antibody (which is used interchangeably with the term "domain antibody" or "dAb"). A single-domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable domain of an antibody. In certain examples, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., US6248516).

Diabodies, Triabodies, Tetrabodies

In some examples, an antigen binding fragment of an antibody of the disclosure is or comprises a diabody, triabody, tetrabody or higher order protein complex such as those described in W098/044001 and/or WO94/007921.

For example, a diabody is a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure VL-X-VH or VH-X-VL, wherein VL is an antibody light chain variable region, VH is an antibody heavy chain variable region, X is a linker comprising insufficient residues to permit the VH and VL in a single polypeptide chain to associate (or form an Fv) or is absent, and wherein the VH of one polypeptide chain binds to a VL of the other polypeptide chain to form an antigen binding site, i.e., to form a Fv molecule capable of specifically binding to one or more antigens. The VL and VH can be the same in each polypeptide chain or the VL and VH can be different in each polypeptide chain so as to form a bispecific diabody (i.e., comprising two Fvs having different specificity).

A diabody, triabody, tetrabody, etc capable of inducing effector activity can be produced using an antigen binding domain capable of binding to a CXCR4-CCR7 heteromultimer and an antigen binding domain capable of binding to a cell surface molecule on an immune cell, e.g., a T cell (e.g., CD3) or a NK cell (e.g., CD16). An exemplary diabody capable of inducing effector function is described in Arndt et ah, 1999 and is readily adaptable to the present disclosure.

Single Chain Fv (scFv) Fragments

In one example, the present disclosure provides a scFv that specifically binds to a CXCR4-CCR7 heteromultimer. The skilled artisan will be aware that scFvs comprise VH and VL regions in a single polypeptide chain and a polypeptide linker between the VH and VL which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv). For example, the linker comprises in excess of 12 amino acid residues with (Gly₄Ser)3 being one of the more favored linkers for a scFv.

The present disclosure also contemplates a disulfide stabilized Fv (or diFv or dsFv), in which a single cysteine residue is introduced into a FR of VH and a FR of VL and the cysteine residues linked by a disulfide bond to yield a stable Fv.

Alternatively, or in addition, the present disclosure encompasses a dimeric scFv, i.e., a protein comprising two scFv molecules linked by a non-covalent or covalent linkage, e.g., by a leucine zipper domain (e.g., derived from Fos or Jun). Alternatively, two scFvs are linked by a peptide linker of sufficient length to permit both scFvs to form and to bind to an antigen, e.g., as described in US20060263367.

The present disclosure also contemplates a dimeric scFv capable of inducing effector activity. For example, one scFv binds to a CXCR4-CCR7 heteromultimer and another scFv binds to a cell surface molecule on an immune cell, e.g., a T cell (e.g., CD3) or a NK cell (e.g., CD16). In one example, the dimeric protein is a combination of a dAb and a scFv. Examples of bispecific antibody fragments capable of inducing effector function are described, for example, in US7235641.

Other Antibodies and Antibody Fragments

The present disclosure also contemplates other antibodies and antibody fragments, such as:

(i) "key and hole" bispecific proteins as described in US5731 168;

(ii) heteroconjugate proteins, e.g., as described in US4676980;

(iii) heteroconjugate proteins produced using a chemical cross-linker, e.g., as described in US4676980; and

(iv) Fab₃ (e.g., as described in EP19930302894).

Fc Regions

The present disclosure encompasses antibodies and/or antigen binding domain fragments comprising or fused to a Fc region of an antibody.

Sequences of Fc regions useful for producing the binding molecules of the present disclosure may be obtained from a number of different sources. In some examples, the Fc or portion thereof of the protein is derived from a human antibody. Moreover, the Fc or portion thereof may be derived from any antibody class, including IgM, IgG, IgD, IgA and IgE, and any antibody isotype, including IgGl, IgG2, IgG3 and IgG4. In one example, the Fc is human isotype IgGl or human isotype IgG2 or human isotype IgG3 or a hybrid of any of the foregoing.

In one example, the Fc region is capable of inducing an effector function. For example, the Fc region is a human IgGl or IgG3 Fc region. In another example, the Fc region is a hybrid of an IgGl and an IgG2 Fc region or a hybrid of an IgGl and an IgG3 Fc region or a hybrid of an IgG2 and an IgG3 Fc region. Exemplary hybrids of human IgGl and IgG2 Fc regions are described in Chappel et ah, 1991.

Methods for determining whether or not a Fc region can induce effector function will be apparent to the skilled artisan and/or described herein.

Effector Function

Suitably, an antibody or antigen binding fragment thereof of the present disclosure has or displays an effector function that facilitates or enables at least partial depletion, substantial depletion or elimination of cells expressing a CXCR4-CCR7 heteromultimer. Such an effector function may be enhanced binding affinity to Fc receptors, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell mediated phagocytosis (ADCP) and/or complement dependent cytotoxicity (CDC).

As will be apparent to the skilled artisan based on the description herein, some examples of the present disclosure make use of an antibody or antigen binding fragment thereof capable of inducing effector function.

In one example, the antibody or antigen binding fragment thereof binds to CXCR4-CCR7 heteromultimer in such a manner that it is capable of inducing an effector function, such as, ADCC or CDC.

In one example, the antibody or antigen binding fragment thereof binds to an epitope within the CXCR4-CCR7 heteromultimer that permits it to induce an effector function, such as ADCC and/or CDC.

For example, the antibody or antigen binding fragment thereof remains bound to the heteromultimer on the surface of the cell for a time sufficient to induce an effector function, such as ADCC and/or CDC. For example, the immunoglobulin is not internalized too quickly to permit ADCC and/or CDC to be induced.

Alternatively, or in addition, the antibody or antigen binding fragment thereof is bound to the heteromultimer on the surface of the cell in a manner permitting an immune effector cell to bind to a Fc region in the antibody or antigen binding fragment thereof and induce an effector function, such as ADCC. For example, the Fc region of the antibody or antigen binding fragment thereof is exposed in such a manner when the antibody or antigen binding fragment thereof is bound to the heteromultimer that is capable of interacting with a Fc receptor (e.g., a FcyR) on an immune effector cell. In the context of the present disclosure, the term "immune effector cell" shall be understood to mean any cell that expresses a Fc receptor and that is capable of killing a cell to which it is bound by ADCC or ADCP, e.g., a NK cell or a T cell.

Each of the above paragraphs relating to effector functions of an antibody or antigen binding fragment thereof shall be taken to apply mutatis mutandis to inducing CDC. For example, the antibody or antigen binding fragment thereof is bound to the heteromultimer on the surface of the cell in a manner permitting complement component Clq to bind to a Fc region in the immunoglobulin and induce CDC.

In one example, the antibody or antigen binding fragment thereof is capable of inducing an enhanced level of effector function.

In one example, the level of effector function induced by the Fc region is enhanced relative to a wild-type Fc region of an IgGl antibody or a wild-type Fc region of an IgG3 antibody.

In another example, the Fc region is modified to increase the level of effector function it is capable of inducing compared to the Fc region without the modification. Such modifications can be at the amino acid level and/or the secondary structural level and/or the tertiary structural level and/or to the glycosylation of the Fc region.

The skilled addressee will appreciate that greater effector function may be manifested in any of a number of ways, for example as a greater level of effect, a more sustained effect or a faster rate of effect.

In one example, the Fc region comprises one or more amino acid modifications that increase its ability to induce enhanced effector function. In one example, the Fc region binds with greater affinity to one or more FcyRs. In one example, the Fc region has an affinity for an FcyR that is more than 1-fold greater than that of a wild-type Fc region or more than 5 -fold greater than that of a wild-type Fc region or between 5 -fold and 300-fold greater than that of a wild-type Fc region. In one example, the Fc region comprises at least one amino acid substitution at a position selected from the group consisting of: 230, 233, 234, 235, 239, 240, 243, 264, 266, 272, 274, 275, 276, 278, 302, 318, 324, 325, 326, 328, 330, 332, and 335, numbered according to the EU index of Kabat. In another example, the Fc region binds to FcyRIIIa more efficiently than to FcyRIIb. For example, the Fc region comprises at least one amino acid substitution at a position selected from the group consisting of: 234, 235, 239, 240, 264, 296, 330, and 1332, numbered according to the EU index of Kabat. In one example, the Fc region comprises at least one amino acid substitution selected from the group consisting of: L234Y, L234I, L235I, S239D, S239E, S239N, S239Q, V240A, V240M, V264I, V264Y, Y296Q, A330L, A330Y, A330I, I332D, and I332E, numbered according to the EU index of Kabat. In a further example, the Fc region induces ADCC at a level greater than that mediated by a wild-type Fc region. For example, the Fc region induces ADCC at a level that is more than 5-fold or between 5-fold and 1000-fold greater than that induced by a wild-type Fc region. In one example, the Fc region comprise at least one amino acid substitution at a position selected from the group consisting of: 230, 233, 234, 235, 239, 240, 243, 264, 266, 272, 274, 275, 276, 278, 302, 318, 324, 325, 326, 328, 330, 332, and 335, numbered according to the EU index of Kabat. In one example, the Fc region comprises the following amino acid substitutions S239D/I332E, numbered according to the EU index of Kabat. This Fc region has about 14 fold increase in affinity for FcyRIIIa compared to a wild-type Fc region and about 3.3 increased ability to induce ADCC compared to a wild-type Fc region.

In one example, the Fc region comprises the following amino acid substitutions S239D/A330L/I332E, numbered according to the EU index of Kabat. This Fc region has about 138 fold increase in affinity for FcyRIIIa compared to a wild-type Fc region and about 323 increased ability to induce ADCC compared to a wild-type Fc region.

Additional amino acid substitutions that increase ability of a Fc region to induce effector function are known in the art and/or described, for example, in US6737056 or US7317091.

In one example, the glycosylation of the Fc region is altered to increase its ability to induce enhanced effector function. In this regard, native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some examples, Fc regions according to the present disclosure comprise a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region, i.e., the Fc region is "afucosylated". Such variants may have an improved ability to induce ADCC. Methods for producing afucosylated antibodies include, expressing the antibody or antigen binding fragment thereof in a cell line incapable of expressing a-l,6-fucosyltransferase (FUT8) (e.g., as described in Yumane-Ohnuki et ah, 2004), expressing the antibody or antigen binding fragment thereof in cells expressing a small interfering RNA against FUT8 (e.g., as described in Mori et ah, 2004), expressing the antibody or antigen binding fragment thereof in cells incapable of expressing guanosine diphosphate (GDP)-mannose 4,6-dehydratase (GMD) (e.g., as described in Kanda et ah, 2007). The present disclosure also contemplates the use of antibody or antigen binding fragment thereof having a reduced level of fucosylation, e.g., produced using a cell line modified to express β— (l,4)-N-acetylglucosaminyltransferase III (GnT-III) (e.g., as described in Umana e/ a/., 1999).

Other methods include the use of cell lines which inherently produce antibodies capable of inducing enhanced Fc-mediated effector function (e.g. duck embryonic derived stem cells for the production of viral vaccines, WO2008/129058; Recombinant protein production in avian EBX® cells, WO 2008/142124).

Antibodies or antigen binding fragments thereof useful in the methods of the present disclosure also include those with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region is bisected by GlcNAc. Such immunoglobulins may have reduced fucosylation and/or improved ADCC function. Examples of such antibody or antigen binding fragment thereof are described, e.g., in US6602684 and US20050123546.

Antibodies or antigen binding fragments thereof with at least one galactose residue in the oligosaccharide attached to the Fc region are also contemplated. Such immunoglobulins may have improved CDC function. Such immunoglobulins are described, e.g., in WO1997/30087 and W01999/22764.

Antibodies or antigen binding fragments thereof can also comprise a Fc region capable of inducing enhanced levels of CDC. For example, hybrids of IgGl and IgG3 produce antibodies having enhanced CDC activity (Natsume et ah, 2008).

Methods for determining the ability of an antibody or antigen binding fragment thereof to induce effector function and known in the art and/or described herein. Additional Modifications

The present disclosure also contemplates additional modifications to an antibody or antigen binding fragment thereof.

For example, the immunoglobulin comprises one or more amino acid substitutions that increase the half-life of the antibody or antigen binding fragment thereof. For example, the antibody or antigen binding fragment thereof comprises a Fc region comprising one or more amino acid substitutions that increase the affinity of the Fc region for the neonatal Fc region (FcRn). For example, the Fc region has increased affinity for FcRn at lower pH, e.g., about pH 6.0, to facilitate Fc/FcRn binding in an endosome. In one example, the Fc region has increased affinity for FcRn at about pH 6 compared to its affinity at about pH 7.4, which facilitates the re -release of Fc into blood following cellular recycling. These amino acid substitutions are useful for extending the half life of an immunoglobulin, by reducing clearance from the blood.

Exemplary amino acid substitutions include T250Q and/or M428L according to the EU numbering system of Kabat. Additional or alternative amino acid substitutions are described, for example, in US20070135620.

Non-Antibody Binding Proteins

Binding molecules contemplated by the present disclosure include, for example a domain which is a derivative of a scaffold selected from the group consisting of CTLA-4 (Evibody); lipocalin; Protein A derived molecules such as Z-domain of

Protein A (Affibody, SpA), A-domain (Avimer/Maxibody); Heat shock proteins such as GroEl and GroES; transferrin (trans-body); ankyrin repeat protein (DARPin); peptide aptamer; C-type lectin domain (Tetranectin); human γ-crystallin and human ubiquitin (affilins); PDZ domains; scorpion toxin kunitz type domains of human protease inhibitors; and fibronectin (adnectin); which has been subjected to protein engineering in order to obtain binding to a ligand other than its natural ligand.

CTLA-4 (Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-family receptor expressed on mainly CD4⁺ T-cells. Its extracellular domain has a variable domain- like Ig fold. Loops corresponding to CDRs of antibodies can be substituted with heterologous sequence to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as Evibodies. For further details see van den Beucken et ah, 2001.

Lipocalins are a family of extracellular proteins which transport small hydrophobic molecules such as steroids, bilins, retinoids and lipids. They have a rigid β-sheet secondary structure with a number of loops at the open end of the conical structure which can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size, and are derived from lipocalins. For further details see US7250297 and/or US20070224633.

An affibody is a scaffold derived from Protein A of Staphylococcus aureus which can be engineered to bind to antigen. The domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details see EP 1641818.

A transferrin is a monomeric serum transport glycoprotein. Transferrins can be engineered to bind different target antigens by insertion of peptide sequences in a permissive surface loop. Examples of engineered transferrin scaffolds include the

Trans-body. For further details see Sturt et ah, 1999. Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33 residue motif consisting of two a-helices and a β-turn. They can be engineered to bind different target antigens by randomizing residues in the first a-helix and a β-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details see US20040132028.

Fibronectin is a scaffold which can be engineered to bind to antigen. Adnectins consists of a backbone of the natural amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the β- sandwich can be engineered to enable an Adnectin to specifically recognize a therapeutic target of interest. For further details see US6818418.

Peptide aptamers are combinatorial recognition molecules that comprise a constant scaffold protein, typically thioredoxin (TrxA) which contains a constrained variable peptide loop inserted at the active site.

Other binding molecules include proteins which have been used as a scaffold to engineer different target antigen binding properties include human γ-crystallin and human ubiquitin (affilins), kunitz type domains of human protease inhibitors, PDZ- domains of the Ras-binding protein AF-6, scorpion toxins (charybdotoxin), C-type lectin domain (tetranectins) are reviewed in Dubel, 2007.

Optionally, any of the foregoing binding molecules are linked to a Fc region, e.g., as described herein. This linkage may extend half life or confer or improve effector function. Peptides or Polypeptides

In one example, a binding molecule is a peptide, e.g., isolated from a random peptide library. To identify a suitable peptide, a random peptide library is generated and screened as described in US5,733,731, US5,591,646 and US5,834,318. Generally, such libraries are generated from short random oligonucleotides that are expressed either in vitro or in vivo and displayed in such a way to facilitate screening of the library to identify a peptide that, is capable of specifically binding to a heteromultimer. Methods of display include, phage display, retroviral display, bacterial surface display, bacterial flagellar display, bacterial spore display, yeast surface display, mammalian surface display, and methods of in vitro display including, mRNA display, ribosome display and covalent display. A peptide that is capable of binding a heteromultimer is identified by any of a number of methods known in the art, such as, for example, standard affinity purification methods as described, for example in Scopes, 1994) purification using FACS analysis as described in US645563.

Small Molecules

In another example, a binding molecule is a small molecule. Such a small molecule may be isolated from a library. Chemical small molecule libraries are available commercially or alternatively may be generated using methods known in the art, such as, for example, those described in US5,463,564.

Techniques for synthesizing small organic compounds will vary considerably depending upon the compound, however such methods will be well known to those skilled in the art.

In one example, informatics is used to select suitable chemical building blocks from known compounds, for producing a combinatorial library. For example, QSAR (Quantitative Structure Activity Relationship) modeling approach uses linear regressions or regression trees of compound structures to determine suitability. The software of the Chemical Computing Group, Inc. (Montreal, Canada) uses high- throughput screening experimental data on active as well as inactive compounds, to create a probabilistic QSAR model, which is subsequently used to select lead compounds. The Binary QSAR method is based upon three characteristic properties of compounds that form a "descriptor" of the likelihood that a particular compound will or will not perform a required function: partial charge, molar refractivity (bonding interactions), and logP (lipophilicity of molecule). Each atom has a surface area in the molecule and it has these three properties associated with it. All atoms of a compound having a partial charge in a certain range are determined and the surface areas (Van der Walls Surface Area descriptor) are summed. The binary QSAR models are then used to make activity models or ADMET models, which are used to build a combinatorial library. Accordingly, lead compounds identified in initial screens, can be used to expand the list of compounds being screened to thereby identify highly active compounds.

Nucleic Acid Aptamers

In another example, a binding molecule is a nucleic acid aptamer (adaptable oligomer). A nucleic acid aptamer is a nucleic acid that is capable of forming a secondary and/or tertiary structure that provides the ability to bind to a molecular target, e.g., a heteromultimer. An aptamer library is produced, for example, by cloning random oligonucleotides into a vector (or an expression vector in the case of an RNA aptamer), wherein the random sequence is flanked by known sequences that provide the site of binding for PCR primers. An aptamer that provides the desired biological activity (e.g., binds specifically to a CXCR4-CCR7 heteromultimer) is selected. Aptamers to the CXCR4-CCR7 heteromultimer may be prepared using methods generally disclosed by Mascini (2009). An aptamer with increased activity is selected, for example, using SELEX (Sytematic Evolution of Ligands by Exponential enrichment). Suitable methods for producing and/or screening an aptamer library are described, for example, in Elloington and Szostak, 1990.

Detecting Binding

As described herein, binding molecules bind specifically to a CXCR4-CCR7 heteromultimer. Some binding molecules do not detectably bind a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer.

Methods for determining the specificity of binding of a binding molecule will be apparent to the skilled artisan.

In one example, the method comprises:

(i) contacting a binding molecule or library thereof with a CXCR4-CCR7 heteromultimer or a cell expressing a CXCR4-CCR7 heteromultimer and identifying and/or isolating binding molecule(s) that bind to the CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer; and

(ii) contacting the identified/isolated binding molecules with a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer or a cell expressing same and identifying and/or isolating a binding molecule that does not detectably bind a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer or a cell expressing same.

Optionally, the method additionally comprises providing or producing the binding molecule.

In another example, the method comprises:

(i) contacting a binding molecule or library thereof with a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer or a cell expressing same and identifying and/or isolating a binding molecule that does not detectably bind a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer or a cell expressing same; and

(ii) contacting the identified/isolated binding molecules with a CXCR4-CCR7 heteromultimer or a cell expressing a CXCR4-CCR7 heteromultimer and identifying and/or isolating binding molecule(s) that bind to the CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer.

In the case of a specific binding molecule, a low level of binding to a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer may be permitted. For example, the binding molecule may bind at a level no greater than 20% above background levels.

Methods for determining binding will be apparent to the skilled artisan and/or described herein.

For example, a display technology (e.g., phage display) is used and phage capable of binding a heteromultimer and/or that do not detectable or significantly bind a homomultimer or a monomer selected using standard phage panning.

In another example a binding molecule is contacted to an immobilized CXCR4- CCR7 heteromultimer or a membrane comprising same or a cell expressing same. Following washing to remove any unbound or non-specifically bound molecules, binding molecules capable of binding to the heteromultimer are selected and/or identified. Optionally, the molecule is labeled (e.g., with a detectable tag, such as a fluorescent label) to facilitate detection. The identified/isolated binding molecules are then contacted with immobilized CXCR4 or CCR7 monomer or homomultimer or membrane comprising same or cell expressing same. Binding molecules that do not bind are then identified/isolated. Processes useful for detecting binding include, enzyme linked immunosorbent assays (ELISAs), fluorescence linked immunosorbent assays (FLISAs), FACS, or Biacore analysis. These methods are known in the art and/or described herein.

In some examples, the method additionally comprises determining the level of background binding. In the case of an antibody, this may be assessed using an isotype control antibody (e.g., an antibody of the same isotype as that being tested however that specifically binds to a different antigen), serum or immunoglobulin from a non- immunized mammal and/or a mammal that has not been immunized with a CXCR4- CCR7 heteromultimer and that does not suffer from a condition associated with or mediated by a CXCR4-CCR7 heteromultimer. These controls permit the determination of the level of cross reactivity or non-specific binding observed with antibodies. In the case of non-antibody molecules, a molecule that specifically binds to a different antigen may be used, for example a molecule having a related structure. For example, in the case of a peptide, a peptide comprising a randomized version of the sequence may be used. In some examples, the method comprises identifying binding molecules that bind to CXCR4 and CCR7 monomer or homomultimer at a level no greater than 10% or 9% or 8% or 7% or 6% or 5% or 4% or 3% or 2% or 1% background.

Detecting Effect on Activity

In some examples, the binding molecule reduces or prevents CXCR-CCR7 heteromultimer activity.

In some examples, the binding molecule does not modulate (or does not detectably modulate) CXCR4-CCR7 heteromultimer activity. By "does not detectably modulate" shall be understood to mean that a binding molecule, e.g., an antibody, does not increase or reduce heteromultimer activity at a level significantly greater than background, e.g., less than 10%, or 8% or 6% or 5% above background.

Methods for determining activity of a chemokine receptor include calcium release assays, phosphotidyl inositol hydrolysis, actin polymerization, adenylate cyclase inhibition, reporter gene assays and/or chemotaxis assays.

Calcium release assays are determined using a calcium sensitive dye, such as Fluo 3, Fluo 4 and Fura-2. The dye is contacted to a cell expressing a CXCR4-CCR7 heteromultimer in the presence or absence of a binding molecule. Optionally, the cell is pretreated with a chelator, e.g., EGTA, to remove intracellular calcium. An increase in the level of fluorescence indicates that the molecule activates CXCR4-CCR7 heteromultimer activity. The assay can also be performed in the presence of a ligand that activates the heteromultimer. In this regard, reference herein to "ligand" for the CXCR4-CCR7 heteromultimer includes any molecule that is able to bind to, and optionally activate, the heteromultimer. Ligands for the CXCR4-CCR7 heteromultimer may include native ligands for each of the monomers (e.g. the ligand may comprise CXCL12, CCL19, and/or CCL21) or ligands not normally attributed to the monomers. In some examples, the ligand may be a ligand that binds to the heteromultimer but not the monomers or homomultimers of either CXCR4 or CCR7. Exemplary ligands are SDF-1, CCL19 and/or CCL21. A binding molecule that reduces fluorescence in the presence of a ligand (compared to a control in which no binding molecule is added) inhibits or reduces activity of the heteromultimer. A binding molecule that does not enhance fluorescence on its own and that does not inhibit fluorescence in the presence of a ligand of the heteromultimer is considered not to modulate activity of the heteromultimer. Phosphotidyl inositol hydrolysis assays can involve culturing cells expressing a CXCR4-CCR7 heteromultimer with myo-[2-³H] inositol in inositol-free medium. After a time sufficient for the labelled inositol to be incorporated, cells are washed and cultured in the presence or absence of a binding molecule and/or a ligand that activates the heteromultimer in inositol free medium. Cells are then lysed and supernatant fractions assayed for inositol phosphates by detecting β-emmissions. A binding molecule that enhances incorporation of labelled inositol in the absence of a ligand that activates the heteromultimer (compared to a control with no binding molecule) enhances activity of the heteromultimer. A binding molecule that reduces incorporation of labelled inositol in the presence of a ligand that activates the heteromultimer (compared to a control in which no binding molecule is added) inhibits or reduces activity of the heteromultimer. A binding molecule that does not enhance incorporation of labelled inositol on its own and that does not inhibit incorporation in the presence of a ligand that activates the heteromultimer is considered not to modulate activity of the heteromultimer.

Actin polymerization is assessed by culturing cells expressing the heteromultimer with the binding molecule and labelled phalloidin (e.g., as available from Molecular Probes, CA, USA). Cells are then assessed for fluorescence, e.g., by immunofluorescence or FACS. A binding molecule that enhances incorporation of labelled phalloidin in the absence of a ligand that activates the heteromultimer (compared to a control with no binding molecule) enhances activity of the heteromultimer. A binding molecule that reduces incorporation of labelled phalloidin in the presence of a ligand that activates the heteromultimer (compared to a control in which no binding molecule is added) inhibits or reduces activity of the heteromultimer. A binding molecule that does not enhance incorporation of labelled phalloidin on its own and that does not inhibit incorporation in the presence of a ligand that activates the heteromultimer is considered not to modulate activity of the heteromultimer. An exemplary actin polymerization assay is described in Airoldi etal, 2006.

Adenylate cyclase activity is detected by detecting cAMP levels. Numerous kits are commercially available for detecting cAMP levels, e.g., from Molecular Devices, Inc. or CisBio, Inc.

Reporter gene assays for detecting activity of GPCRs, including chemokine receptors are also commercially available, e.g., from Promega Corporation. Several of these assays are described in Cheng et ah, 2009 or Kotarski et ah, 2006.

Chemotaxis assays can also be used to assess the effect of a binding molecule on a CXCR4-CCR7 heteromultimer. These assays are based on the functional migration of cells in vitro or in vivo induced by a compound (chemoattractant). Chemotaxis can be assessed by any suitable means, for example, in an assay utilizing a 96-well chemotaxis plate, or using other art-recognized methods for assessing chemotaxis. For example, the use of an in vitro transendothelial chemotaxis assay is described in WO 94/20142. Cells expressing the CXCR4-CCR7 heteromultimer are used in assays to assess chemotaxis toward a suitable ligand, e.g., SDF-1 and/or CCL19 and/or CCL21.

Generally, chemotaxis assays monitor the directional movement or migration of a suitable cell into or through a barrier (e.g., endothelium, a filter), toward increased levels of a compound, from a first surface of the barrier toward an opposite second surface. Membranes or filters provide convenient barriers, such that the directional movement or migration of a suitable cell into or through a filter, toward increased levels of a compound, from a first surface of the filter toward an opposite second surface of the filter, is monitored. In some assays, the membrane is coated with a substance to facilitate adhesion, such as ICAM-1, fibronectin or collagen. Such assays provide an in vitro approximation of cell "homing".

For example, one can detect or measure inhibition of the migration of cells expressing the heteromultimer in a suitable container, from a first chamber into or through a microporous membrane into a second chamber which contains a suitable ligand (e.g., SDF-1 and/or CCL19 and/or CCL21) and binding molecule to be tested, and which is divided from the first chamber by the membrane. To assess migration and inhibition of migration, the distance of migration into the filter, the number of cells crossing the filter that remain adherent to the second surface of the filter, and/or the number of cells that accumulate in the second chamber can be determined using standard techniques (e.g., microscopy and flow cytometry). In one example, the cells are labeled with a detectable tag (e.g., radioisotope, fluorescent label, antigen or epitope label), and migration can be assessed in the presence and absence of the binding molecule by determining the presence of the label adherent to the membrane and/or present in the second chamber using an appropriate method (e.g., by detecting radioactivity, fluorescence, immunoassay). The extent of migration can be determined relative to a suitable control (e.g., compared to background migration determined in the absence of the binding molecule, compared to the extent of migration induced by a second compound (i.e., a standard), compared with migration of untransfected cells in the presence of the binding molecule).

A binding molecule that enhances migration/chemotaxis in the absence of a ligand that activates the heteromultimer (compared to a control with no binding molecule) enhances activity of the heteromultimer. A binding molecule that reduces migration/chemotaxis in the presence of a ligand that activates the heteromultimer (compared to a control in which no binding molecule is added) inhibits or reduces activity of the heteromultimer. A binding molecule that does not enhance migration/chemotaxis on its own and that does not inhibit migration/chemotaxis in the presence of a ligand that activates the heteromultimer is considered not to modulate activity of the heteromultimer.

Other assays for detecting heteromultimer activity include, detection of ERK activation (e.g., as described in Belcheva et ah, 2002), Racl and/or Cdc42 activation (e.g., as described in Dadke et ah, 2003), and phosphotyrosine phospohorylation (e.g., as described in Hu et ah, 1999).

The inventors have also shown that inhibiting the CXCR4-CCR7 heteromultimer increases anoikis, i.e., cell death resulting from cells detaching from an extracellular matrix. Thus, activity of a CXCR4-CCR7 heteromultimer may be detecting by measuring cell death rates (in the case of a binding molecule having effector function, this may be measured in the absence of immune effector cells and/or complement components). For example, cells expressing a CXCR4-CCR7 heteromultimer are cultured on an extracellular matrix in the presence or absence of a binding molecule. The level of cell death is then assessed, e.g., by ethidium bromide fluorescence or a terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end-labeling (TU EL) assay or a caspase activation assay. A binding molecule that reduces cell death is considered to activate the CXCR4-CCR7 heteromultimer. A binding molecule that increases cell death is considered to inhibit or reduce activity of the heteromultimer. A binding molecule that does not modulate cell death is considered not to modulate activity of the heteromultimer.

Conjugates

The present disclosure also provides conjugates of binding molecules described herein. Examples of compounds to which a binding molecule can be conjugated are selected from the group consisting of a radioisotope, a detectable tag, a therapeutic compound, a colloid, a toxin, a nucleic acid, a peptide, a protein, a compound that increases the half life of the protein in a subject and mixtures thereof. Exemplary therapeutic agents include, but are not limited to an anti-angiogenic agent, an anti- neovascularization and/or other vascularization agent, an anti-proliferative agent, a pro- apoptotic agent, a chemotherapeutic agent or a therapeutic nucleic acid.

A toxin includes any agent that is detrimental to (e.g., kills) cells. For a description of these classes of drugs which are known in the art, and their mechanisms of action, see Goodman et ah, (1990). Additional techniques relevant to the preparation of immunotoxin conjugates are provided in for instance US5194594. Exemplary toxins 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. See, for example, W093/21232.

Suitable chemotherapeutic agents for forming immunoconjugates of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-de-hydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BS U), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin), antibiotics (such as dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)).

In the case of a binding molecule that is conjugated to a toxic compound (e.g., a toxin and/or a chemotherapeutic agent), a molecule that is internalized following binding to a CXCR4-CCR7 heteromultimer can be selected. Such binding molecules provide an advantage because they can reduce the effect of the toxic compound on cells/tissues surrounding the cell expressing a heteromultimer. Molecules that are internalized also permit use of compounds that are toxic only when within a cell, such as, a maytansinoid, e.g., DM1 from Immunogen, Inc. Methods for detecting whether or not a binding molecule is internalized are known in the art and described in, for example, Kuo et al, 2009.

In one example, a binding molecule is conjugated or linked to another protein, including another binding molecule. Other proteins are not excluded. Additional proteins will be apparent to the skilled artisan and include, for example, an immunomodulator or a half-life extending protein or a peptide or other protein that binds to serum albumin amongst others. Exemplary serum albumin binding peptides or protein are described in US20060228364 or US20080260757.

A variety of radionuclides are available for the production of radioconjugated proteins. Examples include, but are not limited to, low energy radioactive nuclei (e.g., suitable for diagnostic purposes), such as ¹³C, ¹⁴C _, ¹⁵N, ¾, ¹²⁵I, ¹²³I, "Tc, ⁴³K, ⁵²Fe, ⁶⁷Ga, ⁶⁸Ga, ¹¹ 'in and the like. In one example, the radionuclide is a gamma, photon, or positron-emitting radionuclide with a half-life suitable to permit activity or detection after the elapsed time between administration and localization to an imaging site. The present disclosure also encompasses high energy radioactive nuclei (e.g., for therapeutic purposes), such as ¹²⁵I, ¹³¹I, ¹²³I, ^mIn, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re and ¹⁸⁸Re. These isotopes typically produce high energy a- or β-particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells and are essentially non- immunogenic. Alternatively, high-energy isotopes may be generated by thermal irradiation of an otherwise stable isotope, for example as in boron neutron -capture therapy. Additional radioisotopes and methods for conjugating radioactive isotopes to molecules such as proteins are known in the art and include methods discussed by Slater (2002) Radioisotopes in Biology: A Practical Approach, Oxford University Press, 2002). Radioisotopes may be detected using suitable detectors such as gamma, beta or scintillation counters.

In another example, the protein is conjugated to a "receptor" (such as streptavidin) for utilization in cell pretargeting wherein the conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) that is conjugated to a therapeutic agent (e.g., a radionucleotide).

The binding molecules of the present disclosure can be modified to contain additional nonproteinaceous moieties that are known in the art and readily available. For example, the moieties suitable for derivatization of the protein are physiologically acceptable polymer, for example a water soluble polymer. Such polymers are useful for increasing stability and/or reducing clearance (e.g., by the kidney) and/or for reducing immunogenicity of a protein of the invention. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or propropylene glycol (PPG).

In one example, a binding molecule comprises one or more detectable tags or detectable tags to facilitate detection and/or isolation. In some examples, the detectable tag may be applied to the CXCR4-CCR7 heteromultimer binding molecule. For example, the detectable tag may be bound to the CXCR4-CCR7 heteromultimer binding molecule. Alternatively, the label or tag may be part of the CXCR4-CCR7 heteromultimer binding molecule. For example, the CXCR4-CCR7 heteromultimer binding molecule may include the detectable tag as a fusion partner, a labeled amino acid or labeled nucleotide. Examples of suitable detectable tags include antigens, enzymes, fluorophores, quenchers, radioactive isotopes, luminescent labels, and the like. For example, the molecule comprises a fluorescent tag such as, for example, fluorescein (FITC), 5,6-carboxymethyl fluorescein, Texas red, nitrobenz-2-oxa-l,3- diazol-4-yl ( BD), coumarin, dansyl chloride, rhodamine, 4'-6-diamidino-2- phenylinodole (DAPI), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine (5,6- tetramethyl rhodamine). The absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm). Exemplary fluorescent tags include fluorescent proteins (e.g. GFP, EGFP, etc.), phycobiliproteins (e.g. allophycocyanin, phycocyanin, phycoerythrin or phycoerythrocyanin) or derivatives of any of the foregoing.

Alternatively, or in addition, the biding molecule is labeled with, for example, a fluorescent semiconductor nanocrystal (as described, for example, in US6306610).

Alternatively, or in addition, the binding molecule is labeled with, for example, a magnetic or paramagnetic compound, such as, iron, steel, nickel, cobalt, rare earth materials, neodymium-iron-boron, ferrous-chromium-cobalt, nickel-ferrous, cobalt- platinum, or strontium ferrite.

Antigens that may be used as a detectable tag include, for example, any antigenic component of the CXCR4-CCR7 heteromultimer binding molecule that may be targeted by a secondary antibody. For example, in some examples, a secondary antibody may be used to detect an antigen on the CXCR4-CCR7 heteromultimer binding molecule. The secondary antibody may be fluorescently or enzymatically labeled. In examples where the CXCR4-CCR7 heteromultimer binding molecule is a primary antibody, the secondary antibody may have binding affinity to an antigen on the primary antibody. For example, the antigen may be derived from the host in which the primary antibody was raised. Suitable antigens will be apparent to the skilled artisan and include, for example, influenza virus hemagglutinin (HA), Simian Virus 5 (V5), polyhistidine (e.g., hexa-HIS), c-myc or FLAG. Enzymes that may be used as detectable tags include, for example, enzymes that result in the conversion of a substrate into a detectable product, for example resulting in a change in color or fluorescence. Such enzymes may include, for example, horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, acetylcholinesterase, luciferase, or catalase. Depending on the enzyme and substrate used, detection may be performed with a spectrophotometer, flow cytometer fluorometer or luminometer.

In some examples, the detectable tag may be part of the CXCR4-CCR7 heteromultimer binding molecule (e.g. in the form of a fusion protein or a protein comprising fluorescent amino acids) or conjugated to or bound to the CXCR4-CCR7 heteromultimer binding molecule.

In some examples, a binding molecule is labeled with a quencher. Exemplary quenchers include dimethylaminoazosulfonic acid, a black hole quencher™ or a QXL Quencher™. Agents

Various examples of the present disclosure make use of agents that inhibit the formation and/or activity of a CXCR4-CCR7 heteromultimer in a cell. Exemplary agents include:

(ii) an agent that kills a cell expressing a CXCR4-CCR7 heteromultimer;

(iii) an agent reduces or prevents the activity or expression of CXCR4;

(iv) an agent that kills a cell expressing CXCR4;

(v) an agent reduces or prevents the activity or expression of CCR7; and

(iv) an agent that kills a cell expressing CCR7.

In one example, the agent is a binding molecule as described herein.

In another example, the agent may inhibit the formation or activity of a CXCR4- CCR7 heteromultimer by any one or more of the following actions:

i) blocking expression of one or both of CXCR4 and CCR7;

ii) blocking association between CXCR4 and CCR7 to form a heteromultimer; iii) blocking ligand binding to CXCR4, CCR7 or the CXCR4-CCR7 heteromultimer; and

iv) blocking one or more intracellular signaling pathways associated with CXCR4,

CCR7 or the heteromultimer.

Additional exemplary agents disrupt a CXCR4-CCR7 heteromultimer, thereby reducing the activity of the heteromultimer. In some examples, the agent may comprise an isolated protein, a small molecule, a DNA sequence, an RNA sequence, an aptamer, an antibody, a ligand, a fusion protein, siRNA, an antisense molecule, microRNA, shRNA, etc.

In the case where the agent is an antibody, the antibody may be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, a Fab fragment, or fragments produced by a Fab expression library.

In one example, the agent is a ligand of the heteromultimer (e.g., as described herein). Such agents are useful for, for example, inducing or enhancing activity of the heteromultimer.

Inhibiting Expression

In some examples, the agent inhibits formation and/or activity of a CXCR4- CCR7 heteromultimer by blocking the expression of one or both of CXCR4 and CCR7. Blocking expression of one or both of CXCR4 and CCR7 may reduce the amount of either or both proteins in the cell, thereby inhibiting the formation of the CXCR4- CCR7 heteromultimer in the cell. The agent may inhibit expression of CXCR4 and/or CCR7 at the transcriptional or translational level.

In some examples, the agent may be a ligand for a cellular receptor that downregulates the expression of CXCR4 and/or CCR7. In some examples, the agent may comprise siRNA, an antisense molecule, microRNA, short hairpin RNA (shRNA) directed against CXCR4 and/or CCR7 transcripts (for examples of synthetic siRNA mediated silencing see Caplen et ah, 2001; Elbashir et ah, 2001.

In one example, the agent is a nucleic acid, e.g., an antisense polynucleotide, a ribozyme, a PNA, an interfering RNA, a siRNA, short hairpin RNA a microRNA

Antisense Polynucleotides

The term "antisense polynucleotide" shall be taken to mean a DNA or RNA, or combination thereof that is complementary to at least a portion of a specific mRNA molecule encoding a polypeptide as described herein in any embodiment and capable of interfering with a post-transcriptional event such as mRNA translation. The use of antisense methods is known in the art (see for example, Hartmann and Endres, 1999).

An antisense polynucleotide of the disclosure will hybridize to a target polynucleotide under physiological conditions. Antisense polynucleotides include sequences that correspond to the structural genes or for sequences that effect control over gene expression or splicing. For example, the antisense polynucleotide may correspond to the targeted coding region of CXCR4 or CCR7, or the 5 '-untranslated region (UTR) or the 3 '-UTR or combination of these. It may be complementary in part to intron sequences, which may be spliced out during or after transcription, preferably only to exon sequences of CXCR4 or CCR7. The length of the antisense sequence should be at least 19 contiguous nucleotides, preferably at least 50 nucleotides, and more preferably at least 100, 200, 500 or 1000 nucleotides of a nucleic acid encoding CXCR4 or CCR7. The full-length sequence complementary to the entire gene transcript may be used. The degree of identity of the antisense sequence to the targeted transcript should be at least 90% and more preferably 95-100%.

An exemplary antisense polynucleotide against CXCR4 is described in Kusunoki et al., 2001.

RNA Interference

RNA interference (RNAi) is useful for specifically inhibiting the production of a particular protein. Although not wishing to be limited by theory, Waterhouse et al, (1998) have provided a model for the mechanism by which dsRNA (duplex RNA) can be used to reduce protein production. This technology relies on the presence of dsRNA molecules that contain a sequence that is essentially identical to the mRNA of the gene of interest or part thereof, in this case an mRNA encoding CXCR4 or CCR7. Conveniently, the dsRNA can be produced from a single promoter in a recombinant vector or host cell, where the sense and anti-sense sequences are flanked by an unrelated sequence which enables the sense and anti-sense sequences to hybridize to form the dsRNA molecule with the unrelated sequence forming a loop structure. The design and production of suitable dsRNA molecules for the present invention is well within the capacity of a person skilled in the art, particularly considering Waterhouse et al, (1998), W099/32619, WO99/53050, WO99/49029, and WO01/34815.

The length of the sense and antisense sequences that hybridize should each be at least 19 contiguous nucleotides, preferably at least 30 or 50 nucleotides, and more preferably at least 100, 200, 500 or 1000 nucleotides. The full-length sequence corresponding to the entire gene transcript may be used. The lengths are most preferably 100-2000 nucleotides. The degree of identity of the sense and antisense sequences to the targeted transcript should be at least 85%, preferably at least 90% and more preferably 95-100%.

Exemplary RNAi to reduce expression of CCR7 are described in Lin et al, 2009. Exemplary RNAi to reduce expression of CXCR4 are described in Du et al, 2009. Exemplary small interfering RNA ("siRNA") molecules comprise a nucleotide sequence that is identical to about 19-23 contiguous nucleotides of the target mRNA. Preferably, the siRNA sequence commences with the dinucleotide AA, comprises a GC-content of about 30-70% (preferably, 30-60%, more preferably 40-60% and more preferably about 45%-55%), and does not have a high percentage identity to any nucleotide sequence other than the target in the genome of the mammal in which it is to be introduced, for example as determined by standard BLAST search.

Exemplary siRNA to reduce expression of CCR7 are described in Redondo- Munoz et ah, 2008. Exemplary siRNA to reduce expression of CXCR4 are described in Lapteva et ah, 2005.

Alternative Receptors

In some examples, the expression of CXCR4 or CCR7 may be directly or indirectly modulated by activation of a cellular receptor other than CXCR4 or CCR7. The cellular receptor may include, for example, liver X receptor, which can regulate CCR7 expression on cells (Villablanca et ah, 2009), EGF receptor variant, which can regulate CXCR4 expression on cells such as breast cancer cells (Rahimi et ah, 2009), or other CXCR4 or CCR7 regulating receptors that are known in the art. Blocking Formation of the Heteromultimer

In some examples, the agent inhibits the formation and/or activity of a CXCR4- CCR7 heteromultimer by blocking association between CXCR4 and CCR7 to form a heteromultimer. Blocking association between CXCR4 and CCR7 may reduce the formation of the CXCR4-CCR7 heteromultimer. Accordingly, in some examples, the agent may inhibit the formation of a CXCR4-CCR7 heteromultimer by inhibiting the formation of a heteromultimer from individual monomers or homomultimers. In some examples, the agent may inhibit the formation of a CXCR4-CCR7 heteromultimer by inhibiting the addition of one or more monomers or multimers to an existing multimer (e.g. conversion of a heterodimer to a heterotrimer or heteroquadromer).

CXCR4-CCR7 heteromultimers comprise one or more CXCR4-CCR7 binding interfaces. Accordingly, in some embodiments, the agent may inhibit the formation of a CXCR4-CCR7 heteromultimer by interfering with a binding interface. For example, an agent which is capable of binding to a portion of a CXCR4-CCR7 binding interface may be used to competitively inhibit the formation of the heteromultimer or to disrupt a CXCR4-CCR7 heteromultimer. For example, the agent comprises a region of CXCR4 that interacts with CCR7 or the agent comprises a region of CCR7 that interacts with CXCR4.

In one example, the agent is an antibody or antigen binding fragment thereof that binds to CXCR4 or CCR7 and prevents formation of the heteromultimer. For example, the antibody or antigen binding fragment thereof binds at or near a site of multimerization thereby blocking formation of the heteromultimer. In another example, the antibody or antigen binding fragment thereof binds to CXCR4 or CCR7 and prevents it from changing to a conformation required for heteromultimerization. Inhibiting Ligand Binding

In some examples, the agent may inhibit the activity of a CXCR4-CCR7 heteromultimer by inhibiting ligand binding to the CXCR4-CCR7 heteromultimer. In some embodiments, the agent may bind to a ligand of the CXCR4-CCR7 heteromultimer, thereby preventing it from binding to the CXCR4-CCR7 heteromultimer. For example, a soluble (e.g. non-membrane bound) protein comprising a binding domain for the ligand may be used.

In some embodiments, the agent may bind to the CXCR4-CCR7 heteromultimer. In this regard, the agent may prevent or reduce ligand binding. In one example, the agent will bind in a manner that does not activate the CXCR4-CCR7 heteromultimer. The agent may bind to at least a portion of the ligand binding domain, thereby preventing the ligand from binding, or the agent may bind to another portion of the CXCR4-CCR7 heteromultimer and sterically hinder binding of the ligand.

Exemplary antibodies and antigen binding fragments thereof that reduce or prevent binding of CCL19 or CCL21 to CCR7 are described in WO2009/139853. Exemplary antibodies and antigen binding fragments thereof that reduce or prevent binding of SDF-1 to CXCR4 are described in WO2006/089141, WO2008/060367, WO2009/140124 and WO2010/037831.

Exemplary small molecule that prevent ligand binding to CXCR4 useful in the present disclosure are disclosed in U.S. Patent Nos. 5021409; 6001826; 5583131; 5698546; 5817807; 6506770; 6756391; 7160872; 6872714; 7414065; 6667320 and 7022717. In one example, the agent is a small molecule described in US5021409 and/or 6001826 and/or 5583131, for example, l,l'-[l,4-phenylene-bis(methylene)]-bis- 1,4,8,1 1-tetraazacyclotetradecane, sometimes referred to in the art as AMD3100.

Exemplary peptides that prevent or reduce ligand binding to CXCR4 are described in WO01/85196 and/or WO00/09152. Blocking Downstream Signaling

In some examples, the agent inhibits formation and/or activity of a CXCR4- CCR7 heteromultimer by blocking one or more downstream signaling pathways from the heteromultimer. For example, the agent may block any one or more of G protein coupling, kinase activity (e.g. MAP kinase, Src, J K, JAK, PI3 kinase, etc.), phosphatase activity, arrestin activity (e.g. β-arrestins), activity of PDZ domain containing proteins (e.g. NHERF, PSD-95, MUPP1, Grb2, etc.), intracellular calcium flux, etc. Killing Cells

In a further example, the agent binds to CXCR4 and/or CCR7 and kills a cell. In one example, the agent is an antibody or antigen binding fragment thereof that binds to CXCR4 and/or CCR7 and kills a cell to which it is bound, e.g., by an effector function and/or by virtue of being conjugated to a toxin. Exemplary antibodies and/or antigen binding fragments thereof are described herein. Exemplary antibodies that bind to CCR7 and induce CDC are described in WO2007/003216.

Compositions

In some examples, an agent or binding molecule as described herein can be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, bucally, vaginally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, or by any other convenient dosage form. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

Methods for preparing an agent or binding molecule into a suitable form for administration to a subject (e.g. a pharmaceutical composition) are known in the art and include, for example, methods as described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and U.S. Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

The pharmaceutical compositions of this invention are particularly useful for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ or joint. The compositions for administration will commonly comprise a solution of the compound of the present invention dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of agents/compounds of the present invention in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as mixed oils and ethyl oleate may also be used. Liposomes may also be used as carriers. The vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

The agents/compounds of the present invention can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub- cutaneous, transdermal, or other such routes, including peristaltic administration and direct instillation into a tumor or disease site (intracavity administration). The preparation of an aqueous composition that contains the compounds of the present invention as an active ingredient will be known to those of skill in the art.

Suitable pharmaceutical compositions will generally include an amount of the agents/compounds of the present invention admixed with an acceptable pharmaceutical diluent or excipient, such as a sterile aqueous solution, to give a range of final concentrations, depending on the intended use. The techniques of preparation are generally known in the art as exemplified by Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980, incorporated herein by reference.

Upon formulation, agents/compounds of the present invention will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically/prophylactically effective. Formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but other pharmaceutically acceptable forms are also contemplated, e.g., tablets, pills, capsules or other solids for oral administration, suppositories, pessaries, nasal solutions or sprays, aerosols, inhalants, liposomal forms and the like. Pharmaceutical "slow release" capsules or compositions may also be used. Slow release formulations are generally designed to give a constant drug level over an extended period and may be used to deliver compounds of the present invention. WO2002/080967 describes compositions and methods for administering aerosolized compositions comprising antibodies for the treatment of, e.g., asthma, which are also suitable for administration of an antibody of the present invention.

Suitable dosages of agents/compounds of the present invention will vary depending on the specific agent/compound, the condition to be treated and/or the subject being treated. It is within the ability of a skilled physician to determine a suitable dosage, e.g., by commencing with a sub-optimal dosage and incrementally modifying the dosage to determine an optimal or useful dosage. Alternatively, to determine an appropriate dosage for treatment/prophylaxis, data from the cell culture assays or animal studies are used, wherein a suitable dose is within a range of circulating concentrations that include the ED5₀ of the active compound with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A therapeutically/prophylactically 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 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. Levels in plasma maybe measured, for example, by high performance liquid chromatography.

In some examples, a method of the present disclosure comprises administering a prophylactically or therapeutically effective amount of an agent/binding molecule.

The term "therapeutically effective amount" is the quantity which, when administered to a subject in need of treatment, improves the prognosis and/or state of the subject and/or that reduces or inhibits one or more symptoms of a clinical condition to a level that is below that observed and accepted as clinically diagnostic or clinically characteristic of that condition. . The amount to be administered to a subject will depend on the particular characteristics of the condition to be treated, the type and stage of condition being treated, the mode of administration, and the characteristics of the subject, such as general health, other diseases, age, sex, genotype, and body weight. A person skilled in the art will be able to determine appropriate dosages depending on these and other factors. Accordingly, this term is not to be construed to limit the present disclosure to a specific quantity, e.g., weight or amount of compound(s), rather the present invention encompasses any amount of the compound(s) sufficient to achieve the stated result in a subject.

As used herein, the term "prophylactically effective amount" shall be taken to mean a sufficient quantity of a compound to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. The skilled artisan will be aware that such an amount will vary depending on, for example, the specific agent(s)/compound(s) administered and/or the particular subject and/or the type or severity or level of condition and/or predisposition (genetic or otherwise) to the condition. Accordingly, this term is not to be construed to limit the present disclosure to a specific quantity, e.g., weight or amount of compound(s), rather the present invention encompasses any amount of the compound(s) sufficient to achieve the stated result in a subject. Cellular Compositions

In one example of the present disclosure, cells or populations of cells isolated using a binding molecule of the present disclosure are administered in the form of a composition. In one example, such a composition comprises a pharmaceutically acceptable carrier and/or excipient.

Suitable carriers for this disclosure include those conventionally used, e.g., water, saline, aqueous dextrose, lactose, Ringer's solution, a buffered solution, hyaluronan and glycols are preferred liquid carriers, particularly (when isotonic) for solutions. Suitable pharmaceutical carriers and excipients include starch, cellulose, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like.

In another example, a carrier is a media composition, e.g., in which a cell is grown or suspended. An exemplary media composition does not induce any adverse effects in a subject to whom it is administered.

Exemplary carriers and excipients do not adversely affect the viability of a cell and/or the ability of a cell to reduce, prevent or delay a Treg-associated condition.

In one example, the carrier or excipient provides a buffering activity to maintain the cells at a suitable pH to thereby exert a biological activity, e.g., the carrier or excipient is phosphate buffered saline (PBS). PBS represents an attractive carrier or excipient because it interacts with cells minimally and permits rapid release of the cells. In such a case, the composition may be produced as a liquid for direct application to the blood stream or into a tissue or a region surrounding or adjacent to a tissue, e.g., by injection.

Cells can also be incorporated or embedded within scaffolds that are recipient- compatible and which degrade into products that are not harmful to the recipient. These scaffolds provide support and protection for cells that are to be transplanted into the recipient subjects. Natural and/or synthetic biodegradable scaffolds are examples of such scaffolds. Other suitable scaffolds include polyglycolic acid scaffolds or synthetic polymers such as polyanhydrides, polyorthoesters, and polylactic acid.

In one example, the composition comprises an effective amount or a therapeutically or prophylactically effective amount of cells. For example, the composition comprises about lxlO⁵ cells/kg to about lxlO⁹ cells/kg or about lxlO⁶ cells/kg to about lxlO⁸ cells/kg or about lxlO⁶ cells/kg to about lxlO⁷ cells/kg. The exact amount of cells to be administered is dependent upon a variety of factors, including the age, weight, and sex of the patient, and the extent and severity of the condition to be treated.

The cellular compositions can be administered to the subject by any recognized methods, either systemically or at a localized site. When less invasive procedures are desired, the composition can be injected at a desired location through a needle. For deeper sites, the needle can be positioned using endoscopic ultrasound techniques, radioscintigraphy, or some other imaging technique, alone or in combination with the use of an appropriate scope or cannula. For such applications, the cell population is conveniently administered when suspended in isotonic saline or a neutral buffer.

Isolation or Enrichment of Cells

One exemplary approach to enrich for the desired cells is magnetic bead cell sorting (MACS) or any other cell sorting method making use of magnetism, e.g., Dynabeads®. A conventional MACS procedure is described by Miltenyi et al, 1990). In this procedure, cells are labeled with magnetic beads bound to a binding molecule as described herein, e.g., an antibody or antigen binding fragment thereof and the cells are passed through a paramagnetic separation column or exposed to another form of magnetic field. The separation column is placed in a strong magnet, thereby creating a magnetic field within the column. Cells that are magnetically labeled are trapped in the column; cells that are not pass through. The trapped cells are then eluted from the column.

Cells can be enriched, for example, from a suitable sample using MACS to separate cells expressing a CXCR4-CCR7 heteromultimer. The sample is incubated with immunomagnetic beads that bind to the heteromultimer. Following incubation, samples are washed and resuspended and passed through a magnetic field to remove cells bound to the immunomagnetic beads, and cells bound to the beads collected. These techniques are equally applicable to negative selection, e.g., removal of cells expressing the heteromultimer, e.g., metastatic cells from blood. Such a method involves contacting a population of cells with a magnetic particle labeled with a binding molecule as described herein. Following incubation, samples are washed and resuspended and passed through a magnetic field to remove cells bound to the immunomagnetic beads. The remaining cells depleted of the undesirable cell type(s) are then collected. Such methods are useful for, e.g., immunoadsorption therapy.

In another example, a binding molecule is immobilized on a solid surface and a population of cells is contacted thereto. Following washing to remove unbound cells, cells bound to the compound can be recovered, e.g., eluted, thereby isolating or enriching for cells expressing a CXCR4-CCR7 heteromultimer. Alternatively, cells that do not bind to the binding molecule can be recovered if desired.

In a further example, cells are isolated or enriched using fluorescence activated cell sorting (FACS). FACS is a known method for separating particles, including cells, based on the fluorescent properties of the particles and described, for example, in Kamarch, 1987. Generally, this method involves contacting a population of cells with one or more detectably tagged binding molecules capable of binding to one or more proteins or cell surface markers, wherein if multiple binding molecules that bind to distinct markers are used, they are labeled with different labels, e.g., fluorophores. The cells are entrained in the center of a narrow, rapidly flowing stream of liquid. The flow is arranged so that there is a separation between cells relative to their diameter. A vibrating mechanism causes the stream of cells to break into individual droplets. The system is adjusted so that there is a low probability of more than one cell being in a droplet. Just before the stream breaks into droplets the flow passes through a fluorescence measuring station where the fluorescent character of interest of each cell is measured, e.g., whether or not a labeled binding molecule is bound thereto. An electrical charging ring is placed at the point where the stream breaks into droplets. A charge is placed on the ring based on the immediately prior fluorescence intensity measurement and the opposite charge is trapped on the droplet as it breaks from the stream. The charged droplets then fall through an electrostatic deflection system that diverts droplets into containers based upon their charge, e.g., into one container if a labeled binding molecule is bound to the cell and another container if not. In some systems the charge is applied directly to the stream and the droplet breaking off retains charge of the same sign as the stream. The stream is then returned to neutral after the droplet separates. Heteromultimer Associated Conditions The present disclosure provides methods for diagnosing/prognosing/treating/preventing any condition associated with a cell expressing a CXCR4-CCR7 heteromultimer, for example, caused by a cell expressing a CXCR4-CCR7 heteromultimer.

In one example, the condition selected from the group consisting of cancer, an inflammatory condition, an autoimmune condition, an obstructive condition, an infectious condition, an excitotoxic condition, an immunodeficiency condition, a metabolic condition, pain, a circulatory condition and a degenerative condition.

An "autoimmune condition" herein is a non-malignant condition arising from and directed against an individual's own tissues. Exemplary autoimmune diseases are inflammatory bowel disease (IBD), systemic lupus erythematosus, rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's syndrome, systemic vaculitis, sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura, immune -mediated thrombocytopenia), thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus, immune-mediated renal disease (glomerulonephritis, tiibulointerstitial nephritis), demyelinating diseases of the central and peripheral nervous systems such as multiple sclerosis, idiopathic polyneuropathy, hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C, D, E and other nonhepatotropic viruses), autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis, and sclerosing cholangitis, inflammatory and fibrotic lung diseases (e.g., cystic fibrosis), gluten-sensitive enteropathy, Whipple's disease, WHIM syndrome, autoimmune or immune-mediated skin diseases including bullous skin diseases, erythema multiforme and contact dermatitis, psoriasis, allergic diseases of the lung such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, transplantation associated diseases including graft rejection and graft- versus host disease.

In one example, the condition is autoimmune arthritis, e.g., RA or juvenile chronic arthritis.

In another example, the condition is an autoimmune neurological condition, e.g., a myelin associated condition, e.g., multiple sclerosis.

In another example, the condition is an autoimmune skin condition e.g., a bullous skin diseases, erythema multiforme, contact dermatitis. Alternatively, the skin condition is psoriasis. In another example, the condition is an inflammatory condition. Inflammatory conditions are a class of conditions characterized by movement of leukocytes (e.g., granulocytes) to a localized position in a subject's body, e.g., in a tissue. Inflammatory conditions can be chronic or acute. Inflammatory conditions include many autoimmune conditions, e.g., rheumatoid arthritis and/or Crohn's disease. Other conditions associated with or caused by inflammation include acne vulgaris, asthma, chronic prostatitis, pancreatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, vasculitis, interstitial cystitis, myopathy, cancer, or atherosclerosis.

In one example, the condition is an obstructive condition. Exemplary obstructive conditions include, for example, renal failure (e.g., arising from proliferation of mesangial cells), obstructive nephropathy, obstructive liver disease, obstructive hepatitis and obstructive airway conditions (e.g., asthma, emphysema, bronchitis or chronic obstructive pulmonary disease). Some conditions falling within this category are associated with liver cells, lung cells or mesangial cells.

Infectious conditions include viral infections. For example, the condition is a human immunodeficiency virus infection or a hepatitis C virus infection. In this regard, a binding molecule or agent described herein may reduce or prevent infection and/or reduce or prevent viral replication and/or spread and/or kill a cell infected by the virus.

Excitotoxic conditions are characterized by nerve cell damage by excessive stimulation by neurotransmitters such as glutamate and similar substances. Exemplary excitotoxic conditions include spinal cord injury, stroke, traumatic brain injury, neurodegenerative diseases of the central nervous system (CNS) (such as, multiple sclerosis, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease or Huntington's disease) or status epilepticus.

Immunodeficiency conditions are characterized by insufficient immune cells and/or compounds secreted therefrom in a subject or at a site within a subject to adequately protect the subject, e.g., from infection and/or cancer. Such conditions can be treated by administering immune cells to a subject or inducing immune cells to migrate into circulation or another site at which they are required. Exemplary immunodeficiency conditions include primary (genetic) immunodeficiencies, hypogammaglobulinemia, or immunodeficiency caused by chemotherapy, disease- modifying antirheumatic drugs, immunosuppressive drugs after organ transplants, glucocorticoids, or diseases that diseases directly or indirectly impair the immune system (e.g., leukemia, lymphoma, multiple myeloma, and certain chronic infections, e.g., HIV.

Degenerative conditions refer to conditions that progressively worsen over time, and generally involve tissue/cell destruction. Degenerative disease can affect a variety of tissues including, neural tissue, joints, eyes, spinal discs, blood vessels or bones. In one example, the degenerative condition is a neurodegenerative condition, such as, ALS, Huntington's disease (or other trinucleotide repeat or polyglutamine disorder), Parkinson's disease or Alzheimer's disease. In another example, the degenerative condition is a condition of the heart. In another example, the degenerative condition is a necrotic condition, e.g., as occurs during infarction or as a result of infection or insect/arachnid bite.

A circulatory condition includes conditions associated with insufficient circulation (e.g., angiogenesis/vascularization) or with excessive circulation (e.g., angiogenesis/vascularization). Exemplary conditions associated with insufficient circulation include cardiovascular disease, autoimmune conditions (e.g., as discussed above), antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, ischemia (including ischemia resulting from a transplant) and testicular necrosis. Exemplary conditions associated with excessive circulation include cancer (including solid tumors, leukemias, lymphoma, melanoma, glioma, breast cancer, colonic cancer, gastric cancer, esophageal cancer, renal cell cancer, ovarian cancer, cervical cancer, carcinoid cancer, testicular cancer, prostate cancer, head and neck cancer and hepatocellular carcinoma), cancer metastasis, cancer neovascularization, autoimmune disease (including psoriasis), nephropathy, retinopathy, preeclampsia hepatitis, sepsis and macular degeneration.

In one example, the condition is associated with or caused by a cell selected from the group consisting of an immune cell, a tonsil cell, a thymus cell, an endothelial cell, an endothelial progenitor cell, a mesangial cell, an hepatic cell, a lung cell, a bone marrow CD34⁺ cell, a cord blood CD34⁺ cell, or a lymph node cell.

Exemplary immune cells are of B lymphoblasts, B cells, dendritic cells, plasmacytoid dendritic cells, regulatory T (Treg) cells, monocytes, macrophages, granulocytes and T cells.

In one example, the condition is selected from the group consisting of an inflammatory condition, an autoimmune condition, graft versus host disease, graft rejection, liver disease, liver cirrhosis, kidney disease, renal failure, neutropenia or hepatitis.

A "condition associated with or caused by a B cell" includes autoimmune conditions as well as malignant B cell-associated conditions, such as B cell lymphoma (e.g., non-Hodgkin's lymphoma), including precursor B cell lymphoblastic leukemia/lymphoma and mature B cell neoplasms, such as B cell chronic lymhocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, intermediate-grade and high-grade FL, cutaneous follicle center lymphoma, marginal zone B cell lymphoma (MALT type, nodal and splenic type), hairy cell leukemia, diffuse large B cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large-cell lymphoma (ALCL)

A "condition associated with or caused by a T cell" includes autoimmune conditions (such as multiple sclerosis, neuritis, polymyositis, psoriasis, vitiligo, Sjogren's syndrome, rheumatoid arthritis, Type 1 diabetes, autoimmune pancreatitis, inflammatory bowel diseases, Crohn's disease, ulcerative colitis, celiac disease, glomerulonephritis, scleroderma, sarcoidosis, autoimmune thyroid diseases, Hashimoto's thyroiditis, Graves disease, myasthenia gravis, Addison's disease, autoimmune uveoretinitis, pemphigus vulgaris, primary biliary cirrhosis, pernicious anemia, or systemic lupus erythematosis), graft rejection (e.g., allograft rejection), graft versus host disease, an unwanted delayed-type hypersensitivity reaction (e.g., asthma), a T-cell mediated pulmonary disease, pulmonary fibrosis or idiopathic pulmonary fibrosis. The term "disease associated with or caused by a T cell" also includes malignant T cell-associated conditions, such as leukemias and lymphomas, such as extranodal T cell lymphoma, cutaneous T cell lymphoma (e.g., Sezary syndrome, mycosis fungiodes), anaplastic large cell lymphoma, angioimmunoblastic T cell lymphoma, large granular lymphocytic leukemia, adult T cell leukemia, T-cell prolymphocytic leukemia, T-cell chronic lymphocytic leukemia, acute lymphocytic leukemia, acute myeloid leukemia or chronic myeloid leukemia.

In one example, the T cell is a regulatory T (Treg) cell. Diseases caused or mediated by Treg cells include conditions associated with or caused by an excessive immune response (e.g., by a Thelper cell or a CTL or a T_H17 cell or other cell regulated by one or more T cells). Accordingly, this term encompasses inflammatory conditions and/or autoimmune conditions. Exemplary Treg-associated conditions include, an inflammatory disorder of the nervous system (e.g., multiple sclerosis), or a mucosal inflammatory disease (e.g., inflammatory bowel disease, asthma or tonsillitis), or an inflammatory skin disease (e.g., dermatitis, psoriasis or contact hypersensitivity) or autoimmune arthritis (e.g., rheumatoid arthritis). Other exemplary Treg-associated conditions include conditions characterized by excessive Treg numbers and/or activity. For example, an immune response against a graft or graft versus host disease or host versus graft disease is also a Treg-associated condition. This term also includes cancer, e.g., in which Treg cells suppress the activity of immune cells against cancerous cells, thereby permitting the disease to develop.

In one example, the T cell is a T_H17 cell. Conditions caused or mediated by

T_H17 cells include an autoimmune disease, discussed above as well as graft rejection, graft versus host disease, and cardiovascular disease. Exemplary T_H17 mediated or associated conditions include psoriasis, inflammatory bowel disease, arthritis (e.g., rheumatoid arthritis), multiple sclerosis inflammatory bowel disease (e.g., Crohn's disease) and graft versus host disease.

In one example, the condition is a dendritic cell-mediated or associated condition. Exemplary dendritic cell-mediated or associated conditions are graft versus host disease, graft rejection, host versus graft disease, allergy, asthma, autoimmune disease.

In one example, the condition is a plasmacytoid dendritic cell (pDC)-associated or mediated condition. pDC associated or mediated conditions include autoimmune conditions (e.g., as discussed above), such as lupus (e.g., SLE), Sj5gren's syndrome, dermatitis (e.g., contact dermatitis), sclerosis (e.g., systemic sclerosis), allergy, or asthma.

In one example, the condition is an EPC-associated or mediated condition. In one example, an EPC-associated or mediated condition is characterized by insufficient EPC numbers and/or activity. Exemplary conditions include cardiovascular disease, autoimmune conditions (e.g., as discussed above), antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis, ischemia (including ischemia resulting from a transplant) and testicular necrosis. In another example, the condition is associated with excessive EPC numbers and/or activity (including excessive neovascularization). Exemplary conditions include cancer (including solid tumors, leukemias, lymphoma, melanoma, glioma, breast cancer, colonic cancer, gastric cancer, esophageal cancer, renal cell cancer, ovarian cancer, cervical cancer, carcinoid cancer, testicular cancer, prostate cancer, head and neck cancer and hepatocellular carcinoma), cancer metastasis, cancer neovascularization, autoimmune disease (including psoriasis), nephropathy, retinopathy, preeclampsia hepatitis, sepsis and macular degeneration.

In one example, the condition is a mesangial cell-associated or mediated condition. Mesangial cells are cells that surround blood vessels in the kidney and regulate blood flow through capillaries. Proliferation and/or expansion of mesangial cells can lead to glomerulonephritis, which can lead to renal disease and/or kidney failure due to obstructed blood filtration. Accordingly, mesangial cell-associated or mediated conditions include glomerulonephritis, renal disease and/or kidney failure.

In a further example, the condition is a liver disease, such as an inflammatory liver disease. For example, the disease is hepatitis and/or cirrhosis and/or liver fibrosis.

In a further example, the condition is graft versus host disease, host versus graft disease or graft rejection.

In one example, the condition is cancer or a metastasis thereof. The term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Examples of cancer include, but are not limited to, an adenocarcinoma, a squamous cell carcinoma, a digestive/gastro intestinal cancer, an endocrine cancer, an eye cancer, a musculoskeletal cancer, a breast cancer, a neurologic cancer, a genitourinary cancer, a germ cell cancer, a head and neck cancer, a hematologic/blood cancer, a respiratory cancer, a skin cancer, an AIDS-related malignancy or a gynelogic cancer.

An adenocarcinoma is a cancer of an epithelium that originates in glandular tissue. Exemplary adenocarcinomas include forms of colorectal cancer, lung cancer, cervical cancer, prostate cancer, urachus cancer, vulval cancer, breast cancer, esophageal cancer, pancreatic cancer and gastric cancer.

Digestive/gastro intestinal cancers include anal cancer; bile duct cancer; extrahepatic bile duct cancer; appendix cancer; carcinoid tumor, gastrointestinal cancer; colon cancer; colorectal cancer including childhood colorectal cancer; esophageal cancer including childhood esophageal cancer; gallbladder cancer; gastric (stomach) cancer including childhood gastric (stomach) cancer; hepatocellular (liver) cancer including childhood hepatocellular (liver) cancer; pancreatic cancer including childhood pancreatic cancer; sarcoma, rhabdomyosarcoma; rectal cancer; and small intestine cancer.

Endocrine cancers include islet cell carcinoma (endocrine pancreas); adrenocortical carcinoma including childhood adrenocortical carcinoma; gastrointestinal carcinoid tumor; parathyroid cancer; pheochromocytoma; pituitary tumor; thyroid cancer including childhood thyroid cancer; childhood multiple endocrine neoplasia syndrome; and childhood carcinoid tumor.

Eye cancers include intraocular melanoma; and retinoblastoma.

Musculoskeletal cancers include Ewing's family of tumors; osteosarcoma/malignant fibrous histiocytoma of the bone; rhabdomyosarcoma including childhood rhabdomyosarcoma; soft tissue sarcoma including childhood soft tissue sarcoma; clear cell sarcoma of tendon sheaths; and uterine sarcoma. Neurologic cancers include childhood brain stem glioma; brain tumor; childhood cerebellar astrocytoma; childhood cerebral astrocytoma/malignant glioma; childhood ependymoma; childhood medulloblastoma; childhood pineal and supratentorial primitive neuroectodermal tumors; childhood visual pathway and hypothalamic glioma; other childhood brain cancers; adrenocortical carcinoma; central nervous system lymphoma, primary; childhood cerebellar astrocytoma; neuroblastoma; craniopharyngioma; spinal cord tumors; central nervous system atypical teratoid/rhabdoid tumor; central nervous system embryonal tumors; and supratentorial primitive neuroectodermal tumors including childhood and pituitary tumor.

Genitourinary cancers include bladder cancer including childhood bladder cancer; renal cell (kidney) cancer; ovarian cancer including childhood ovarian cancer; ovarian epithelial cancer; ovarian low malignant potential tumor; penile cancer; prostate cancer; renal cell cancer including childhood renal cell cancer; renal pelvis and ureter, transitional cell cancer; testicular cancer; urethral cancer; vaginal cancer; vulvar cancer; cervical cancer; Wilms tumor and other childhood kidney tumors; endometrial cancer; and gestational trophoblastic tumor;

Germ cell cancers include childhood extracranial germ cell tumor; extragonadal germ cell tumor; ovarian germ cell tumor; and testicular cancer.

Head and neck cancers include lip and oral cavity cancer; childhood oral cancer; hypopharyngeal cancer; laryngeal cancer including childhood laryngeal cancer; metastatic squamous neck cancer with occult primary; mouth cancer; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer including childhood nasopharyngeal cancer; oropharyngeal cancer; parathyroid cancer; pharyngeal cancer; salivary gland cancer including childhood salivary gland cancer; throat cancer; and thyroid cancer.

Hematologic/blood cell cancers include leukemia (e.g., acute lymphoblastic leukemia in adults and children; acute myeloid leukemia, e.g., in adults and children; chronic lymphocytic leukemia; chronic myelogenous leukemia; and hairy cell leukemia); a lymphoma (e.g., AIDS-related lymphoma; cutaneous T-cell lymphoma; Hodgkin's lymphoma including Hodgkin's lymphoma in adults and children; Hodgkin's lymphoma during pregnancy; non-Hodgkin's lymphoma including non-Hodgkin's lymphoma in adults and children; non-Hodgkin's lymphoma during pregnancy; mycosis fungoides; Sezary syndrome; Waldenstrom's macroglobulinemia; and primary central nervous system lymphoma); and other hematologic cancers (e.g., chronic myeloproliferative disorders; multiple myeloma/plasma cell neoplasm; myelodysplasia syndromes; and myelodysplastic/myeloproliferative disorders). Respiratory cancers include non-small cell lung cancer; small cell lung cancer; malignant mesothelioma including malignant mesothelioma in adults and children; malignant thymoma; childhood thymoma; thymic carcinoma; bronchial adenomas/carcinoids including childhood bronchial adenomas/carcinoids; pleuropulmonary blastoma.

Skin cancers include Kaposi's sarcoma; Merkel cell carcinoma; melanoma; basal cell carcinoma and childhood skin cancer.

In one example, the cancer is selected from the group consisting of a breast cancer, a lymphoma, a leukemia, a head and neck cancer, a thyroid cancer, a gastric cancer, an endometrial cancer, a lung cancer, a cervical cancer, a melanoma, a non- melanoma skin cancer, a pancreatic cancer, a prostate cancer, an esophageal cancer, a nasopharyngeal cancer, a colorectal cancer, an osteocarcinoma cancer, a kidney cancer, an ovarian cancer, a myeloma, a neuroblastoma, a myosarcoma and a testicular cancer. In one example, the cancer is breast cancer or a metastatic breast cancer.

In one example, a cancer cell is a cancer stem cell.

In one example, a method of the present disclosure is used to kill or reduce or prevent metastasis of a cancer stem cell.

In one example, a condition to be diagnosed/prognosed/treated/prevented is a metastasis of a cancer, e.g., a cancer mentioned in one or both of the foregoing paragraphs.

In one example an agent or binding molecule is used to diagnose/prognose/treat/prevent a breast cancer, leukemia, a gastro-intestinal tract cancer, a pancreatic cancer, a colo-rectal cancer, a esophageal cancer, a head and neck cancer, or a non-small cell lung cancer. In some examples, the agent/binding molecule may be used to diagnose/prognose/treat/prevent a metastatic cancer.

In one example, a condition to be diagnosed/prognosed/treated/prevented is a metastasis of a breast cancer or a metastatic breast cancer.

Methods of Diagnosis/Prognosis

As will be apparent from the description herein, the present disclosure provides numerous methods for diagnosing/prognosing any of a variety of conditions.

These methods involve detecting a CXCR4-CCR7 heteromultimer and/or a cell expressing same. Protein Detection Assays

In some examples, detecting the presence of a CXCR4-CCR7 heteromultimer in a cell may comprise contacting the cell with one or more CXCR4-CCR7 heteromultimer binding molecules and detecting binding of the one or more binding molecules to the CXCR4-CCR7 heteromultimer. Detection may involve, for example, immunoprecipitation, flow cytometry, immunostaining, histochemistry or other detection techniques that are known in the art.

The amount, level or presence of a protein is determined using any of a variety of techniques known to the skilled artisan such as, for example, a technique selected from the group consisting of, immunohistochemistry, immunofluorescence, an immunoblot, a Western blot, a dot blot, an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-tof-MS), electrospray ionization (ESI-MS) (including tandem mass spectrometry, e.g. LC-ESI-MS/MS and MALDI-tof/tof-MS), biosensor technology, evanescent fiber-optics technology or protein chip technology.

In one example, the assay used to determine the amount or level of a heteromultimer is a semi-quantitative method.

In another embodiment the assay used to determine the amount or level of a heteromultimer is a quantitative method.

In one example, the protein is detected with an immunoassay. Preferably, using an assay selected from the group consisting of, immunohistochemistry, immunofluorescence, ELISA, fluorescence-linked immunosorbent assay (FLISA) Western blotting, RIA, a biosensor assay, a protein chip assay and an immunostaining assay (e.g. immunofluorescence).

Standard solid-phase ELISA or FLISA formats are particularly useful in determining the concentration of a heteromultimer from a variety of samples.

In one form such an assay involves immobilizing a biological sample onto a solid matrix, such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide). A binding molecule (e.g., an antibody) is brought into direct contact with the immobilized biological sample, and forms a direct bond with any of its target protein present in said sample. This binding molecule is generally labeled with a detectable tag, such as, for example, a fluorescent label (e.g. FITC or Texas Red) or a fluorescent semiconductor nanocrystal (as described in US6306610) in the case of a FLISA or an enzyme (e.g. horseradish peroxidase (HRP), alkaline phosphatase (AP) or β-galactosidase) in the case of an ELISA, or alternatively a second labeled binding molecule can be used that binds to the first binding molecule. Following washing to remove any unbound binding molecule the label is detected either directly, in the case of a fluorescent label, or through the addition of a substrate, such as for example hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indol-beta-D-galaotopyranoside (x-gal) in the case of an enzymatic label. Such ELISA- or FLISA-based systems are particularly suitable for quantification of the amount of a protein in a sample, by calibrating the detection system against known amounts of a protein standard to which the binding molecule binds, such as for example, an isolated and/or recombinant heteromultimer or a cell expressing same.

In another form, an ELISA or FLISA comprises of immobilizing a binding molecule (e.g., an antibody) on a solid matrix, such as, for example, a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate dipstick or a glass support. A sample is then brought into contact with the binding molecule, and the heteromultimer or cell expressing same is bound or 'captured'. The bound protein is then detected using a second labeled compound that binds to a different protein or a different site in the same protein. Alternatively, a third labeled antibody can be used that binds the second (detecting) antibody.

Various forms of "sandwich"-type assays will be apparent to the skilled person based on the disclosure herein, such as:

o Capture with a binding molecule that binds to CCR7 and detect with a binding molecule that binds to CXCR4;

o Capture with a binding molecule that binds to CXCR4 and detect with a binding molecule that binds to CCR7;

o Capture with a binding molecule that binds to CXCR4 or CCR7 and detect with a binding molecule that binds specifically to the heteromultimer; or

o Capture with a binding molecule that binds specifically to the heteromultimer and detect with a binding molecule that binds to CXCR4 or CCR7.

It will be apparent to the skilled person that the assay formats described herein are amenable to high throughput formats, such as, for example, automation of screening processes or a microarray format as described in Mendoza et al. (1999). Furthermore, variations of the above-described assay will be apparent to those skilled in the art, such as, for example, a competitive ELISA.

In an alternative example, a heteromultimer is detected within or on a cell, using methods known in the art, such as, for example, immunohistochemistry or immunofluorescence. Methods using immunofluorescence are exemplary, because they are quantitative or at least semi-quantitative. Methods of quantitating the degree of fluorescence of a stained cell are known in the art and described, for example, in Cuello (1984).

Biosensor devices generally employ an electrode surface in combination with current or impedance measuring elements to be integrated into a device in combination with the assay substrate (such as that described in US5567301). A binding molecule is incorporated onto the surface of a biosensor device and a biological sample contacted to the device. A change in the detected current or impedance by the biosensor device indicates heteromultimer binding to the binding molecule. Some forms of biosensors known in the art also rely on surface plasmon resonance to detect protein interactions, whereby a change in the surface plasmon resonance surface of reflection is indicates heteromultimer binding (US5485277 and US5492840).

Biosensors are of particular use in high throughput analysis due to the ease of adapting such systems to micro- or nano-scales. Furthermore, such systems are conveniently adapted to incorporate several detection reagents, allowing for multiplexing of diagnostic reagents in a single biosensor unit. This permits the simultaneous detection of several proteins or peptides in a small amount of body fluids.

Evanescent biosensors are also preferred as they do not require the pretreatment of a biological sample prior to detection of a heteromultimer. An evanescent biosensor generally relies upon light of a predetermined wavelength interacting with a fluorescent molecule, such as for example, a fluorescent binding molecule attached near the probe's surface, to emit fluorescence at a different wavelength upon binding of the heteromultimer to the binding molecule.

Micro- or nano-cantilever biosensors are also preferred as they do not require the use of a detectable tag. A cantilever biosensor utilizes a binding molecule capable of specifically detecting a heteromultimer that is bound to the surface of a deflectable arm of a micro- or nano-cantilever. Upon binding of the heteromultimer the deflectable arm of the cantilever is deflected in a vertical direction (i.e. upwards or downwards). The change in the deflection of the deflectable arm is then detected by any of a variety of methods, such as, for example, atomic force microscopy, a change in oscillation of the deflectable arm or a change in pizoresistivity. Exemplary micro-cantilever sensors are described in US20030010097.

The present disclosure also contemplates the use of BRET/FRET or quenching to detect binding of a binding molecule that binds to CXCR4 and a binding molecule that binds to CCR7 to thereby detect a heteromultimer. For example, stimulation may be provided by a fluorescence resonance energy transfer (FRET) partner or a bioluminescence resonance energy transfer (BRET) partner (i.e. donor molecules). For example, a CXCR4-CCR7 heteromultimer binding molecule specific for CXCR4 may comprise a fluorophore (FRET donor molecule) or bioluminescent molecule (BRET donor molecule) and a CXCR4-CCR7 heteromultimer binding molecule specific for CCR7 may comprise a FRET or BRET acceptor molecule. Typically, the emission spectrum of the donor molecule overlaps with the excitation spectrum of the acceptor molecule. Alternatively, the CXCR4-CCR7 heteromultimer binding molecule specific for CCR7 may comprise the donor molecule and the CXCR4-CCR7 heteromultimer binding molecule specific for CXCR4 may comprise the acceptor molecule. When the donor molecule comes into close proximity to the acceptor molecule (e.g. when both binding molecules bind to the same CXCR4-CCR7 heteromultimer and/or are within approximately ΙΟθΑ), the acceptor molecule may become excited by the donor molecule and fluoresce. As the acceptor molecules have different emission spectra to the donor molecule, emission from the acceptor molecule may be specifically detected.

In accordance with the proceeding paragraph, in some examples the method comprises:

contacting the cell with a first CXCR4-CCR7 heteromultimer binding molecule which binds to CXCR4 in the CXCR4-CCR7 heteromultimer in the cell;

contacting the cell with a second CXCR4-CCR7 heteromultimer binding molecule which binds to CCR7 in the CXCR4-CCR7 heteromultimer in the cell; and detecting binding of the first binding molecule and the second binding molecule to the CXCR4-CCR7 heteromultimer in the cell.

In some examples, a detectable tag conjugated to one of the binding molecule may comprise a quencher. Quenchers are able to absorb excitation energy from fluorophores or bioluminescent molecules and may be used to suppress their emission when in close proximity. In this regard, the reaction is similar to a FRET or BRET reaction, except that the readout is a loss of fluorescence.

As will be apparent to the skilled artisan from the description herein, various diagnostic and/or prognostic methods are applicable to ascertaining the metastatic potential of a cancer in a subject. For example, the present disclosure provides, a method for ascertaining the metastatic potential of a cancer in a subject, the method comprising collecting a cell from the cancer and ascertaining the metastatic potential of the cell by performing a diagnostic/prognostic method described herein. For example, a method for ascertaining the metastatic potential of a cancer cell comprises detecting the presence of a CXCR4-CCR7 heteromultimer in the cell, wherein the presence of the CXCR4-CCR7 heteromultimer in the cell is indicative of the metastatic potential of the cell.

In some examples, a cancer cell comprising a CXCR4-CCR7 heteromultimer is indicative that the cancer cell has a higher metastatic potential than a cancer cell of the same type that lacks a CXCR4-CCR7 heteromultimer (or a standard produced using date from a population of such cells). In some examples, a cancer cell with a high level of expression of a CXCR4-CCR7 heteromultimer is indicative that the cancer cell that has a higher metastatic potential than a cancer cell of the same type with a lower level of expression of a CXCR4-CCR7 heteromultimer(or a standard produced using date from a population of such cells).

In some examples, a cancer cell lacking a CXCR4-CCR7 heteromultimer is indicative that the cancer cell has a lower metastatic potential than a cancer cell of the same type that comprises a CXCR4-CCR7 heteromultimer(or a standard produced using date from a population of such cells). In some examples, a cancer cell with a low level of expression of a CXCR4-CCR7 heteromultimer is indicative that the cancer cell that has a lower metastatic potential than a cancer cell of the same type with a higher level of expression of a CXCR4-CCR7 heteromultimer(or a standard produced using date from a population of such cells).

As many cancers are associated with progressive mutations which can influence the severity and/or functionality and/or progression of the cancer, it will be appreciated that a cancer comprising cells that do not express the CXCR4-CCR7 heteromultimer, and thus have a low metastatic potential, may express the CXCR4-CCR7 heteromultimer at a later time. Ascertaining the metastatic potential of a cancer or a cell thereof according to examples of the present disclosure is therefore a dynamic method which may be used to assess the metastatic potential of the cancer or a cell thereof at the time the method is performed. It is envisaged that in some examples, the method will be performed before, during or after treatment of the cancer or on an ongoing basis to monitor metastatic potential of cancer cells. For example, some therapies (e.g., chemotherapy or radiation therapy) may induce metastases arising from some cancers (e.g., breast cancer) and the method may be used to monitor changes in the cancer during therapy indicating an increased risk of metastasis.

In some examples, the method may be used to ascertain the metastatic potential of a breast cancer, leukemia, gastro-intestinal tract cancer, pancreatic cancer, a colorectal cancer, a esophageal cancer, a head and neck cancer, or a non-small cell lung cancer. In some examples, the method may be used to ascertain the metastatic potential of a breast cancer. Imaging Methods

As will be apparent to the skilled artisan from the foregoing, the present disclosure also contemplates imaging methods using a binding molecule. For imaging, a binding molecule is generally conjugated to a detectable tag, which can be any molecule or agent that can emit a signal that is detectable by imaging. However, a secondary labeled binding molecule that specifically binds to a binding molecule that specifically binds to a heteromultimer may also be used. Exemplary detectable tags include a protein, a radioisotope, a fluorophore, a visible light emitting fluorophore, infrared light emitting fluorophore, a metal, a ferromagnetic substance, an electromagnetic emitting substance a substance with a specific magnetic resonance (MR) spectroscopic signature, an X-ray absorbing or reflecting substance, or a sound altering substance.

The binding molecule (and, if used the labeled secondary compound) can be administered either systemically or locally to the tumor, organ, or tissue to be imaged, prior to the imaging procedure. Generally, the binding molecule is administered in one or more doses effective to achieve the desired optical image of a tumor, tissue, or organ. Such doses may vary widely, depending upon the particular binding molecule employed, condition to be imaged, tissue, or organ subjected to the imaging procedure, the imaging equipment being used, and the like.

In some examples, the binding molecule is used as an in vivo optical imaging agent of tissues and organs in various biomedical applications including, but not limited to, imaging of tumors or metastases (including micrometastases), tomographic imaging of organs, monitoring of organ functions, coronary angiography, fluorescence endoscopy, laser guided surgery, photoacoustic and sonofluorescence methods, and the like. Exemplary conditions in which a binding molecule is useful for imaging are described herein and shall be taken to apply mutatis mutandis to the present example of the disclosure. In one example, the binding molecule is useful for detecting the presence of tumors or metastases and/or other abnormalities (e.g., retinopathy and/or nephropathy) by monitoring where the heteromultimer is concentrated in a subject.

In another example, the compound is useful for laser-assisted guided surgery. Examples of imaging methods include magnetic resonance imaging (MRI), MR spectroscopy, radiography, computerized tomography (CT), ultrasound, planar gamma camera imaging, single-photon emission computed tomography (SPECT), positron emission tomography (PET), other nuclear medicine-based imaging, optical imaging using visible light, optical imaging using luciferase, optical imaging using a fluorophore, other optical imaging, imaging using near infrared light, or imaging using infrared light.

Certain examples of the methods of the present disclosure further include imaging a tissue during a surgical procedure on a subject.

A variety of techniques for imaging are known to those of ordinary skill in the art. Any of these techniques can be applied in the context of the imaging methods of the present disclosure to measure a signal from the detectable tag. For example, optical imaging is one imaging modality that has gained widespread acceptance in particular areas of medicine. Examples of optical imaging agents include, for example, fluorescein, a fluorescein derivative, indocyanine green, Oregon green, a derivative of Oregon green, rhodamine green, a derivative of rhodamine green, an eosin, an erytlirosin, Texas red, a derivative of Texas red, malachite green, nanogold sulfosuccinimidyl ester, cascade blue, a coumarin derivative, a naphthalene, a pyridyloxazole derivative, cascade yellow dye, dapoxyl dye.

Gamma camera imaging is contemplated as a method of imaging that can be utilized for measuring a signal derived from the detectable tag. One of skill in the art will be familiar with techniques for application of gamma camera imaging. In one example, measuring a signal can involve use of gamma-camera imaging of an ¹¹ 'in or ^99mTc conjugate, in particular ^mIn- octreotide or ^99mTc-somatostatin analogue.

CT is contemplated as an imaging modality in the context of the present disclosure. By taking a series of X-rays from various angles and then combining them with a computer, CT makes it possible to build up a three-dimensional image of any part of the body. A computer is programmed to display two-dimensional slices from any angle and at any depth. The slices may be combined to build three-dimensional representations.

In CT, intravenous injection of a radiopaque contrast agent conjugated to a binding molecule, which binds to a heteromultimer identified herein can assist in the identification and delineation of soft tissue masses when initial CT scans are not diagnostic. Similarly, contrast agents aid in assessing the vascularity of a soft tissue lesion. For example, the use of contrast agents may aid the delineation of the relationship of a tumor and adjacent vascular structures.

CT contrast agents include, for example, iodinated contrast media. Examples of these agents include iothalamate, iohexol, diatrizoate, iopamidol, ethiodol, and iopanoate. Gadolinium agents have also been reported to be of use as a CT contrast agent, for example, gadopentate. MRI is an imaging modality that uses a high-strength magnet and radio- frequency signals to produce images. In MRI, the sample to be imaged is placed in a strong static magnetic field and excited with a pulse of radio frequency (RF) radiation to produce a net magnetization in the sample. Various magnetic field gradients and other RF pulses then act to code spatial information into the recorded signals. By collecting and analyzing these signals, it is possible to compute a three-dimensional image which, like a CT image, is normally displayed in two-dimensional slices. The slices may be combined to build three-dimensional representations.

Contrast agents used in MRI or MR spectroscopy imaging differ from those used in other imaging techniques. Examples of MRI contrast agents include gadolinium chelates, manganese chelates, chromium chelates, and iron particles. For example, a protein of the invention is conjugated to a compound comprising a chelate of a paramagnetic metal selected from the group consisting of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, molybdenum, ruthenium, cerium, indium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, and ytterbium. A further example of imaging agents useful for the present invention is halocarbon- based nanoparticle such as PFOB or other fluorine-based MRI agents. Both CT and MRI provide anatomical information that aid in distinguishing tissue boundaries and vascular structure.

Imaging modalities that provide information pertaining to information at the cellular level, such as cellular viability, include PET and SPECT. In PET, a patient ingests or is injected with a radioactive substance that emits positrons, which can be monitored as the substance moves through the body.

SPECT is closely related to PET. The major difference between the two is that instead of a positron-emitting substance, SPECT uses a radioactive tracer that emits high-energy photons. SPECT is valuable for diagnosing multiple illnesses including coronary artery disease, and already some 2.5 million SPECT heart studies are done in the United States each year.

For PET, the binding molecule is commonly labeled with positron-emitters such as ⁿC, ¹³N, ¹⁵0, ¹⁸F, ⁸²Rb, ⁶²Cu, and ⁶⁸ Ga. Compounds that bind to a protein set forth in any one or more of Tables 1-6 are labeled with positron emitters such as ^99mTc, ²⁰¹T1, and ⁶⁷Ga, ^mIn for SPECT.

Non-invasive fluorescence imaging of animals and humans can also provide in vivo diagnostic information and be used in a wide variety of clinical specialties. For instance, techniques have been developed over the years including simple observations following UV excitation of fluorophores up to sophisticated spectroscopic imaging using advanced equipment (see, e.g., Andersson-Engels et al, 1997). Specific devices or methods known in the art for the in vivo detection of fluorescence, e.g., from fluorophores or fluorescent proteins, include, but are not limited to, in vivo near- infrared fluorescence (see, e.g., Frangioni, 2003), the Maestro™ in vivo fluorescence imaging system (Cambridge Research & Instrumentation, Inc.; Woburn, MA), in vivo fluorescence imaging using a flying-spot scanner (see, e.g., Ramanujam et al, 2001), and the like.

Other methods or devices for detecting an optical response include, without limitation, visual inspection, CCD cameras, video cameras, photographic film, laser- scanning devices, fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or signal amplification using photomultiplier tubes.

In some examples, an imaging agent is tested using an in vitro or in vivo assay prior to use in humans, e.g., using a model described herein.

Samples

To the extent that the method of the present invention is performed in vitro, on an isolated tissue sample, rather than as an in vivo based screen, reference to "sample" should be understood as a reference to any sample of biological material derived from an animal such as, but not limited to, a body fluid (e.g., blood or synovial fluid or cerebrospinal fluid or bone marrow), cellular material (e.g. tissue aspirate), tissue biopsy specimens or surgical specimens. The term "sample" includes extracts and/or derivatives and/or fractions of said sample, e.g., serum, plasma, peripheral blood mononuclear cells (PBMC), a buffy coat fraction. Preferably, the sample comprises EPCs or is likely to comprise EPCs.

The sample which is used according to the method of the present invention may be used directly or may require some form of treatment prior to use. For example, a biopsy or surgical sample may require homogenization or other form of cellular dispersion prior to use. Furthermore, to the extent that the sample is not in liquid form, (if such form is required or desirable) it may require the addition of a reagent, such as a buffer, to mobilize the sample.

Collecting a cell from a subject, e.g., from a cancer in a subject, may comprise taking one or more cells from, for example, a tumour, a draining lymph node, bone marrow, a lymph sample or a blood sample depending on the type of condition (e.g., cancer and/or the state of cancer progression). Cells (e.g., cancer cells) may be isolated and identified by methods known in the art. In some examples, cancer cells may be identified by detecting generic cancer markers on cells or cancer-specific markers. For example, a breast cancer cell may be identified by immunological methods (e.g. flow cytometry, immunohistology, etc.) assessing the levels of cell surface expression of the estrogen receptor (ER), progesterone receptor (PR) or HER2/neu proteins, while prostate cancer cells may be identified by assessing the level of overexpression of prostate specific membrane antigen on prostate cells.

In some examples, negative selection can be used to isolate epithelial cells from samples of bone marrow, peripheral blood, pleural effusions or peritoneal effusions, by depleting all hematopoietic cells from these samples by immunomagnetic separation with antibodies against hematopoietic lineage markers. Positive immunomagnetic enrichment with anti-cytokeratin antibodies may be used to purify epithelial cancer cells (i.e. from carcinomas of any origin) from various samples.

When the one or more cancer cells are collected as part of a biopsy, the biopsy may be suitably processed to allow the presence of the CXCR4-CCR7 heteromultimer to be detected. Such processing will depend on the detection method used, but may include tissue homogenisation or digestion to disperse individual cells or sectioning of a frozen or paraffin embedded sample. When the one or more cells are collected in a fluid or dispersed into a fluid, the one or more cells may be used for flow cytometric analysis or applied to a slide using a cytospin and stained for immunohisotchemical analysis.

In some examples, one or more cells (e.g., cancer cells collected from a cancer) may also be cultured. In some examples, culturing the cancer cells may increase the number of cells (e.g., cancer cells) enabling easier processing of the cells (e.g., cancer cells) and/or detection of a CXCR4-CCR7 heteromultimer on the cells (e.g., cancer cells). In some examples, culturing the cells (e.g., cancer cells) from a sample containing a mixed cell population may increase the number of cells of interest (e.g., cancer cells) relative to other cells (e.g., non-cancer cells) in the sample. Methods of culturing cells are known in the art.

As will be apparent from the description and/or claims herein, diagnostic/prognostic assays described herein may require the use of a suitable control, e.g. a normal or healthy individual or a typical population, e.g., for quantification or standards derived therefrom.

In some examples, a suitable standard is a control data set comprising measurements of the level of the heteromultimer for a typical population of normal and/or healthy subjects. In one example, a control sample is not included in an assay. Instead, a suitable standard sample is derived from an established data set previously generated from a typical population. Data derived from processing, analyzing and/or assaying a test sample is then compared to data obtained for the sample population.

Methods of Treatment

The present disclosure encompasses the treatment or prevention of a variety of conditions using binding molecules and/or agents described herein.

Methods of the present disclosure are useful for treating, ameliorating or preventing the symptoms of conditions, or for improving the prognosis of a mammal. The quality of life in a mammal suffering from a condition may be improved, and the symptoms of the condition may be reduced or eliminated following treatment with the binding molecule or agent. Methods of the present disclosure are also useful for delaying development of or preventing a condition in an individual at risk of developing the condition (or a relapse thereof).

It will be apparent to the skilled artisan based on the description herein that the present disclosure also provides a method for treating or preventing a cancer in a subject wherein the cancer expresses a CXCR4-CCR7 heteromultimer, the method comprising administering to the subject a therapeutically effective amount of an agent (including a binding molecule) according to the present disclosure. In some examples, the method may be used to treat or prevent breast cancer, leukemia, gastro-intestinal tract cancer, pancreatic cancer, colo-rectal cancer, esophageal cancer, head and neck cancer, or non-small cell lung cancer. In some examples, the method may be used to treat or prevent a metastatic cancer.

As will be apparent to the skilled artisan, some methods of the present disclosure comprise administering a therapeutically effective dose of a binding molecule or agent or a prophylactically effective amount of a binding molecule or agent.

For in vivo administration of the binding molecules described herein, normal dosage amounts may vary from about lOng/kg up to about lOOmg/kg of a subject's body weight or more per day. Exemplary dosages are O.Olmg/kg to 50mg/kg, such as from O. lmg/kg to 30mg/kg, for example, lmg/kg to 20mg/kg, for example from 5mg/kg to lOmg/kg. For repeated administrations over several days or longer, depending on the severity of the condition to be treated/pre vented, the treatment can be sustained until a desired suppression of symptoms is achieved. In some examples, the binding molecule is administered at an initial (or loading) dose which is higher than subsequent (maintenance doses). For example, the biding molecule is administered at an initial dose of between about lmg/kg to about 30mg/kg. The binding molecule is then administered at a maintenance dose of between about O.OOOlmg/kg to about lmg/kg. The maintenance doses may be administered every 7- 35 days, such as, every 14 or 21 or 28 days.

In some examples, a dose escalation regime is used, in which a binding molecule is initially administered at a lower dose than used in subsequent doses. This dosage regime is useful in the case of subject's initially suffering adverse events

In the case of a mammal that is not adequately responding to treatment, multiple doses in a week may be administered. Alternatively, or in addition, increasing doses may be administered.

Dosages for a particular binding molecule or agent may be determined empirically in subjects who have been given one or more administrations of the immunoglobulin. To assess efficacy of a binding molecule or agent, a clinical symptom of a disease or condition can be monitored.

Administration of a binding molecule or agent according to the methods of the present disclosure can be continuous or intermittent, depending, for example, on the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of a binding molecule may be essentially continuous over a preselected period of time or may be in a series of spaced doses.

In one example, the binding molecule or agent is administered so as to achieve a reduction in a score assessing the severity of a condition. For example, treatment with the binding molecule achieves a reduction in one or two or three points according to an accepted score for assessing severity of the condition.

In one example, the binding molecule or agent is administered in combination with another therapeutic agent. In this regard, the binding molecule/agent and the other therapeutic agent can be administered in a single composition or in distinct compositions.

In one example, the binding molecule or agent is administered in combination with a chemotherapeutic agent. Such a combination is useful for treating, e.g., cancers and/or metastases thereof. Exemplary chemotherapeutic agents are known in the art and/or described in WO2011/008696 and/or include teniposide; cryptophycins duocarmycin spongistatin; chlorambucil, chlorophosphamide, actinomycin, bleomycins, doxorubicin, epirubicin, methotrexate, gemcitabine 5-fluorouracil (5-FU); paclitaxel, docetaxel cisplatin, oxaliplatin or vincristine.

In another example, the binding molecule is administered in combination with a an anti-inflammatory, or a pain killer or a corticosteroid, such as prednisone and/or prednisolone; or an antimalarial compound, such as hydroxychloroquine or chloroquinine; or methotrexate; or azathioprine; or cyclophosphamide; or an anti-CD20 antibody (e.g., rituximab or ofatumumab); or an anti-TNF antibody (e.g., infliximab or adalimumab or golimumab); or a CTLA-4 antagonist (e.g., abatacept, CTLA4-Ig); or an anti-BLys antibody (e.g., belimumab).

Inducing an Immune Response

The present disclosure also provides methods for inducing an immune response. In one example, the method comprises administering ligands of CXCR4 and CCR7 (e.g., SDF-1 and CCL19 and/or CCL21) to a subject. In one example, the ligands (e.g., SDF-1 and CCL19 and/or CCL21) are administered to a subject with an antigen. In another example, the method comprises administering a binding agent of the disclosure to a subject. In one example, the binding molecule is conjugated to an antigen against which an immune response is to be raised, and the binding molecule delivers the antigen to a dendritic cell.

In one example, the antigen is a cancer antigen or a tumor antigen. Any cancer or tumor antigen known to one skilled in the art may be used in accordance with the immunogenic compositions of the invention including, but not limited to, KS 1/4 pan- carcinoma antigen, ovarian carcinoma antigen (CA125), prostatic acid phosphate, prostate specific antigen, melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate specific membrane antigen, carcinoembryonic antigen (CEA), TAG-72, CO 17-1 A; human B-lymphoma antigen-CD20, GD2, ganglioside GM2, EGFR (Epidermal growth factor receptor), HER2 antigen. Additional tumor antigens are described, for example, in Novellino et al, 2005.

In another example, the immunogen is a cancer cell or a lysate thereof.

In one example, the cancer is breast cancer.

In another example, the cancer is brain cancer, e.g., glioma.

In a further example, the cancer is gastric cancer.

In another example, the cancer is prostate cancer.

In another example, the cancer is melanoma. In another example, the cancer is lymphoma, e.g., Hodgkin's lymphoma or a leukemia.

Antigens used in methods of this invention may also be an infectious disease agent including, but not limited to, influenza virus hemagglutinin, hepatitis B surface antigen, hepatitis B virus core protein and/or hepatitis B virus surface antigen or a fragment or derivative thereof.

In one example, the antigen is a DNA encoding a polypeptide antigen.

The present disclosure also encompasses isolating an immune cell using a method of the invention (e.g., a DC), loading the immune cell with antigen by contacting the cell with one or more antigens and administering the antigen loaded cell to a subject.

Suppressing an Immune Response

The present invention also provides a method of reducing or suppressing an immune response in a subject by administering a binding molecule or agent of the present disclosure to the subject.

In another example, the method comprises contacting the blood of a subject with a binding molecule of the present disclosure to thereby remove immune cells and suppress or prevent an immune response.

In yet another example, the present disclosure provides a method for suppressing an immune response comprising isolating a Treg cell by performing a method disclosed herein and administering the Treg cell to a subject. In one example, the cell is isolated based on the expression of the CXCR4-CCR7 heteromultimer and CD4 and/or CD25. In one example, the cell is additionally isolated based on the expression of a marker disclosed in WO2010/105298, such as, protease inhibitor 16 (PI 16). The present disclosure also provides a Treg cell or population enriched therefor produced by the method and/or expressing the CXCR4-CCR7 heteromultimer and/or CD4 and/or CD25.

In one example, the subject suffers from or is at risk of developing an autoimmune condition (e.g., as discussed herein) and/or the subject requires immunosuppression (e.g., is undergoing or about to undergo a transplant or suffers from graft-versus-host disease).

In one example, the subject suffers from type 1 diabetes.

In another example, the subject suffers from multiple sclerosis.

In a further example, the subject suffers from inflammatory bowel disease. In a preferred example, the subject suffers from arthritis, e.g., rheumatoid arthritis.

In a further example, the cells are administered with a graft (e.g., a cell graft or a tissue graft or an organ graft) to thereby suppress or reduce a graft-versus-host or host-versus-graft immune response.

In one example, a method of reducing or preventing an immune response or treating an autoimmune condition additionally comprises detecting the immune response, e.g., based on the presence of autoantibodies. Inducing Angiogenesis

In one example, a method of the present disclosure comprises isolating an EPC or a population of cells enriched for EPCs based on expression of a CXCR4-CCR7 heteromultimer. In one example, the method additionally comprises isolating the cell on the basis of expression of CD34 and/or CD14 and/or CD133 and/or CDla and/or CD45 and/or CD31 and/or VEGFR2. The present disclosure also provides an EPC or a population of cells enriched for EPCs expressing the CXCR4-CCR7 heteromultimer and, optionally, CD34 and/or CD14 and/or CD133 and/or CDla and/or CD45 and/or CD31 and/or VEGFR2.

In one example, the EPC or population of cells enriched for EPCs are administered to a subject to induce or enhance angiogenesis. In one example, the EPCs are administered to treat a condition, such as, a cardiovascular disease, an autoimmune disease or sepsis.

In another example, a binding molecule that enhances activity of a CXCR4- CCR7 heteromultimer or ligands of CXCR4 and CCR7 (e.g., SDF-1 and CCL19 and/or CCL21) are administered to a subject at a site at which angiogenesis is required/desired. In one example, the binding molecule or ligands are administered to treat a condition, such as, a cardiovascular disease, an autoimmune disease or sepsis.

Inhibiting Angiogenesis

In one example, a binding molecule that inhibits the activity or formation of a

CXCR4-CCR7 heteromultimer or disrupts the heteromultimer or kills a cell expressing same is used to kill endothelial cells and/or EPCs. Such binding molecules are useful for treating conditions characterized by excessive angiogenesis, such as, psoriasis, nephropathy, cancer neovascularization, cancer or retinopathy. Stem Cell Mobilization

The present disclosure additionally provides methods of inducing or enhancing stem cell mobilization comprising administering a binding molecule as disclosed herein.

Alternatively, or additionally, the method comprises administering ligands of

CXCR4 and CCR7 (e.g., SDF-1 and CCL19 and/or CCL21) to a site in a subject to which stem cells are to be mobilized. For example, the subject suffers from a condition requiring vascularization, such as ischemia, and the ligands are administered to the site requiring vascularization.

In one example, the present disclosure additionally comprises collecting or isolating the mobilized stem cells.

The present disclosure also encompasses cells isolated by this process. The disclosure also provides an isolated stem cell expressing the CXCR4-CCR7 heteromultimer, e.g., an isolated hematopoietic stem cell expressing a CXCR4-CCR7 heteromultimer.

The mobilized stem cells, e.g., hematopoietic stem cells can be administered to a subject, e.g., following myeloablation therapy for cancer or an autoimmune condition, such as rheumatoid arthritis.

In another example, the stem cells are mobilized following nonmyeloablative chemotherapy (e.g., for leukemia and/or lymphoma) or for the treatment of neutropenia.

In one example, the binding molecule is administered with another compound that induces stem cell mobilization, such as, G-CSF, filgrastim, PEG-filgrastim, AMD3100 or an anti-integrin α₄βι antibody.

Inducing Anoikis

The present disclosure also provides a method of inducing and/or enhancing anoikis or cell death, e.g., as a result of detachment of a cell from extracellular matrix. For example, the method comprises contacting a cell with a binding molecule/agent that inhibits or prevents CXCR4-CCR7 activity. In one example, the cell is an anchorage-dependent cell. In one example, the cell is a cancer cell.

Treatment of Metastasis

One exemplary form of the present disclosure is the treatment or prevention of metastasis by administering a binding molecule or agent as described herein. In one example, the method additionally comprises determining a subject at risk of suffering a metastasis, i.e., suffering from a cancer with a high metastatic potential. For example, the method additionally comprises performing a method described herein to determine a subject suffering from a cancer with a high metastatic potential.

In one example, the method comprises administering a binding molecule or an agent as described herein in combination with a chemotherapy agent and/or in combination with radiation therapy and/or in combination with another form of cancer treatment (e.g., an anti-CD20 antibody, an anti-CD52 antibody, an anti-VEGF antibody, an anti-EGFR antibody or an anti-HER2 antibody). One area in which such a method may be useful in the treatment is in the treatment of cancers that have a higher risk of metastasis during treatment (e.g., breast cancer). Thus, the binding molecule and/or agent administered according to the present disclosure can reduce the risk of metastasis, while the cancer is treated.

In another example, a binding molecule and/or agent is administered as described herein to a subject already suffering from a metastasis, e.g., a subject suffering from leukemia or lymphoma with cells in circulation.

In one example, an imaging method as described herein is performed during or after treatment to detect the presence of metastases. If metastases are detected, treatment is repeated.

In one exemplary form of the disclosure, the metastasis is a metastasis of breast cancer.

Screening Methods

The present disclosure also provides numerous methods for identifying binding molecules that bind specifically to a CXCR4-CCR7 heteromultimer or that inhibit the formation or activity of a CXCR4-CCR7 heteromultimer.

In one example, the present disclosure provides a method for identifying an agent for treating or preventing a condition in a subject, the method comprising identifying an agent that inhibits the formation or activity of a CXCR4-CCR7 heteromultimer in a cell.

In another example, the present disclosure provides a method for identifying an agent for treating or preventing cancer in a subject, the method comprising identifying an agent that inhibits the formation or activity of a CXCR4-CCR7 heteromultimer in a cell. In this regard, as described herein, the present inventors have determined an association between the presence of a CXCR4-CCR7 heteromultimer in a cancer cell and the metastatic potential of the cell. Accordingly, in some examples, an agent that inhibits the formation or activity of a CXCR4-CCR7 heteromultimer in a cell may be suitable for treating or preventing a cancer.

The method of identifying an agent that inhibits the formation or activity of a CXCR4-CCR7 heteromultimer may comprise designing and/or screening for agents that exhibit any one or more of the activities listed herein.

Methods for detecting the presence of a CXCR4-CCR7 heteromultimer include those discussed herein. Accordingly, in some embodiments, cells may be exposed to candidate agents and changes in the formation or activity of the CXCR4-CCR7 heteromultimer determined by methods including, for example, those discussed previously herein.

For example, identifying an agent that blocks expression of one or both of CXCR4 and CCR7 may comprise exposing the cell to an agent and detecting whether the agent reduces or inhibits the expression of either or both proteins. Detecting the expression of CXCR4 and/or CCR7 may be performed by measuring the level of CXCR4 and/or CCR7 mRNA or protein in the cell using methods known in the art.

Methods for identifying agents that block association between CXCR4 and CCR7 may include rational drug or protein design, screening methods and/or raising antibodies against CXCR4 and/or CCR7 epitopes. Methods are known in the art for determining the structure of binding interfaces and designing molecules capable of binding thereto. Such methods may include, for example, x-ray crystallography, protein modeling, protein nuclear magnetic resonance spectroscopy (NMR) and protein-protein docking. Screening candidate agents for binding to the binding interface between CXCR4 and CCR7 may be performed in silico or in vitro and may utilize chemical and/or protein databases or libraries. Alternatively, antibodies may be raised against a CXCR4 monomer and/or a CCR7 monomer and screened to determine which antibodies are able to bind to the CXCR4-CCR7 heteromultimer binding interface and therefore inhibit the formation of the heteromultimer.

Similarly, methods for identifying agents that block ligand binding to CXCR4, CCR7 or the CXCR4-CCR7 heteromultimer may include, for example, rational drug or protein design or screening methods as described above. In some embodiments, the method may include raising antibodies against ligands for CXCR4, CCR7 or CXCR4- CCR7 heteromultimer epitopes. Antibodies that bind to the ligands may be used to sequester the ligands thereby preventing binding to the CXCR4-CCR7 heteromultimer. Alternatively, antibodies that are capable of binding to the CXCR4-CCR7 heteromultimer and inhibit activation of the heteromultimer may be identified and used to block ligand binding. Methods for detecting ligand binding will be apparent to the skilled artisan. For example, labeled ligand, e.g., SDF-1 and/or CCL19 and/or CCL21 is contacted to a cell expressing a CXCR4-CCR7 heteromultimer or a cell membrane comprising the heteromultimer in the presence or absence of the test compound/agent and the level of binding assessed. A binding molecule/agent that reduces the level of ligand bound compared to the level in the absence of the binding molecule/agent is considered to inhibit ligand binding to the heteromultimer.

Methods for identifying agents that block one or more intracellular signaling pathways associated with CXCR4, CCR7 or the heteromultimer may include contacting cells with candidate agents and measuring one or more intracellular signaling pathways including, for example, changes in Ca²⁺ mobilization, cAMP levels and/or activation or inhibition of kinases or phosphatases. For example, tyrosine phosphorylation or dephosphorylation may be measured using phosphospecific ELISA or FACS analysis. Methods for detecting various activities of CXCR4 and/or CCR7 are described herein and are to be taken to apply mutatis mutandis to the present example of the disclosure.

In one example, an agent that inhibits the formation or activity of a CXCR4- CCR7 heteromultimer or disrupts a CXCR4-CCR7 heteromultimer is determined using an assay that utilizes BRET/FRET. In one example, CXCR4 is fused to or conjugated to a fluorescent or bioluminescent donor, e.g., as described herein. CCR7 is fused or conjugated to a fluorescent or bioluminescent acceptor, e.g., as described herein. Exemplary fluorescent donors and acceptors include cyan fluorescent protein (CFP) as the donor alongside yellow fluorescent protein (YFP) as the acceptor. Two common implementations of BRET comprise Renilla luciferase (RLuc) with either coelenterazine h (BRET ; λ = -475 nm) or coelenterazine 400a (Clz400a) substrate

2

(BRET ; _em = -395 nm) as the donor system coupled to either of the GFP mutants, YFP (BRET¹; = -530 nm) or GFP² (BRET²; = -510 nm). When the CXCR4-

CCR7 heteromultimer forms, a detectable signal is produced. The present disclosure also contemplates methods in which CCR7 is fused or conjugated to a donor and CXCR4 is linked or fused to an acceptor.

In another example, CXCR4 is linked or fused to a donor. A molecule that interacts with CXCR4 and/or CCR7 upon heterodimerization (e.g., β-arrestin or a mutant thereof) is linked or fused to an acceptor. When the heteromultimer forms, the signal emitted from the donor stimulates the acceptor and a detectable signal is produced. The present disclosure also contemplates CCR7 being fused or linked to a donor rather than CXCR4. The BRET/FRET systems described herein are useful for detecting binding molecules that inhibit formation of the heteromultimer or disrupt the heteromultimer or enhance formation of the heteromultimer. For example, a binding molecule or agent that prevents the detectable signal or prevents an increase in the detectable signal (e.g., in the presence of SDF-1 and/or CCL19 and/or CCL21) inhibits the formation of a heteromultimer. A binding molecule or agent that reduces the detectable signal inhibits the formation of a heteromultimer or disrupts the heteromultimer. A binding molecule that enhances the detectable signal (e.g., in the presence of SDF-1 and/or CCL19 and/or CCL21) induces or enhances formation of the heteromultimer.

In one example, the method additionally comprises determining the effect of the identified binding molecule/agent on activity of a CCR7 monomer or homomultimer and/or a CXCR4 monomer or homomultimer and/or a heteromultimer in which CXCR4 and CCR7 do not contact one another. In this way it is possible to identify binding molecules/agents having effects specific to the heteromultimer or to CXCR4 or CCR7. Methods for determining the effect of a binding molecule/agent on activity of CXCR4 and/or CCR7 are known in the art and/or described herein.

The present disclosure also provides methods for identifying binding molecules/agents that specifically modulate the activity of CXCR4 or CCR7, the method comprising:

(ii) contacting the molecule identified at (i) to a CXCR4-CCR7 heteromultimer or cell expressing the CXCR4-CCR7 heteromultimer and identifying a molecule that does not detectably modulate the activity of the CXCR4-CCR7 heteromultimer.

Any of the assays described herein are useful for such a method. For example, an assay is performed to detect activation of CXCR4 or CCR7 in cells expressing monomers and/or homomultimers and a molecule/agent that activates the relevant monomer/homomultimer is identified. This molecule/agent is then tested in a BRET/FRET test described herein or in a binding assay and a molecule/agent that has no detectable effect on heteromultimer formation and/or that does not bind the heteromultimer is identified. This method permits identification of selective binding molecules/agents thereby reducing off target effects in therapy and/or improving diagnostic specificity. As described herein, the present disclosure also provides various methods for identifying binding molecules that specifically bind to a CXCR4-CCR7 heteromultimer.

This present disclosure also encompasses for the provision of information concerning the identified or isolated molecule/agent. Accordingly, the screening methods are further modified by:

(i) optionally, determining the structure of the molecule/agent; and

(ii) providing the molecule/agent or the name or structure of the molecule/agent such as, for example, in a paper form, machine-readable form, or computer-readable form.

Naturally, for molecules/agents that are known, albeit not previously tested, for their function using a screen provided by the present disclosure, determination of the name and/or structure of the molecule/agent is implicit. This is because the skilled artisan will be aware of the name and/or structure of the molecule/agent at the time of performing the screen.

As used herein, the term "providing the molecule/agent" shall be taken to include any chemical or recombinant synthetic means for producing the molecule/agent or alternatively, the provision of a molecule/agent that has been previously synthesized by any person or means. This clearly includes isolating the molecule/agent.

In one example, the molecule/agent or the name or structure of the molecule/agent is provided with an indication as to its use e.g., as determined by a screen described herein.

The screening assays can be further modified by:

(i) optionally, determining the structure of the molecule/agent;

(ii) optionally, providing the name or structure of the molecule/agent such as, for example, in a paper form, machine -readable form, or computer-readable form; and

(iii) providing the molecule/agent.

In one example, the synthesized/produced molecule/agent or the name or structure of the molecule/agent is provided with an indication as to its use e.g., as determined by a screen described herein.

In one example, the molecule/agent is provided in a library of molecules/agents, each of which or a subset of which may be separated from other members (i.e., physically isolated). In such cases, a molecule/agent is isolated from the library by its identification, which then permits a skilled person to produce that molecule/agent in isolation, e.g., in the absence of other members of the library. In some examples, the screening methods described herein comprise determining the effect of an isolated and/or identified molecule/agent on CXCR4- CCR7 activity (e.g., as described herein) and/or on cells expressing same and/or in a model of a disease associated with the heteromultimer. Such an assay may be performed in vitro and/or in vivo.

In Vitro Assays

Assays for determining the effect of a binding molecule/agent on activity of CXCR4 and/or CCR7 and/or a CXCR4-CCR7 heteromultimer are described elsewhere herein. For example, an agent/binding molecule is tested using a chemotaxis assay.

The effect of a binding molecule/agent on metastasis can be determined using an invasion assay. For example, a cancer cell expressing a CXCR4-CCR7 heteromultimer is cultured on one side of a membrane, e.g., an extracellular matrix membrane. A ligand (e.g., SDF-1 and/or CCL19 and/or CCL21) is placed on the other side of the membrane that the number of cells that can penetrate the membrane is assessed. This assays is performed in the presence of absence of a binding molecule/agent. A binding molecule/agent that reduces the number of cells penetrating the membrane is considered useful for treating or preventing metastasis. In one example, the membrane comprises endothelial cells cultured thereon to mimic tissue in vivo.

In one example, the level of ADCC activity is assessed using a ⁵¹Cr release assay, an europium release assay or a ³⁵S release assay. In each of these assays, cells expressing a heteromultimer (e.g., cancer cells or metastatic cells) are cultured with one or more of the recited compounds for a time and under conditions sufficient for the compound to be taken up by the cell. In the case of a ³⁵S release assay, cells expressing the heteromultimer can be cultured with ³⁵S-labeled methionine and/or cysteine for a time sufficient for the labeled amino acids to be incorporated into newly synthesized proteins. Cells are then cultured in the presence or absence of the binding molecule/agent and in the presence of immune effector cells, e.g., peripheral blood mononuclear cells (PBMC) and/or NK cells. The amount of ⁵¹Cr, europium and/or ³⁵S in cell culture medium is then detected, and an increase in the presence of the binding molecule/agent compared to in the absence of immunoglobulin indicates that the binding molecule/agent has effector function. Exemplary publications disclosing assays for assessing the level of ADCC induced by an immunoglobulin include Hellstrom, et al. 1986 and Bruggemann, et al, 1987.

Other assays for assessing the level of ADCC induced by an immunoglobulin include ACTI™ nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. CA, USA) or CytoTox 96® non-radioactive cytotoxicity assay (Promega, WI, USA).

Alternatively, or additionally, effector function of an immunoglobulin is assessed by determining its affinity for one or more FcyRs, e.g., as described in US7317091.

Clq binding assays may also be carried out to confirm that the binding molecule/agent is able to bind Clq and may induce CDC. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al, 1996.

An exemplary in vitro method for determining the effect of a compound on an autoimmune condition and/or graft rejection and/or graft versus host disease is a mixed lymphocyte reaction (MLR) or a mixed lymphocyte culture (MLC). Such a method involves culturing a mixture of cells, e.g., T cells and/or dendritic cells expressing a CXCR4-CCR7 heteromultimer and allotypically different T cells. Several measures may then be performed to measure suppression of an immune response in the presence or absence of a binding molecule/agent or a cell isolated by a method described herein, e.g., cell proliferation is then measured using a standard method, e.g., ¹³H thymidine incorporation (which indicates proliferation of active T cells indicating that the binding molecule/agent/cells are not active); and/or cytokine secretion by T cells which can indicate (immunosuppression or otherwise). Such a method is useful for identifying binding molecules/agents/cells having immunosuppressive activity. An exemplary MLR is described in Wang et al, 2008.

Alternatively, or in addition an in vitro method for determining the effect of a compound on an autoimmune condition and/or graft rejection and/or graft versus host disease is a 5,6-carboxy fluorescein diacetate succinimidyl ester (CFSE) suppressor assay. In such an assay CD4+CD25- cells are labeled with CFSE. CD4⁺CD25^" CFSE labeled T cells expressing a CXCR4-CCR7 heteromultimer are then cultured with irradiated PBMCs in the presence of varying amounts of binding molecule/agent/cells isolated according to the present disclosure. After a sufficient time, proliferation of the CD4+ CD25- CFSE labeled T cells is analyzed by flow cytometry. Each CFSE signal peak represents one division cycle. The ability of the binding molecule/agent/cells to suppress cell proliferation is assessed by comparing CFSE signal peaks of CD4⁺CD25^" CFSE labeled T cells with and without the presence of the compound/agent/cells. An exemplary CFSE suppressor assay is described in Venken et al, (2007). As will be apparent to the skilled artisan, methods of screening may involve detecting levels of cell death, cell proliferation and/or cell survival. Such methods are known in the art. In Vivo Assays

The in vivo assay used to assess a binding molecule/agent/cell will be dependent on the condition to be treated/prevented.

For example, to assess the effect of a binding molecule/agent on cancer or metastasis, the molecule/agent is administered to a test subject at the time of, prior to or following administration of tumor cells. Optionally, the tumor cells are labeled to facilitate detection (e.g., as described herein). The presence/absence and/or size of any resulting tumor and/or metastases is then assessed and compared to subjects to which the cells but not the binding molecule/agent has been administered. A binding molecule/agent that reduces tumor size and/or prevents tumor formation and/or prevents metastasis or reduces the number of metastases is considered to be useful for treating cancer or metastasis. The level of angiogenesis can also be assessed to determine the effect of the compound on angiogenesis/EPCs.

In another example, a test binding molecule/agent is administered at the time of or prior to administration of a composition comprising an immunogenic compound. The immune response is then measured against the immunogenic compound, e.g., antibody response (e.g., by ELISA/FLISA) or a T cell response (e.g., by ELISPOT or Fluorospot). Alternatively, or in addition, the binding molecule/agent and composition are administered to a subject suffering from or developing a condition treatable by an immune response, e.g., an infection or a tumor. Suitable models are known in the art and/or described herein.

Various models of autoimmune conditions are also known in the art. For example, binding molecules/agents/cells can also be administered to NOD mice to test their ability to suppress, prevent, treat or delay diabetes (e.g., as described in Tang et al, (2004)) and/or to a mouse model of GVHD (e.g., as described in Trenado (2002)) and/or to a mouse model of psoriasis (e.g., Wang et al, 2008) and/or to a model of rheumatoid arthritis e.g., a SKG strain of mouse (Sakaguchi et al), rat type II collagen arthritis model, mouse type II collagen arthritis model or antigen induced arthritis models in several species (Bendele, 2001)) and/or a model of multiple sclerosis (for example, experimental autoimmune encephalomyelitis (EAE; Bradl and Linington, 1996)) and/or inflammatory airway disease (for example, OVA challenge or cockroach antigen challenge (Chen et al, 2007; Lukacs et al, 2001) and/or models of inflammatory bowel disease (e.g., dextran sodium sulphate (DSS)-induced colitis or Muc2 deficient mouse model of colitis (Van der Sluis et al, 2006) or CD45Rb adoptive transfer model of colitis (e.g., Kanai et al, 2006)).

In vivo models of inflammation are available which can be used to assess the effects of binding molecules/agents/cells in vivo as therapeutic agents. For example, leukocyte infiltration upon intradermal injection of a chemokine (e.g., CCL19 and/or CCL21 and/or SDF-1) and a binding molecule/agent/cell into a suitable animal, such as rabbit, mouse, rat, guinea pig or rhesus macaque can be monitored (see, for example, Van Damme et al. (1992); Jose et al. (1994)).

In one example, skin biopsies are assessed histologically for infiltration of leukocytes (e.g., eosinophils, granulocytes). In another example, labeled cells (e.g., stably transfected cells expressing a CXCR4-CCR7 heteromultimer) capable of chemotaxis and extravasation are administered to the animal. A binding molecule/agent/cell to be assessed can be administered, either before, simultaneously with or after the labeled cells are administered to the test animal. A decrease of the extent of infiltration in the presence of binding molecule/agent/cell as compared with the extent of infiltration in the absence of binding molecule/agent/cell is indicative of inhibition.

In another example, a binding molecule/agent is administered to a subject and the number of target cells in a sample assessed. Target cells will be apparent to the skilled artisan based on the disclosure herein. For example, the number of metastatic cells in a blood sample or a tissue sample is assessed. Alternatively, the number of B cells or T cells or DCs or pDCs or Treg cells or EPCs or hematopoietic stem cells in a sample (e.g., a blood sample or a plasma sample) is assessed.

Kits

The present disclosure also provides therapeutic/prophylactic/diagnostic kits comprising binding molecules/agents/cells of the present disclosure for use in the present detection/isolation/diagnostic/prognostic/treatment/prophylactic methods. Such kits will generally contain, in suitable container means, binding molecules/agents/cells of the present disclosure. The kits may also contain other compounds, e.g., for detection/isolation/diagnosis/imaging or combined therapy. For example, such kits may contain any one or more of a range of anti-inflammatory drugs and/or chemotherapeutic or radiotherapeutic drugs; anti-angiogenic agents; anti-tumor cell antibodies; and/or anti-tumor vasculature or anti-tumor stroma antibodies or coaguligands or vaccines. Exemplary kits comprise a binding molecule that specifically binds to a CXCR4-CCR7 heteromultimer.

In one example, the kit additionally comprises a reagent to facilitate detection (a detectable tag and/or a substrate of a detectable tag. Such kits may additionally comprise a positive control.

In another example, the kit is for isolating a cell. In such kits the binding molecule may be labeled with a detectable tag to facilitate FACS. The binding molecule may also be labeled with a magnetic or paramagnetic particle to facilitate MACS. The binding molecule may also be immobilized on a solid or semi-solid substrate to facilitate isolation.

In a further example, the kit is for treatment or prevention of a condition. In such kits the binding molecule/agent may be provided in solution or in a lyophilized form, optionally with a solution for resuspension. The binding molecule/agent may be conjugated to a therapeutic compound or the kit may include a therapeutic compound for conjugation thereto. As discussed above, the kit may also comprise additional therapeutic or prophylactic compounds.

Alternatively or in addition, a kit for therapy or prophylaxis comprises a cell according to the present disclosure.

The present disclosure includes the following non-limiting examples. It is to be understood that the following description is for the purpose of describing exemplary forms of the disclosure only and is not intended to be limiting with respect to the above description.

Example 1: Down-Regulation of CCR7 Abrogates Lung Colonization in a SCID Mouse Metastatic Model

CCR7 was knocked down in highly invasive human breast cancer cells MDA- 231 by shRNA. The ability of this knock down to sufficiently inhibit CCR7 mediated functional responses in these cells was confirmed by the inability of both CCR7 ligands, CCL19 and CCL21, to stimulate Ca²⁺ mobilization and chemotaxis (not shown).

A dual color fluorescence approach was then employed to determine the effect of the CCR7 downregulation on lung metastasis in vivo. Control or CCR7 shRNA expressing cells were tagged with GFP and wild type MDA-231 cells were tagged with RFP. Cells with different color tags were mixed and injected into the tail vein of SCID mice. After 10 weeks of growth, a striking difference was observed between the two groups. While wild type and control shRNA expressing cells produced metastases with similar effectiveness (Figure 1 - bottom panel), CCR7 knockdown severely abrogated formation of lung lesions lungs with only green color (wild type) metastasis detected in this group (Figure 1 - top panel). No differences in cell growth were detected in an in vitro proliferation assay (not shown).

Example 2: CXCR4-CCR7 Heteromultimers Determine Pro-Metastatic Function of these Receptors in Breast Cancer Cells

Breast cancer cells were seeded on glass cover slips, grown to sub-confluence and fixed in a 3.7% solution of formaldehyde in PBS. Cells were then simultaneously stained with primary human anti-CXCR4 mouse monoclonal antibodies and mouse anti-CCR7 (cross-reacting with human CCR7) rat monoclonal antibodies overnight at 4°C followed by incubation with respective goat anti-mouse FITC-conjugated and donkey anti-rat Alexa-647 conjugated secondary antibodies for lhr at room temperature. Dual-fluorescent images were recorded with a confocal fluorescent microscope and CXCR4 and CCR7 co-localization regions were detected using co- localization analysis software.

As shown in Figure 2, endogenous CXCR4 and CCR7 showed specific membrane co-localization in MDA-231, but only a proportion of both CXCR4 and CCR7 was found to co-localize suggesting the presence of separate and common receptor pools potentially sequestered in various membrane sub-domains. Similar results were obtained using MDA-361 cells (data not shown).

Fluorescence Resonance Energy Transfer by acceptor photobleaching (FRET) was next employed to further investigate the interaction between endogenous CXCR4 and CCR7. Breast cancer cells were again grown on cover slips, fixed and stained with CXCR4 and CCR7 specific antibodies. Cy3 and Cy5 conjugated secondary antibodies were then used to generate a donor-acceptor fluorescence pair. Cell images were processed and recorded using FRET Wizard algorithm from Leica Application Suite advanced fluorescence software according to manufacturer recommendations.

FRET has been highly correlated with direct molecular interaction and FRET imaging analysis has been used to investigate coupling of GPCRs, including chemokine receptors. Figure 3 shows the difference in donor (Cy3) fluorescence after complete bleaching of the acceptor (Cy5). FRET intensity measurements in MDA-361 cells (Figure 3) and MDA-231 cells (not shown), showed a specific and significant increase, thus showing a direct interaction between CXCR4 and CCR7 in these cells, indicating heteromultimerization between these two chemokine receptors in the cells. FRET data were also supported by immunoprecipitation of endogenous receptors from MDA-361 (Figure 4 and Figure 5) and MDA-231 (not shown) breast cancer cells.

To perform the immunoprecipitation, breast cancer cells were lysed in buffer containing 1% Triton-X-100, 150 mM NaCl, 10 mM Tris-HCl, 10 mM sodium vanadate, 10 mM sodium fluoride, 10 mM protease inhibitor cocktail (Sigma) and 10 mM PMSF, pH 8.5. Immunocomplexes were formed from 500 μg of protein lysate with 1 μg of monoclonal mouse anti-human CXCR4 antibodies, captured by incubation with protein G-coupled magnetic beads and separated on a magnetic column (both from Miltenyi Biotech, Bergisch Gladbach, Germany). Columns were washed several times with lysis buffer and bound protein complexes were eluted with pre -heated sample buffer as recommended by the magnetic beads supplier. Samples were then resolved by SDS-PAGE and subjected to Western blot to detect CCR7 and ϋβ protein bands.

CXCR4, CCR7 and G proteins were found to specifically co-precipitate together from metastatic cancer cell lysates, indicating the presence of a higher order receptor complex in these cells. The complex was present in unstimulated metastatic cancer cells (Figure 4, lane 2) demonstrating constitutive association of the receptors in metastatic cancer cells. Interestingly, addition of two receptor ligands (CXCL12 and CCL21) differentially regulated CXCR4 - CCR7 heteromultimerization (Fig.4, lanes 3- 6) suggesting distinct allosteric modifications of the multimer depending on which receptor is being ligated. The ability of these two receptors to form multimers with respect to the cells invasive phenotype was investigated and it was found that CXCR4 and CCR7 do not interact in non-metastatic MDA-134 and MDA-453 cells (Figure 5) nor are the receptors functional. In contrast, CXCR4 and CCR7 heteromultimers exist in metastatic cells (MDA-231 & MDA-361) and the receptors are functional. These results demonstrate the requirement for a CXCR4-CCR7 heteromultimer for receptor functionality and provide a molecular basis for a receptor "on-switch" that is associated with metastatic transformation.

A challenge in proving the existence and functional significance of receptor heteromultimers in native tissues is to demonstrate that the direct physical interaction of the two receptors is important for the modification of their signaling and/or function. To address this question breast cancer cells were generated and characterized in which either CXCR4 or CCR7 has been knocked down by retrovirally-mediated RNAi. Apoptosis and intracellular calcium changes were investigated.

Control cells or specific CXCR4 or CCR7 shRNA-transduced breast cancer cells were seeded on polyHEMA-coated dishes and cultured for 48 hrs. Cells were fixed in 3.7% formaldehyde and apoptotic cells were detected and quantified by FACS analysis using the Terminal Deoxynucleotidyl Transferase-mediated dUTP TRITC Nick-end Labelling Assay (TUNEL Assay) kit (Roche) according to the manufacturer's instructions.

To measure changes in intracellular calcium, suitably modified breast cancer cells (10⁶/ml) were incubated for 15 minutes at with 2 μΜ of Fura-2AM at 37°C (Molecular Probes Eugene, OR) and stimulated with CXCL12 at 150 ng/ml final concentration and/or CCL21 at 375 ng/ml final concentration. Changes in intracellular calcium were quantified using an Aminco-Bowman Series 2 luminescence spectrometer.

Using metastatic MDA-231 cells, a significant decrease in surface numbers of one chemokine receptor was observed to lead to the loss of function of the other receptor. Thus, either of the remaining receptors could not support inhibition of anoikis (Figure 6) or induction of intracellular calcium mobilization (Figure 7) when compared with control cells expressing a vector only control. Importantly, when exogeneous CXCR4 was reintroduced into CXCR4 RNAi-expressing cells the function of CCR7 was restored (Figure 7B), providing further evidence for the requirement of a CXCR4- CCR7 heteromultimer for their function in metastatic breast cancer cells. Example 3: Antibodies Against CXCR4 Competitively Inhibit Binding of Antibodies Against CCR7 to CXCR4-CCR7 Heteromultimers

Cells shown herein-above to express a CXCR4-CCR7 heteromultimer (MDA- 361 and MDA-231 cells) were simultaneously stained with rat anti-human CCR7 antibody (APC conjugated, clone 3D 12, eBioscience) with or without increasing amounts of mouse anti-human CXCR4 antibody (clone 1D9, BD Biosciences) and analyzed by FACS. ZR-75-1 and T47D breast cancer cells were also stained with the anti-CCR7 antibody in the presence of increasing amounts of anti-CXCR4 antibody. Since these cells generally do not have a migratory phenotype (e.g., they generally do not cause metastases when injected into animals), they are expected not to express significant levels of the heteromultimer. As shown in Figures 8A and B the amount of anti-CCR7 antibody bound to MDA-361 and MDA-231 cells was reduced in the presence of anti-CXCR4 antibodies. This effect was not observed in ZR-75-1 and T47D cells.

Inhibition of the anti-CCR7 monoclonal antibody by the anti-CXCR4 monoclonal antibody for the binding on the surface of cancer cells indicates that the binding epitopes for these antibodies are located in close proximity or overlapping thus providing further evidence for the formation of the CXCR4-CCR7 heteromultimer. The inhibition was observed only in cells shown to express the heteromultimer (and associated with conditions caused by cell migration, such as, metastasis) and not in cells that are not expected to express significant levels of the heteromultimer, e.g., as a result of their lack of migratory phenotype. Studies with other monoclonal antibodies reactive with CXCR4 and CCR7 respectively, but with a different binding epitopes to 1D9 and 3D12 described above demonstrated that the binding competition for the two clones described above is specific and highly selective (not shown). REFERENCES

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Claims

CLAIMS:

1. An isolated or recombinant CXCR4-CCR7 heteromultimer.

2. The heteromultimer of claim 1 comprising at least one CXCR4 monomer with contact with at least one CCR7 monomer.

3. An expression construct comprising a nucleic acid encoding CXCR4 operably linked to a promoter and a nucleic acid encoding CCR7 operably linked to a promoter.

4. A recombinant cell comprising the heteromultimer of any one of claims 1 to 3.

5. The cell of claim 4, wherein the CXCR4 and/or CCR7 is a recombinant protein.

6. An isolated population of cells enriched for cells expressing a CXCR4-CCR7 heteromultimer.

7. The cell of claim 4 or 5, which is selected from the group consisting of a cancer cell, an immune cell, a mesodermal cell, an endothelial cell, an epithelial cell, an ectodermal cell and a stem cell or the population of cells of claim 6 enriched for cells selected from the group consisting of cancer cells, immune cells, mesodermal cells, endothelial cells, epithelial cells, ectodermal cells and stem cells.

8. The cell of claim 4 or 5, which is a cancer cell or the population of cells of claim 6 enriched for cancer cells.

9. A binding molecule that specifically binds to a CXCR4-CCR7 heteromultimer.

10. The binding molecule of claim 9, which does not detectably bind to a CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer.

11. The binding molecule of claim 9 or 10, which is a small molecule, an aptamer, a protein, a ligand or an antibody or antigen binding fragment thereof.

12. The binding molecule of claim 9 or 10, which is an antibody or an antigen binding fragment thereof.

13. The binding molecule of any one of claims 9 to 12, which kills a cell to which it binds.

14. The binding molecule of claim 13, which is an antibody or antigen binding fragment thereof capable of inducing effector function to thereby kill a cell to which it binds and/or that is conjugated to a toxic compound that kills a cell to which is binds.

15. A composition comprising the heteromultimer of claim 1 or 2 or the expression construct of claim 3 or the cell of any one of claims 5, 7 or 8, the population of cells of any one of claims 6 to 8 or the binding molecule of any one of claims 9 to 14 and, optionally, a pharmaceutically acceptable carrier.

16. A method for detecting a CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer, the method comprising contacting a sample with the binding molecule of any one of claims 9 to 14 such that the molecule binds to the heteromultimer, if present, and detecting the bound heteromultimer.

17. A method for diagnosing and/or prognosing a condition in a subject, the method comprising performing the method of claim 16 to detect a CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer or the level of the heteromultimer or the cell in a sample from a subject, wherein detection of CXCR4- CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer or the level of the heteromultimer or the cell or failure to detect CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer is diagnostic or prognostic of the condition.

18. A method for monitoring the efficacy of treatment of a condition, the method comprising performing the method of claim 16 to detect a CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer or the level of the heteromultimer or the cell in a sample from a subject receiving treatment for the condition, wherein detection of CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer or the level of the heteromultimer or the cell or failure to detect CXCR4-CCR7 heteromultimer or a cell expressing the CXCR4-CCR7 heteromultimer is indicative of whether or not the subject is responding to treatment .

19. A method for ascertaining the metastatic potential of a cancer cell, the method comprising detecting the presence or amount of a CXCR4-CCR7 heteromultimer in/on the cell or in a sample that contains or has contained the cell, wherein the presence or amount of the CXCR4-CCR7 heteromultimer in/on the cell or in a sample that contains or has contained the cell is indicative of the metastatic potential of the cell.

20. A method for treating a condition comprising obtaining the results of a method of any one of claims 17 to 19 and administering an agent selected from the group consisting of:

(ii) an agent that kills a cell expressing a CXCR4-CCR7 heteromultimer;

(iii) an agent reduces or prevents the activity or expression of CXCR4;

(iv) an agent that kills a cell expressing CXCR4;

(v) an agent reduces or prevents the activity or expression of CCR7; and

(iv) an agent that kills a cell expressing CCR7.

21. A method of treating or preventing a condition, the method comprising administering the cell of any one of claims 5, 7 or 8, the population of cells of any one of claims 6 to 8 or the binding molecule of any one of claims 9 to 14 or the composition of claim 15 to a subject in need thereof.

22. The method of any one of claims 16 to 18, 20 or 21, wherein the condition is selected from the group consisting of cancer, an inflammatory condition, an autoimmune condition, an obstructive condition, an infectious condition, an excitotoxic condition, an immunodeficiency condition, a metabolic condition, pain, a circulatory condition and a degenerative condition.

23. The method of any one of claims 16 to 18, 20 or 21, wherein the condition is cancer metastasis.

24. A method for treating or preventing a cancer or metastasis thereof in a subject wherein the cancer expresses a CXCR4-CCR7 heteromultimer, the method comprising administering to the subject the binding molecule of any one of claims 9 to 14, wherein the molecule reduces or prevents the activity of a CXCR4-CCR7 heteromultimer and/or kills a cell expressing a CXCR4-CCR7 heteromultimer.

25. A method for identifying an agent for treating or preventing a condition in a subject, the method comprising identifying an agent that inhibits the formation or activity of a CXCR4-CCR7 heteromultimer in a cell.

26. The method of claim 25 comprising contacting a CXCR4-CCR7 heteromultimer with an agent and detecting the amount or activity of the heteromultimer, wherein a reduction in the amount or activity of the heteromultimer is indicative of an agent that inhibits the formation or activity of a CXCR4-CCR7 heteromultimer in a cell.

27. A method for identifying a binding molecule, the method comprising identifying binding molecule that specifically binds to a CXCR4-CCR7 heteromultimer.

28. The method according to claim 27 comprising identifying a binding molecule that binds to binds to a CXCR4-CCR7 heteromultimer and that does not detectably bind to CXCR4 monomer and/or a CXCR4 homomultimer and/or a CCR7 monomer and/or a CCR7 homomultimer.

29. The method of claim 27 or 28 additionally comprising identifying a binding molecule that kills a cell to which it binds.

30. A method for identifying a binding molecule that specifically binds to CXCR4 or CCR7, the method comprising:

(i) identifying a binding molecule that binds to a CXCR4 monomer and/or a CXCR4 homomultimer or a CCR7 monomer and/or a CCR7 homomultimer; and

(ii) contacting the molecule identified at (i) to a CXCR4-CCR7 heteromultimer and identifying a molecule that does not detectably bind to the heteromultimer.

31. A method for identifying a binding molecule that specifically modulates the activity of CXCR4 or CCR7, the method comprising:

32. The method of any one of claims 27 to 31, wherein the binding molecule is an antibody or antigen binding fragment thereof.

33. The method of any one of claims 25 to 32, additionally comprising providing the agent or binding molecule.

34. The method of claim 33, additionally comprising formulating the agent or binding molecule into a pharmaceutical composition.