HK1124760A - Anti-ccr7 receptor antibodies for the treatment of cancer - Google Patents

Description

Antibodies against the CCR7 receptor for the treatment of cancer

Technical Field

The present invention relates generally to cancer therapy. More specifically, the invention relates to the treatment of cancer whose tumor cells are CCR7 receptor expressing cells with an antibody to the CCR7 receptor capable of selectively killing, attenuating the migration and/or blocking the spread of said tumor cells.

Background

Cancer is a disease characterized by uncontrolled cell division (or increased survival or resistance to apoptosis) and the ability of the cell to invade other adjacent tissues (invasion) and spread to other areas of the body where the cell is not normally present (metastasis), through lymphatic and blood vessels, through the circulation of the blood stream, and then to invade normal tissues elsewhere in the body. Tumors are classified as benign and malignant according to whether they can spread by invasion and metastasis: amphoteric tumours are tumours which are unable to spread by invasion and metastasis, i.e. they can only grow locally; while malignant tumors are tumors that can spread by invasion and metastasis.

Cancers can be classified into several types according to the cell type and cell location from which they are derived, i.e., cancers (calicomas), which occur from cells covering the external and internal surfaces of the body, such as the skin, digestive tract, or glands; leukemias, which begin with blood-forming tissues such as bone marrow and cause a large number of abnormal blood cells to be produced and enter the bloodstream; lymphoid cancer, which is a cancer originating from lymph nodes and tissues of the body's immune system; melanoma, which occurs in melanocytes; sarcoma (sarcoma) which is derived from the connective tissue of bone or muscle; and teratomas, which are derived from germ cells.

Cancer can be treated by surgery, chemotherapy, radiation therapy, immunotherapy, or other methods. The choice of treatment depends on the location and grade of the tumor and the stage of the disease.

A significant problem to be solved in tumor treatment protocols is the desire to "kill all cells". This means that a more effective treatment regimen is closer to killing cells that are all referred to as "clonogenic" malignant cells, i.e., cells that have the ability to grow uncontrollably and result in the formation of tumor masses that can be removed by treatment.

Another tumor treatment strategy is the use of "immunotoxins", in which anti-tumor cell antibodies are used to deliver the toxin to the tumor cells. However, immunotoxin therapy, like the above-described chemotherapy methods, also has significant drawbacks. For example, antigen-negative or antigen-deficient cells can survive and reconstitute tumors or cause further metastasis. Even if the primary cancer is completely removed, the malignant tumor is often metastatic. The formation of malignant metastases, starting from the primary tumor, at locations in the body, either distant or near, is one of the most serious consequences of cancer and no satisfactory treatment is currently available. Currently available cancer therapies have limited effectiveness in preventing metastasis or produce serious undesirable side effects.

Although the specific delivery of therapeutic agents such as anti-cellular agents, toxins and coagulation factors to tumor masses represents a significant advance in cancer treatment approaches, there remains room for additional or even alternative therapies. The identification of additional targets that allow tumor destruction specifically in vivo would naturally help to expand the number of target selections.

New therapeutic strategies for cancer treatment are evolving towards the use of specific therapies, including monoclonal antibodies (mabs) against different antigens expressed by tumor cells, which may hopefully cure the disease.

Antigen selection in immunotherapy must take into account the tumor specificity of the antigen, the antigen density on the surface of tumor cells, and antigen modulation or internalization of antigen-antibody complexes, which can reduce the ability to produce cell death. In most cases, complement-dependent cytolysis (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) are thought to be responsible for clinical use of uncoupled mAbs, although induction of apoptosis or cell cycle arrest may also play an important role in other cases.

Cancer cells may express certain molecular receptors. Different studies have shown that CC chemokine receptor 7(CCR7) is expressed in different tumor cells, such as B-cell chronic lymphocytic leukemia, non-hodgkin lymphoma, breast cancer, malignant breast tumors, and the like. Furthermore, it appears that the CCR7 receptor plays a role in lymph node metastasis in different cancers such as gastric cancer, melanoma, non-small cell lung cancer, T cell leukemia cells, and the like. Thus, the chemokine receptor (CCR7) can be selected as a potential target for mAb therapy of cancer.

CCR7 is a 7 transmembrane domain G protein-coupled receptor (GPCR). The G protein-coupled receptor family (GPCRs) includes receptors for hormones, neurotransmitters, growth factors and viruses [ Yoshie O, Imai T, Nomiyama h.novel lymphoma-specific CC chemokines and dtheir receptors (novel lymphocyte-specific CC chemokines and their receptors). J leukocbiol biol.1997; 62: 634-644; kim CH, Pelus LM, White JR, Applebaum E, Johanson K, Broxmeyer HE. CK beta-11/macrophage in nematotropein-3 beta/EBI1-ligand chemoattractant is an effective chemoattractant for T and B cells J Immunol.1998; 160: 2418-; dieu MC, Vanberviet B, Vicari A, et al.selective recovery of immature and mature dendritic cells by differentiation chemokines expressed induced dendritic cells (different chemokines expressed at different anatomical locations selectively recruit immature and mature dendritic cells). J Exp Med.1998; 188: 373-386; willimann K, Legler DF, Loetscher M, et al, the chemokine SLC expressed in T cell areas of lymphnodes and mucosal lymphoids tissue and activated T cells CCR7 (chemokine SLC is expressed in the T cell area of lymph nodes and mucosal lymphoid tissue and attracts activated T cells through CCR 7.) Eur JImmunol.1998; 28: 2025-2034; yoshida R, Nagira M, lmai T, et al, EBI1-ligand chemical (ELC) antibodies a branched spectra of lymphocytes: activated T cells linear up-regulated CCR7 and effective transplantation ELC (EBI1 ligand chemokine (ELC) attracts multiple lymphocytes: activated T cells strongly upregulate CCR7 and migrate efficiently to ELC.) Int Immunol.1998; 10: 901-910; sallusto F, Schaerli P, Loetscher P, et al, Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. Eur JImmunol.1998; 28: 2760-2769].

A specific example of a leukemia in which tumour cells express the CCR7 receptor is Chronic Lymphocytic Leukemia (CLL), the most common type of human adult leukemia. The leukemia is B cell leukemia consisting of strongly anti-apoptotic CD5⁺Accumulation of a single clone of B cells is characterized by the resistance to apoptosis due to dysregulation of extracellular or intracellular signaling involved in programmed cell death. Despite its very low proliferation index, peripheral blood lymphocyte counts reached greater than 5X 10³Value of/. mu.LAnd leukemia cells showed a clear tendency to invade lymph nodes, pancreas and bone marrow.

Treatment of CLL is based on the use of purine analogues as first-line regimens, in particular fludarabine, alone or in combination. To date, the only therapeutic combination that resulted in a higher complete remission rate than fludarabine was rituximab, an anti-CD 20 monoclonal antibody, in combination with fludarabine or fludarabine and cyclophosphamide. Furthermore, the molecular improvement of bone marrow aspirate in CLL patients using the above combination creates the possibility that CLL could potentially be cured without stem cell transplantation. Obtaining the best initial response and removing CLL cells from the inoculum of patients receiving autologous transplantation constitute some of the major therapeutic challenges for CLL.

Mantle Cell Lymphoma (MCL) is an aggressive subtype of B-cell non-hodgkin's lymphoma. Cells were characterized as CD20+ CD5+ CD 23-and t (11; 14) and cyclin D1 over-expressed. rituximab-CHOP (cyclophosphamide, hydroxydaunorubicin, Oncovin, and prednisone) and subsequent stem cell transplantation or by rituximab-HyperCVAD (cyclophosphamide, vincristine, doxorubicin hydrochloride, and dexamethasone) are commonly used. However, MCL is not cured in most cases, clearly indicating the need for new treatments.

CLL patients showing clinical lymphadenopathy have recently been demonstrated to have higher migratory responses of CLL cells to CCR7 ligand, the homeostatic chemokines CCL19(MIP3- β) and CCL21(6Ckine) in vitro. Therefore, blocking CLL cells from entering secondary lymphoid tissues using the CCR7 monoclonal antibody may be another advantage. Also from this perspective, non-lymphoid tumors expressing ectopic CCR7 have the ability to metastasize to secondary lymphoid organs, whereas tumors lacking this molecule or other chemokine receptors involved in homing to secondary lymphoid organs exhibit minimal nodal spread.

Thus, there is a great need for additional cancer therapies, particularly cancer and tumor cells that express the CCR7 receptor. Advantageously, the therapy should allow for specific tumor destruction in vivo, for example by killing, attenuating the migration and/or blocking the spread of tumor cells.

Disclosure of Invention

The present invention is based on the discovery that the CCR7 receptor is highly expressed in certain tumor cells, plays a major role in lymphocyte entry into secondary lymphoid organs including Lymph Nodes (LN), and its expression is limited to naive T and B lymphocytes, as well as mature Dendritic Cells (DC), thus the CCR7 receptor becomes an interesting target for the use of monoclonal antibodies in the treatment of cancer, particularly cancers in which the tumor cells express the CCR7 receptor. The inventors have surprisingly observed that monoclonal antibodies to CCR7, i.e. antibodies that recognize an epitope on the CCR7 receptor and are capable of binding to the CCR7 receptor, are capable of killing CLL and MCL cells, i.e. tumor cells expressing the CCR7 receptor, in vitro, but are substantially incapable of killing non-tumor cells expressing CCR7, such as T lymphocytes.

Accordingly, the present invention relates generally to the use of an antibody or antigen-binding fragment thereof that binds to the CCR7 receptor for killing tumor cells expressing the CCR7 receptor or inducing apoptosis of tumor cells expressing the CCR7 receptor for the preparation of a pharmaceutical composition for the treatment of cancer; in particular embodiments, the cancer to be treated is characterized by tumor cells expressing the CCR7 receptor.

Thus, in one aspect, the invention relates to the use of an antibody, or antigen-binding fragment thereof, that binds to the CCR7 receptor for the preparation of a pharmaceutical composition for killing tumor cells expressing the CCR7 receptor or inducing apoptosis in tumor cells expressing the CCR7 receptor.

In another aspect, the invention relates to a method of killing a tumor cell expressing CCR7 or inducing apoptosis of a tumor cell expressing CCR7, comprising contacting the cell with an antibody or antigen binding fragment thereof that binds to the CCR7 receptor.

In another aspect, the invention relates to a method of killing tumor cells expressing the CCR7 receptor or inducing apoptosis in tumor cells expressing the CCR7 receptor in an individual in need of such treatment, comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to the CCR7 receptor.

In another aspect, the invention relates to a method of attenuating migration of tumor cells expressing a CCR7 receptor to secondary lymphoid tissue and/or blocking dissemination of tumor cells into secondary lymphoid tissue, comprising contacting said tumor cells with an antibody or antigen-binding fragment thereof that binds to a CCR7 receptor.

In another aspect, the invention relates to a method of attenuating migration of tumor cells expressing a CCR7 receptor to secondary lymphoid tissue and/or blocking dissemination of tumor cells into secondary lymphoid tissue in an individual in need of such treatment, comprising administering to said individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to a CCR7 receptor.

In another aspect, the invention relates to a method of identifying a compound for killing tumor cells expressing the CCR7 receptor or inducing apoptosis in tumor cells expressing the CCR7 receptor, comprising:

a) contacting a cell expressing the CCR7 receptor with a candidate compound that binds to an antibody or fragment thereof directed against the CCR7 receptor, and

b) determining whether said candidate compound kills said cells expressing the CCR7 receptor,

the compound for killing the cell expressing the CCR7 receptor is a compound which is potentially used for killing a tumor cell expressing a CCR7 receptor or inducing apoptosis of a tumor cell expressing a CCR7 receptor.

Brief description of the drawings

In FIG. 1, panel A shows that the surface expression of CCR7 is higher on CLL cells than on normal B and T lymphocytes. Peripheral blood samples from 11 CLL patients and 6 healthy donors were analyzed by Flow Cytometry (FCM) to differentiate between different lymphocyte populations, whose corresponding CCR7 surface density was measured as Mean Fluorescence Intensity (MFI). Bars are expressed as mean ± standard deviation; in panel B, the anti-CCR 7 monoclonal antibody did not cause receptor pinocytosis. Peripheral Blood Mononuclear Cells (PBMCs) from CLL patients were incubated with anti-CCR 7 monoclonal antibody (2 μ g/ml), IgM monoclonal antibody (clone 2H4) and IgG2a monoclonal antibody (clone 150503), or with CCL19(1 μ g/ml), one of the physiological receptors of CCR7 as a positive control, for different times ranging from 30 seconds to 60 minutes. CCR7MFI was then determined in electronically gated CD19+ CD5+ CCL cells using FCM. The curves represent the expression of CCR7 versus the basal CCR7 MFI. Representative examples are shown.

Figure 2 shows that both anti-CCR 7 monoclonal antibodies mediate strong and specific complement dependent lysis (CDC) of CLL cells. PBMCs from CLL patients were incubated with anti-CCR 7 monoclonal antibody or its Isotype Control (IC) and then exposed to rabbit complement for 1 hour. Cell lysis was determined by 7-AAD incorporation and FCM analysis in electronically gated CLL cells and normal T cells. Bars represent mean ± standard deviation of 11 cases. (A) anti-CCR 7IgM monoclonal antibody (2. mu.g/mL), (B) anti-CCR 7IgG monoclonal antibody (2. mu.g/mL).

Fig. 3A shows CCR7 surface expression (black line) in tumor cells from representative Mantle Cell Lymphoma (MCL) patients as measured by flow cytometry including the corresponding IC (grey line), and fig. 3B shows CDC of MCL cells in the presence of anti-CCR 7 monoclonal antibody or their respective ICs. Mean ± standard deviation of 4 cases are shown.

Figure 4 depicts dose-response curves for CDC. CDC assays were performed with PBMCs from CLL patients (n ═ 5) and healthy donors (n ═ 2) with anti-CCR 7 monoclonal antibodies at concentrations ranging from 0.5 μ g/mL to 16 μ g/mL. The percentage of CDC-induced cell lysis was determined in electronically gated CLL cells, T cells from CLL patients, and B and T cells from healthy donors by 7-AAD incorporation and FCM analysis. The line represents the mean value of the cell lysis. (A) An anti-CCR 7IgM monoclonal antibody, (B) an anti-CCR 7IgG monoclonal antibody.

Figure 5 shows that CDC capacity correlates with CCR7 expression levels. The CCR7 surface density of CLL cells correlated with the percentage of lysis (r 0.602, P0.025, n 11) due to CDC mediated by 2 μ g/mL of anti-CCR 7IgM monoclonal antibody after 1 hour of incubation with rabbit complement.

Figure 6 shows that murine anti-CCR 7IgG monoclonal antibodies do not mediate ADCC by CLL cells. CLL cells, previously incubated with anti-CCR 7IgG monoclonal antibody (2 μ g/mL), IC or rituximab (as positive controls), were exposed to human Natural Killer (NK) cells for 4 hours. Dead cells were then assayed in electronically gated CLL cells by 7-AAD incorporation and FCM analysis. Bars show mean ± standard deviation of 6 experiments.

FIG. 7 shows that anti-CCR 7 monoclonal antibody does not induce lymphocyte proliferation. PBMCs from healthy donors were cultured with anti-CCR 7 monoclonal antibody, IC, anti-CD 3 monoclonal antibody plus IL2 or medium alone for 72 hours. Pass [ 2 ]³H]Thymidine incorporation measures DNA synthesis within the last 16 hours of culture. Bars represent mean ± standard deviation of 6 cases replicated three times.

Figure 8 shows that anti-CCR 7 monoclonal antibodies block migration of CLL and MCL cells in response to CCL19 or CCL 21. Chemotaxis assays were performed as described in the methods section after pre-incubation of cells for 30 minutes with 2 μ g/mL of anti-CCR 7IgG (black bars), IgM (grey bars) monoclonal antibody or no monoclonal antibody (white bars). (A) Comparing migration of CLL cells in response to CCL19(1 μ g/mL), basal migration, and migration in the presence of CXCL12(100ng/mL), CXCL12 is a ligand for chemokine receptor CXCR4 that is not affected by anti-CCR 7 monoclonal antibodies. Representative cases are shown. (B) MCL cells were compared for migration and basal migration in response to the CCR7 ligands CCL19 (1. mu.g/mL) and CCL21 (1. mu.g/mL). Representative cases are shown.

Detailed Description

The present invention relates generally to the use of an antibody or antigen-binding fragment thereof that binds to the CCR7 receptor for the preparation of a pharmaceutical composition for the treatment of cancer, in particular cancer in which tumor cells express the CCR7 receptor, by killing or inducing apoptosis of said tumor cells.

Thus, in one aspect, the present invention relates to the use of an antibody or antigen-binding fragment thereof that binds to the CCR7 receptor for the preparation of a pharmaceutical composition for killing or inducing apoptosis of CCR receptor expressing tumor cells.

The tumor cells to be treated for cancer are tumor cells expressing the CCR7 receptor. Illustrative, non-limiting examples of cancer types whose tumor cells express the CCR7 receptor include Chronic Lymphocytic Leukemia (CLL), Mantle Cell Lymphoma (MCL), follicular lymphoma, large B-cell lymphoma, AIDS-related lymphoma, lymphoplasmacytic lymphoma (lymphoplasmacytic lymphoma), burkitt's lymphoma, B-cell acute lymphoblastic lymphoma, hodgkin's disease, adult T-cell leukemia/lymphoma, mycosis fungoides, outbreak of chronic myeloproliferative disease, outbreak of myelodysplastic syndrome, cancers such as breast cancer, non-small cell lung cancer, melanoma, gastric cancer, or squamous cell carcinoma of the head and neck, and colon cancer.

Tumor cells expressing the CCR7 receptor can be identified by routine methods; for example, the surface expression of the CCR7 receptor can be analyzed by flow cytometry according to Lopez-Giral S. et al (Journal of Leuukocyte Biology, Vol76, Aug.2004, 462-471).

As used herein, "treating cancer" refers to inhibiting or controlling the proliferation of tumor cells, which are tumor cells that express the CCR7 receptor. The term includes, among other things, killing the tumor cells, inducing apoptosis of the tumor cells, attenuating migration of the tumor cells to secondary lymphoid tissue, and/or blocking diffusion of the tumor cells into secondary lymphoid tissue.

According to the present invention, antibodies or antigen binding fragments thereof specific for the CCR7 receptor, i.e. binding to the CCR7 receptor, sometimes referred to herein as antibodies of the present invention, can be used to kill tumor cells expressing the CCR7 receptor or to induce apoptosis of tumor cells expressing the CCR7 receptor. Therefore, the antibody of the invention can be used for preparing a pharmaceutical composition for killing tumor cells expressing CCR7 receptor or inducing apoptosis of tumor cells expressing CCR7 receptor. Accordingly, the present invention provides alternative methods for treating cancer, in particular cancers whose tumor cells express the CCR7 receptor.

The term "killing tumor cells" as used herein refers to the mechanism of specifically eliminating tumor cells, such as tumor cells expressing the CCR7 receptor, by specifically lysing the cells. The lysis or cytotoxicity is typically mediated by a complement protein (CDC) that recruits the tumor cells or effector cells such as NK cells (ADCC) that are capable of specifically targeting and causing lysis of the tumor cells, respectively, upon treatment of the tumor cells with an anti-CCR 7 antibody.

The term "inducing apoptosis of tumor cells" as used herein refers to the mechanism by which tumor cells expressing the CCR7 receptor undergo apoptosis, i.e., programmed cell death, upon treatment with an anti-CCR 7 antibody.

The term "antibody of the present invention" as used herein refers to gamma globulin or a fragment thereof showing specific binding activity to a target molecule, i.e., a CCR receptor (antigen). Thus, the antibodies of the invention are capable of binding to an epitope of CCR 7; typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope, however, epitopes comprising non-contiguous amino acids may require, for example, at least 15, 25, or 50 amino acids. The term "antibody of the invention" for example includes polyclonal antibodies, monoclonal antibodies, engineered or modified antibodies, chimeric antibodies, humanized antibodies, primatized antibodies, human antibodies, e.g., Fab, F (ab')₂Antibody fragments of single chain fv (scfv) fragments, diabodies (diabodies) bispecific antibodies, and heteroconjugate antibodies. In addition, the antibodies of the invention may also be conjugated to other compounds, such as therapeutic agents, toxins, and the like. These antibodies can be produced by a variety of methods, including hybridoma culture, recombinant expression in bacterial and mammalian cell cultures, and recombinant expression in transgenic animals. Can also be expressed in a display systemThe sequence library of (a) is selected for the production of antibodies, the display system being, for example, filamentous phage, bacteria, yeast or ribosomes. Rich guidance in the literature regarding the selection of specific production methods is for example Chadd and Chamow, curr. 188-194(2001). The choice of preparation method depends on several factors, including the desired antibody structure, the importance of the carbohydrate moiety on the antibody, ease of culture and purification, and cost. Many different antibody structures can be produced using standard expression techniques, including full-length antibodies, antibody fragments such as Fab and Fv fragments, and chimeric antibodies comprising components from different species. Small antibody fragments, such as Fab and Fv fragments, which have no effector function and limited pharmacokinetic activity, can be produced in bacterial expression systems. Single-chain Fv fragments exhibit low immunogenicity and are rapidly cleared from the blood.

Antibodies of the invention that specifically bind to an epitope of the CCR7 receptor can be used therapeutically, as well as in immunochemical assays such as immunofluorescent assays, flow cytometry, Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. A variety of immunoassays can be used to identify antibodies with the desired specificity. Various protocols for competitive binding or immunoradiometric assays are known in the art. These immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to a CCR7 receptor immunogen.

The antibody of the invention may be a polyclonal antibody. These polyclonal antibodies can be produced, for example, in a mammal, such as a non-human mammal, after one or more injections of an immunizing agent, preferably an adjuvant. Typically, the immunizing agent and/or adjuvant is injected into the mammal by a series of subcutaneous or intraperitoneal injections. The immunizing agent may include a CCR7 receptor or fragment thereof or fusion protein thereof or may include cells expressing a CCR7 receptor. Alternatively, crude protein preparations enriched for the CCR7 receptor or fragment thereof can be used to generate antibodies. These proteins, fragments or formulations are introduced into a non-human mammal in the presence of a suitable adjuvant. Other forms of immunogen administration are as transmembrane proteins on the cell surface (methods are described, for example, in Spiller et al.J.Immunol.methods, 224: 51-60 (1999)). These cells may be those which naturally express the antigen on their cell membrane or in which each of this expression can be obtained after transfection of the cells with a DNA construct containing the DNA sequence encoding the antigen, the DNA sequence necessary for its adequate expression in the cells, and other DNA sequences. This approach is possible not only when the cell membrane is the natural site of antigen expression, but even when the antigen, once synthesized in the cell, is directed to these locations by a signal peptide added to the antigen coding sequence. If the serum contains polyclonal antibodies to the undesired epitope, the polyclonal antibodies can be purified by immunoaffinity chromatography.

Alternatively, the antibody may be a monoclonal antibody. Monoclonal antibodies can be produced by hybridomas in which a mouse, hamster, or other suitable host animal is immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind the immunizing agent, such as Kohler and milstein, Nature 256: 495(1975). The immunizing agent typically includes a CCR7 receptor or fragment thereof or fusion protein thereof and optionally includes a vector or crude protein preparation enriched for the CCR7 receptor or fragment thereof or expressing the CCR7 receptor. These proteins, fragments or formulations are introduced into a non-human mammal in the presence of a suitable adjuvant. Other forms of immunogen administration are as transmembrane proteins on the cell surface (methods are described, for example, in Spiller et al.J.Immunol.methods, 224: 51-60 (1999)). These cells may be those which naturally express the antigen on their cell membrane or in which each of this expression can be obtained after transfection of the cells with a DNA construct containing the DNA sequence encoding the antigen, the DNA sequence necessary for its adequate expression in the cells, and other DNA sequences. This approach is feasible not only when the cell membrane is the natural site of antigen expression, but even when the antigen, once expressed in the cell, is directed to these locations by a signal peptide added to the antigen coding sequence. Alternatively, lymphocytes may be immunized in vitro. Typically, spleen cells or lymph node cells are used if non-human mammalian origin is desired, and peripheral blood lymphocytes ("PBLs") are used if human origin is desired. The lymphocytes are fused with an immortalized cell line using a suitable fusion agent such as polyethylene glycol to produce hybridoma cells. Typically, the immortalized cell line is a myeloma cell of rat, mouse, bovine or human origin. The hybridoma cells are cultured in a suitable medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. Clones are isolated using limiting dilution methods and the culture medium (supernatant) in which the hybridoma cells are cultured can be analyzed for the presence of monoclonal antibodies against the CCR7 receptor by conventional techniques, such as by cell flow or by immunoprecipitation or by other in vitro binding assays, such as RIA or ELISA. Can also be used as ascites tumor in vivo culture clone in animal body.

Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) or by immunofluorescence techniques, such as fluorescence microscopy or flow cytometry.

Monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification methods, such as protein a agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

Monoclonal antibodies can also be prepared by recombinant DNA methods, for example as described in US 4,816,567. DNA encoding the monoclonal antibodies of the invention can be isolated from CCR7 receptor-specific hybridoma cells and sequenced using conventional methods, for example, by oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies. Hybridomas are a preferred source of these DNAs. Once isolated, the DNA may be inserted into an expression vector, which is then transfected into a host cell, such as a monkey COS cell, a Chinese Hamster Ovary (CHO) cell, or a myeloma cell that does not produce additional immunoglobulin, to obtain synthesis of the monoclonal antibody in the recombinant host cell.

Another method for generating specific antibodies or antibody fragments reactive against a target molecule is to screen an expression library encoding immunoglobulin genes or parts thereof expressed in bacteria, yeast, filamentous phage, ribosomes or ribosomal subunits, and other display systems. These methods typically use large libraries of antibody sequences or antibody fragment sequences from a variety of sources, such as healthy donors, patients, or healthy or unhealthy animals. These sequences are cloned and expressed in an appropriate system and selected by their binding affinity to the antigen. Various methods of selecting antibodies or fragments with desired properties are described, such as neutralization, agonism, etc. (Fern. ndez, Curr. Op. Biotech., 15: 364-. In one embodiment, antibodies and antibody fragments characteristic of the hybridomas of the present invention can also be produced by recombinant methods by extracting messenger RNA, constructing a cDNA library, and selecting clones encoding antibody molecule fragments.

Engineered or modified antibodies:

many methods use molecular biology and genetic techniques, e.g., a good understanding of genetics and immunoglobulin structure, to construct different modifications of immunoglobulin molecules to improve their properties for clinical or other uses. Some of them tend to reduce the immunogenicity of the molecule in the species to be used and the resulting molecules have sequences that are more homologous to that species. Monoclonal antibodies of human origin have been obtained using various methods, avoiding ethically impermissible behavior in healthy humans. In other methods, molecular weight and size are reduced, for example, to improve distribution of the molecules to solid tumors. Other possibilities are to combine a binding domain for more than one target molecule in a molecule (bispecific or trispecific, etc.), or to combine an antibody or fragment with another molecule with a desired function, such as a toxic agent, a hormone, a growth factor, an immunomodulator (immunosuppressant or immunostimulant), a cytostatic agent, etc. Generally, all resulting molecules retain at least one antibody variable domain that confers high specificity and affinity as a characteristic of antigen-antibody binding. Some examples of such constructs are:

chimeric antibody:

these refer to antibodies constructed with the variable regions of antibodies from certain species (usually mammals that produce monoclonal antibodies) and the constant regions of other species (the species for which chimeric antibodies will be used). The aim of such construction is to obtain antibodies with the original monoclonal antibody but which are less immunogenic and better tolerated in the individual to be treated, which have an increased serum half-life and which can be recognized for the effector immune mechanism, i.e. complement, Fc receptors of cytotoxic cells or specific receptors of other immunoglobulins which show species specificity.

Humanized antibody:

"humanized antibody" refers to an antibody derived from a non-human antibody, usually a murine antibody, which retains the antigen binding properties of the parent antibody but is less immunogenic in humans. This can be achieved by a variety of methods, including (a) grafting the entire non-human variable region onto a human constant region to produce a chimeric antibody; (b) grafting only non-human Complementarity Determining Regions (CDRs) into the human framework and constant regions with or without retention of critical framework residues; and (c) grafting intact non-human variable domains, but masking them with human-like moieties by substituting surface residues.

Methods for humanizing non-human antibodies have been described in the art. Preferably, the humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain.

Humanization can be essentially carried out according to the method of Winter and coworkers (Jones et al, Nature, 321: 522-525 (1986); Reichmann et al, Nature, 332: 323-327 (1988); Verhoeyen et al, Science, 239: 1534-1536(1988)) by replacing the corresponding sequences of a human antibody with hypervariable region sequences. In practice, humanized antibodies are typically human antibodies in which certain hypervariable region residues and possibly certain Framework Region (FR) residues are replaced by residues from analogous sites in rodent antibodies. The choice of human variable region domains for the light and heavy chains used to make the humanized antibody is very important to reduce immunogenicity, preserve specificity and affinity for the antigen. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is used to screen a complete library of known human variable region domain sequences. The human sequence closest to the rodent sequence is accepted as the human Framework Region (FR) of the humanized antibody (Suns et al, J Immunol, 151: 2296(1993), Chothia et al, J Mol Biol, 196: 901 (1987)). Another approach uses specific framework regions derived from the consensus sequence of all human antibodies of a specific subset of light or heavy chains. The same framework regions can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al, J.Immunol., 151: 2623 (1993)).

More importantly, antibodies are humanized, retaining high affinity for the antigen and other favorable biological properties. To achieve this, according to a preferred method, humanized antibodies are prepared by a method of analyzing the parent sequence and various conceptual humanized products using three-dimensional models of the parent and humanized sequences.

Primatized antibodies:

a further step in this approach to make antibodies more similar to humans is to prepare what are referred to as primatized antibodies, i.e., recombinant antibodies engineered to contain the variable heavy and light chain domains of monkey (or other primate) antibodies, particularly cynomolgus monkey antibodies, and which contain human constant domain sequences, preferably human immunoglobulin gamma 1 or gamma 4 constant structures (or PE variants). In Newman et al, Biotechnology, 10: 1458-1460 (1992); the preparation of these antibodies is described in US 5,658,570 and US 6,113,898. These antibodies have been reported to exhibit high homology to human antibodies, i.e., 85-98%, exhibit human effector function, have reduced immunogenicity, and may exhibit high affinity to human antigens. Newman, Biotechnology, 10: 1455-1460(1992) discloses another efficient method for producing recombinant antibodies.

Human antibody:

"human antibody" refers to an antibody containing fully human light and heavy chains and constant regions, produced by any known standard method.

As an alternative to humanization, human antibodies can be produced. For example, transgenic animals (e.g., mice) immunized to produce a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can now be generated. For example, deletion of the antibody heavy chain joining region PH gene in chimeric and germline mutant mice has been described to result in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays in these germline mutant mice will result in the production of human antibodies upon immunization. See, e.g., Jakobovits et al, proc.mad.acad.sci.usa, 90: 2551 (1993); jakobovits et al, Nature, 362: 255-258(1993).

Alternatively, human antibodies and antibody fragments from a donor's immunoglobulin variable (V) domain gene library can be generated in vitro using phage display technology (McCafferty et al, Nature 348: 552-553 (1990)). According to this technique, antibody V domains are cloned in-frame into the major or minor coat protein genes of filamentous phage such as M13 or fd and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of genes encoding antibodies exhibiting those properties. Thus, the phage mimics certain characteristics of B cells. Phage display can be performed in a variety of formats; for a review of this, see for example Johnson, Kevin s.and Chiswell, David j, Current Opinion in structural Biology 3: 564-571(1993).

Human antibodies can also be produced by activated B cells in vitro or SCID mice whose immune system is reconstituted with human cells.

Once the human antibody is obtained, its encoding DNA sequence can be isolated, cloned and introduced into an appropriate expression system, i.e., a cell line, preferably from a mammal, which then expresses and releases the human antibody into a culture medium from which the antibody can be isolated.

Antibody fragment:

antibody fragments are fragments of antibodies, e.g., Fab, F (ab')₂Fab' and scFv. Various techniques for producing antibody fragments have been developed. Traditionally, these fragments have been derived by proteolytic digestion of intact antibodies, but recently these fragments can be produced directly by recombinant host cells. In other embodiments, the antibody of choice is a single chain fv (scfv) fragment, which may also be monospecific or bispecific.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a remaining "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment produces F (ab') which has two antigen binding sites and is still capable of cross-linking the antigen₂And (3) fragment.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and antigen binding site. This region consists of a dimer of one heavy and one light chain variable domain in close, non-covalent linkage. In this configuration the three hypervariable regions of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. Overall, these six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising three hypervariable regions specific for an antigen) has the ability to recognize and bind an antibody, albeit with a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chain and the first constant domain of the heavy Chain (CHI). Fab 'fragments differ from Fab fragments in that a few residues including one or more cysteines from the antibody hinge region are added to the Fab' fragment at the carboxy terminus of the heavy chain CH1 domain. Fab '-SH is the designation herein for Fab' having at least one free thiol group to a cysteine of the constant domain. F (ab ') Z antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

"Single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of monoclonal Antibodies, vol.113, Rosenburg and Moore eds, Springer-Verlag, N.Y., pp.269-315 (1994).

The term "bivalent small antibody (diabodies)" refers to small antibody fragments having two antigen binding sites, those fragments comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is short enough that two domains on the same strand cannot pair, the domain is forced to pair with the complementary domain of the other strand and two antigen binding sites are created. Bivalent small molecule antibodies are described, for example, in EP 404,097, WO 93/11161 and Hollinger et al, proc.natl.acad.sci.usa, 90: 6444-.

Functional fragments of antibodies that bind to the CCR7 receptor encompassed by the present invention retain at least one of the binding and/or modulating functions of the full length antibody from which the fragment is derived. Preferred functional fragments retain the antigen binding function (e.g., the ability to bind to the mammalian CCR7 receptor) of the corresponding full-length antibody. Particularly preferred functional fragments retain the ability to inhibit one or more characteristic functions of the mammalian CCR7 receptor, such as binding activity, signaling activity, and/or stimulation of a cellular response. For example, in one embodiment, the functional fragment is capable of inhibiting the interaction of CCR7 with one or more of its ligands and/or is capable of inhibiting one or more receptor-mediated functions.

Bispecific antibodies:

bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. An exemplary bispecific antibody can bind to two different epitopes of a B cell surface marker. Other such antibodies may bind to a first B cell marker and further bind to a second B cell surface marker. Alternatively, the anti-B cell marker binding arms may be combined with arms that bind to trigger molecules on leukocytes, such as T cell receptor molecules (e.g., CD2 or CD3) or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16) to focus cellular defense mechanisms to B cells. Bispecific antibodies can also be used to localize cytotoxic agents to B cells. These antibodies have a B-cell marker binding arm and an arm that binds a cytotoxic agent (e.g., saporin, anti-interferon alpha, vinca alkaloid, ricin a chain, methotrexate, or radioisotope hapten). Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab)₂Bispecific antibodies).

Methods for making bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, wherein the two chains have different specificities (Millstein et al, Nature, 305: 537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is difficult and the product yield is low. In WO 93/08829 and Traunecker et al, EMBO J, 10: 3655-3659(1991) disclose a similar method.

According to different approaches, antibody variable domains (antibody-antigen binding sites) with the desired binding specificity are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2, and CH3 regions. Preferably, a first heavy chain constant region (CHI) is present in at least one of the fusions, which contains the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and are co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in an embodiment when unequal proportions of the three polypeptide chains are used in the construction to provide optimal yields. However, when equal ratios of at least two polypeptide chains result in high yields or when the ratio is not particularly important, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector.

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin and the other to biotin. Such antibodies are proposed, for example, to target immune system cells to unwanted cells. Heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable cross-linking agents and a number of cross-linking techniques are well known in the art and US 4,676,980 discloses suitable cross-linking agents.

Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical bonds.

Antibody coupling:

cytotoxic chemotherapy or radiotherapy of cancer suffers from serious, sometimes fatal, side effects resulting from toxicity to sensitive normal cells because the treatment is not selective for malignant cells. One way to avoid these problems is to couple the therapeutic agent to an antibody or other ligand that recognizes the tumor-associated antigen. This increases exposure of malignant cells to ligand-targeted therapeutic agents and reduces exposure of normal cells to ligand-targeted therapeutic agents. See Allen, Nature, 2: 750-763(2002).

The therapeutic agent may also be an immunosuppressive agent, i.e., a substance that inhibits or masks the immune system of the mammal being treated herein. This would include substances that inhibit cytokine production, down-regulate or inhibit autoantigen expression or mask MHC antigens.

The therapeutic agent may also be a cytotoxic agent, i.e., a substance that inhibits or prevents the function of cells and/or causes cell destruction. The term is intended to include radioisotopes, chemotherapeutic agents, i.e., chemical compounds useful in the treatment of cancer, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof.

The therapeutic agent may also be a cytokine, hormone, growth factor, necrosis factor, i.e. a protein or polypeptide released by a group of cells that acts as an intercellular mediator (mediators) on another cell or even on the same cell group. As used herein, the term cytokine includes proteins and polypeptides from natural sources or from recombinant cell cultures and biologically active equivalents of the native sequence cytokines.

The therapeutic agent may also be a prodrug, which refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells than the parent drug and is capable of being enzymatically activated or converted to a more active parent form.

Conjugates of an antibody and one or more small molecule toxins. In a preferred embodiment of the invention, the antagonist is conjugated to one or more toxin molecules. Enzymatically activated toxins and fragments thereof that can be used include diphtheria a chain, non-binding active fragments of diphtheria toxin, exotoxin a chain (from Pseudomonas aeruginosa), ricin a chain, abrin a chain, gelonin a chain, alpha broom toxin (sarcin), aleurites fordii protein, dianthin (dianthin) protein, pokeweed (phytolacca americana) protein (PAPI, PAPII and PAP-S), momordica charantia (momordia charrantia) inhibitor, curcin, croton toxin, saponaria officinalis (sapaonaria officinalis) inhibitor, gelonin, ogellin, tricin, phenomycin (phenomycin), enomycin (neomycin) and tricothecene.

The invention also relates to antibodies conjugated to compounds having nucleolytic activity (e.g., ribonucleases or endonucleases such as deoxyribonucleases; dnases) or other compounds capable of disrupting cellular structures or organelles and thereby killing the cells or reducing cell viability.

The antibodies of the invention may also be conjugated to a prodrug activator that converts the prodrug into an active anticancer drug. The reagent component of these conjugates includes any agent capable of acting on the prodrug to convert it to its more active, cytotoxic form.

Alternatively, a fusion protein comprising at least the antigen-binding region of an antagonist of the invention linked to at least a functionally active portion of an enzyme of the invention can be constructed using recombinant DNA techniques well known in the art (see, e.g., Neuberger et al, Nature, 312: 604-608 (1984)).

Conjugates of the antibody and cytotoxic agent may be made with a variety of bifunctional protein coupling agents or linkers. The linker may be a "cleavable linker" that facilitates release of the cytotoxic drug within the cell. For example, an acid labile linker, a peptidase sensitive linker, a dimethyl linker, or a linker containing a disulfide bond may be used.

Alternatively, fusion proteins comprising an antibody and a cytotoxic agent can be prepared, for example, by recombinant techniques or peptide synthesis.

Other potentially useful modifications:

also relates to amino acid sequence modifications of the protein or peptide antagonists described herein. For example, it may be desirable to increase the binding affinity and/or other biological properties of an antibody. Amino sequence variants of an antibody are prepared by introducing appropriate nucleotide changes into antibody-encoding nucleic acids, or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues in the antagonist amino acid sequence. Any combination of deletions, insertions, and substitutions are made to complete the final construct, provided that the final construct has the desired characteristics. Amino acid changes can also alter post-translational processing of the antagonist, for example, changing the number or position of glycosylation sites.

Amino acid sequence insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues in length from one residue to a polypeptide containing one hundred or more residues. Examples of terminal insertions include antagonists having an N-terminal methionine residue or antagonists fused to a cytotoxic polypeptide. Other insertional variants of the antagonist molecule include fusions to the N or C terminus of an antagonist of the enzyme, or polypeptides that increase the serum half-life of the antagonist.

Another type of variant is an amino acid substitution variant. In these variants, at least one amino acid residue of the antagonist molecule is substituted with a different residue. The sites of most interest for substitutional mutagenesis of antibody antagonists include hypervariable regions, but alterations of the FR are also contemplated.

Another type of amino acid variant of an antibody is to alter the original glycosylation pattern of the antagonist. Alteration refers to deletion of one or more sugar moieties found in the antagonist, and/or addition of one or more glycosylation sites not present in the antagonist. Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of the sugar moiety to the side chain of asparagine. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for the enzymatic attachment of the sugar moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of a monosaccharide or monosaccharide derivative, one of N-acetylgalactosamine, galactose or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. The addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Changes (for O-linked glycosylation sites) can also be made by adding or replacing one or more serines or threonines to the sequence of the original antibody. Nucleic acid molecules encoding amino acid sequence variants of antibodies are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an antagonist, either an earlier prepared variant or a non-variant version.

It may be desirable to modify the antibodies used in the present invention to enhance effector function, e.g., in order to enhance ADCC and/or CDC of the antibody. This can be accomplished by introducing one or more amino acid substitutions into the Fc region of the antibody.

The formation of glycosyl groups added to the amino acid backbone of a glycoprotein, e.g., an antibody, by several monosaccharides or monosaccharide derivatives results in a different composition on the same antibody that can be produced in cells from different mammals or tissues. Furthermore, it has been shown that the different composition of the glycosyl groups can influence the efficacy of mediating antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) of antibodies. It is therefore possible to improve those properties by studying the glycosylation pattern of antibodies from different sources. Examples of such methods are Niwa et al, Cancer res.2004 Mar 15; 64(6): 2127-33.

Alternatively or additionally, cysteines may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in this region. The homodimeric antibody thus produced may have improved internalization capacity and/or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al, j.exp med.176: 1191-1195(1992) and shop, B.J.Immunol.148: 2918-2922(1992). Homodimeric antibodies with enhanced anti-tumor capacity can also be prepared using heterobifunctional cross-linkers, such as Wolff et al cancer research 53: 2560, 2565 (1993). Alternatively, antibodies with a dual Fc region can be engineered and may therefore have enhanced complement lysis and ADCC capabilities. See Stevensonet al anti-Cancer Drug Design 3: 219-230(1989).

To increase the serum half-life of the antibody, a salvage receptor binding epitope may be incorporated into the antibody (particularly an antibody fragment), for example as described in US 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

Preferably, an antibody of the invention, e.g., a monoclonal antibody, Fv fragment, Fab fragment, or other binding composition derived from a monoclonal antibody of the invention, has a high affinity for CCR 7. The affinity of monoclonal antibodies and related molecules for the CCR7 receptor can be measured by conventional techniques.

The affinity of an antibody for an antigen can be defined as the effectiveness of the antibody to bind the antigen. Antigen-antibody binding is reversible, so that when two molecules are diluted in the same solution for a sufficient time, the solution reaches equilibrium, with constant concentrations of antigen-antibody complex (AgAb), free antigen (Ag) and free antibody (Ab). Thus the ratio [ AgAb]/[Ag]^*[Ab]Is also a constant, defined as the binding constant known as Ka, which can be used to compare the affinity of some antibodies to their respective epitopes.

A common method of measuring affinity is to experimentally determine the binding curve. This involves measuring the amount of antibody-antigen complex as a function of free antigen concentration. There are two common methods of making this measurement: (i) classical equilibrium dialysis using Scatchard analysis and (ii) surface plasmon resonance methods, wherein an antibody or antigen is bound to a conductive surface and the antigen or antibody, respectively, affects the electrical properties of this surface.

It is often only necessary to determine the relative affinities of two or more monoclonal antibodies that bind the same epitope. In this case, a competition assay may be performed in which a serial dilution of one of the monoclonal antibodies is incubated with a constant amount of ligand, followed by the addition of a second monoclonal antibody labeled with a suitable tracer. After binding to this monoclonal antibody and washing of unbound antibody, the concentration of the second monoclonal antibody was measured and plotted against the concentration of the first monoclonal antibody and analyzed by the Scatchard method. Examples are Tamura et al, j.immunol.163: 1432-1441(2000).

In addition, the antibodies of the invention can be used to detect the CCR7 receptor. These detection methods are advantageously applied in the diagnosis and/or prognosis of cancers in which the tumour cells are cells expressing the CCR7 receptor.

In addition, the antibodies of the invention can be used to identify cells expressing the CCR7 receptor by standard techniques such as immunofluorescence, flow cytometry, affinity chromatography, or immunoprecipitation. For example, the antibodies of the invention can aid in the identification of tumor cells that express the CCR7 receptor. In addition, the antibodies of the invention can be used to identify tumor cells expressing the CCR7 receptor to assess their levels in a particular tissue. Thus, the antibodies of the invention can be used diagnostically to detect the level of cells expressing the CCR7 receptor in a tissue as part of a clinical examination procedure, for example, to determine the effect of a given treatment regimen. Detection can be aided by coupling (i.e., physically linking) the antibody to a labeling group.

As described below, in particular embodiments, the antibodies of the invention can be used in combination with additional therapeutically active compounds such as cytokines, analgesics, immunosuppressive agents and/or chemotherapeutic agents. Co-administration includes co-administration using separate formulations or a single pharmaceutical formulation and sequential administration in any order, wherein there is preferably a time period during which both (or all) active agents exert their biological activities simultaneously.

In other aspects, the invention relates to a pharmaceutical composition, sometimes referred to as a pharmaceutical composition of the invention, for administration to a subject, comprising an antibody of the invention, i.e., an antibody that binds to the CCR7 receptor, or an antigen-binding fragment thereof, or a prodrug thereof, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. The pharmaceutical composition can be used to kill tumor cells expressing the CCR7 receptor or induce apoptosis of tumor cells expressing the CCR7 receptor by being administered to an individual suffering from cancer. The term "individual" as used herein refers to all animals classified as mammals, including but not limited to livestock or farm animals, primates, and humans. The individual is preferably a human male or female of any age or race.

The term "pharmaceutically acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the compositions is contemplated. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants include ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl phenyl ammonium chloride; hexane diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); small molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG).

The antibodies of the invention may be administered in the same formulation, or may be administered in different formulations. Administration can be simultaneous or sequential, and can be effected in any order.

Supplementary active ingredients may also be incorporated into the pharmaceutical compositions of the present invention. Thus, in particular embodiments, the compositions of the present invention may also contain more than one active compound, preferably those having complementary activities that do not adversely affect each other, as desired for the particular indication being treated. For example, it may be desirable to further provide a chemotherapeutic agent, cytokine, analgesic, or immunosuppressive agent. The effective amount of these other active agents will depend on, among other factors, the amount of the antibody of the invention present in the pharmaceutical composition, the type of disease or disorder or the method of treatment.

In embodiments, the antibodies of the invention are prepared with carriers that protect the compound from rapid clearance from the body, such as controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing these formulations will be apparent to those skilled in the art. Materials are also available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells using monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example as described in U.S.4,522,811.

The route of administration of the antibodies (or fragments thereof) of the invention may be oral, parenteral, by inhalation, or topical.

The term "parenteral" as used herein includes intravenous, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. Parenteral administration in subcutaneous and intramuscular forms is generally preferred.

"inhalation" refers to intranasal and oral inhalation administration. Suitable dosage forms for such administration, for example aerosol formulations or metered dose inhalers, may be prepared by conventional techniques.

In addition, "local" refers to non-systemic administration and includes application of the antibodies of the invention to the exterior of the epidermis, the oral cavity, and instillation of the antibodies into the ear, eye and nose and sites not significantly entering the bloodstream. "systemic administration" refers to oral, intravenous, intraperitoneal, and intramuscular administration. The amount of antibody required for therapeutic or prophylactic action will, of course, vary depending on the antibody selected, the nature and severity of the disease state being treated and the patient.

In addition, the antibody may be suitably administered by pulsatile injection, e.g., using a gradually decreasing dose of the antibody. Preferably, the formulation is administered by injection, most preferably intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic.

Thus, in particular embodiments, the pharmaceutical compositions of the present invention may be in a form of administration suitable for oral administration, solid or liquid. Suitable dosage forms for oral administration may be tablets, capsules, syrups or solutions and may contain conventional excipients known in the art, for example binding agents such as syrup, gum arabic, gelatin, sorbitol or polyvinylpyrrolidone; fillers, for example lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; tableting lubricants, such as magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycolate or microcrystalline cellulose; or a pharmaceutically acceptable wetting agent such as sodium lauryl sulfate. Solid oral compositions may be prepared by conventional methods of mixing, filling or tableting. Repeated mixing operations can be used to distribute the active agent throughout the composition using a large amount of filler. Such operations are conventional in the art. Tablets are prepared, for example, by wet or dry granulation and optionally coating according to methods well known in general pharmaceutical practice, in particular with enteric coating.

In another embodiment, the pharmaceutical compositions of the present invention may be adapted for parenteral administration, for example, as sterile solutions, suspensions or lyophilized products in suitable unit dosage forms. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water is appropriate) or dispersions and sterile powders for the in situ preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CremophorEM (BASF, Parsippany, n.j.) or Phosphate Buffered Saline (PBS). In all cases, the compositions must be sterile and fluid to the extent that easy syringability exists. The compositions must be stable under the conditions of manufacture and storage and should be preserved against contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, pharmaceutically acceptable polyols such as glycerol, propylene glycol, liquid polyethylene glycols, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, which maintains fluidity by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferred to include isotonic agents, for example, sugars, polyols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains the essential dispersing media and the necessary other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

In particular embodiments, the pharmaceutical composition is administered Intravenously (IV) or Subcutaneously (SC). Sufficient excipients such as fillers, buffers or surfactants may be used. The above formulations will be prepared using standard methods described or indicated in the spanish pharmacopoeia and the united states pharmacopoeia and similar references.

It is particularly advantageous to formulate pharmaceutical compositions, i.e., oral or parenteral compositions, in unit dosage forms that are easy to administer and consistent in dosage. Unit dosage form as used herein refers to physically discrete units suitable as unitary dosages for the individual to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier. The specifications of the unit dosage forms of the invention are dictated by and directly dependent upon the unique characteristics of the active compounds and the particular therapeutic effect to be achieved, as well as the limitations inherent in the art of compounding these active compounds for the treatment of individuals.

Generally, an effective amount of an antibody of the invention to be administered will depend on the relative potency of the compound selected, the severity of the condition being treated and the weight of the patient. However, the active compound is generally administered one or more times daily, for example 1, 2, 3 or 4 times daily, usually at a total daily dose of from 0.001mg/kg body weight/day to 1000mg/kg body weight/day, preferably from about 0.01mg/kg body weight/day to about 100mg/kg body weight/day, most preferably from about 0.05mg/kg body weight/day to 10mg/kg body weight/day.

In addition to administering antibodies to patients, the present invention contemplates administration of antibodies by gene therapy. WO96/07321 relates to the generation of intracellular antibodies using gene therapy.

The pharmaceutical composition can be included in a container, package, or dispenser with instructions for administration.

The antibodies and pharmaceutical compositions of the invention may be used in combination with other drugs to provide combination therapy. The other agents may form part of the same composition, or as separate compositions for administration at the same time or at different times.

The antibodies and pharmaceutical compositions of the invention will be useful in the treatment of medical conditions, such as conditions or diseases associated with cancer, in particular in the treatment of cancer characterized by tumor cells expressing the CCR7 receptor, more particularly in killing tumor cells expressing the CCR7 receptor or inducing apoptosis of tumor cells expressing the CCR7 receptor. Illustrative non-limiting cancers in which the tumor cells express the CCR7 receptor that are susceptible to treatment of the present invention include Chronic Lymphocytic Leukemia (CLL), Mantle Cell Lymphoma (MCL), follicular lymphoma, large B-cell lymphoma, AIDS-related lymphoma, lymphoplasmacytic lymphoma, burkitt's lymphoma, B-cell acute lymphoblastic lymphoma, hodgkin's disease, adult T-cell leukemia/lymphoma, mycosis fungoides, outbreak of chronic myeloproliferative disease, outbreak of myelodysplastic syndrome, breast cancer, non-small cell lung cancer, melanoma, gastric cancer, or squamous cell carcinoma of the head and neck, and colon cancer. In preferred embodiments, cancers susceptible to treatment of the invention include breast cancer, non-small cell lung cancer, melanoma, gastric cancer, or squamous cell carcinoma of the head and neck and colon cancer. In a most preferred embodiment, cancers susceptible to treatment of the invention include follicular lymphoma, adult T-cell leukemia/lymphoma, burkitt's lymphoma, outbreaks of chronic myeloproliferative disease, and outbreaks of myelodysplastic syndrome. In other most preferred embodiments, the cancer treated according to the invention comprises CLL and MCL.

In particular embodiments, the antibodies of the invention may be combined with other treatments of the medical conditions described herein, such as chemotherapy, radiation therapy, immunotherapy or surgical procedures, including alkylating agents, antimetabolites, anti-hormones, therapeutic agents for a variety of conditions, such as analgesics, diuretics, antidiuretic agents, antivirals, antibiotics, cytokines, nutritional supplements, anemia therapeutic agents, coagulation therapeutic agents, bone therapeutic agents, psychiatric and psychological therapeutic agents, and the like.

To achieve autologous bone marrow or stem cell transplantation, bone marrow or peripheral blood stem cells may be collected from the patient after treatment with an anti-CCR 7 antibody.

Cytokines may also be used to treat patients in order to upregulate expression of CCR7 or other target proteins on the surface of cancerous B cells prior to administration of the antibodies of the present invention. Cytokines may also be administered simultaneously with, prior to, or after the administration of the clearing antibody or radiolabeled antibody in order to stimulate immune effector function.

In one embodiment, a chemotherapeutic regimen may be used to supplement the therapies disclosed herein, and may be administered simultaneously with the radiolabeled antibody or sequentially in any order. The chemotherapy regimen may be selected from the group consisting of CHOP (cyclophosphamide, doxorubicin (also known as hydroxydaunorubicin), vincristine (also known as vincristine sulfate) and prednisone), ICE (demethoxydaunorubicin, cytarabine and etoposide), Mitozantrone, cytarabine, DVP (daunorubicin, vincristine and prednisone), ATRA (all-trans retinoic acid), demethoxydaunorubicin, hoelzer chemotherapy regimen, La chemotherapy regimen, abdd (bleomycin, dacarbazine, doxorubicin and vincristine), CEOP (cyclophosphamide, epidaunorubicin, vincristine and prednisolone), 2-CdA (2-chlorodeoxyadenosine), FLAG & IDA (fludarabine, cytarabine, filgrastim and oxytocin), (with or without subsequent G-CSF (granulocyte colony stimulating factor) therapy), VAD (vincristine, doxorubicin and dexamethasone), M & P (levo-sarcolysin and prednisone), C (cyclophosphamide) -once weekly, ABCM (doxorubicin hydrochloride, bleomycin, cyclophosphamide and mitomycin-C), MOPP (nitrogen mustard, vincristine, prednisone and procarbazine) and DHAP (dexamethasone, cytarabine and cisplatin). The preferred chemotherapeutic regimen is CHOP.

Thus, in other aspects, the invention relates to a method of treating cancer, in particular cancer in which tumour cells express the CCR7 receptor, comprising administering to a subject in need of such treatment a therapeutically effective amount of an antibody of the invention, i.e. an antibody or antigen-binding fragment thereof that binds to the CCR7 receptor, or administering a therapeutically effective amount of a pharmaceutical composition of the invention. In particular embodiments, the cancer is characterized by tumor cells expressing the CCR7 receptor. Illustrative non-limiting cancers to be treated according to the invention include CLL, MCL, follicular lymphoma, large B-cell lymphoma, AIDS-related lymphoma, lymphoplasmacytic lymphoma, burkitt's lymphoma, B-cell acute lymphoblastic lymphoma, hodgkin's disease, adult T-cell leukemia/lymphoma, mycosis fungoides, outbreaks of chronic myeloproliferative disease, outbreaks of myelodysplastic syndrome, breast cancer, non-small cell lung cancer, melanoma, gastric cancer, or squamous cell carcinoma of the head and neck, and colon cancer. In a preferred embodiment, the cancers treated according to the invention include breast cancer, non-small cell lung cancer, melanoma, gastric cancer, or squamous cell carcinoma of the head and neck and colon cancer. In a most preferred embodiment, the cancers treated according to the present invention include follicular lymphoma, adult T-cell leukemia/lymphoma, burkitt's lymphoma, outbreaks of chronic myeloproliferative disease, and outbreaks of myelodysplastic syndrome. In other most preferred embodiments, the cancer treated according to the invention comprises CLL and MCL.

In other aspects, the invention relates to methods of killing tumor cells expressing the CCR7 receptor or inducing apoptosis in tumor cells expressing the CCR7 receptor comprising contacting the cells with an antibody of the invention, i.e., an antibody or antigen-binding fragment thereof that binds the CCR7 receptor. In particular embodiments, the tumor cells are tumor cells that express the CCR7 receptor, such as CLL and MCL cells.

In other aspects, the invention relates to a method of killing tumor cells expressing the CCR7 receptor or inducing apoptosis of tumor cells expressing the CCR7 receptor in an individual in need of such treatment comprising administering to said individual a therapeutically effective amount of an antibody of the invention, i.e. an antibody or antigen-binding fragment thereof that binds to the CCR7 receptor.

In other aspects, the invention relates to methods of attenuating migration of tumor cells expressing a CCR7 receptor to secondary lymphoid tissue and/or blocking dissemination of tumor cells into secondary lymphoid tissue comprising contacting said tumor cells with an antibody of the invention, i.e., an antibody or antigen-binding fragment thereof that binds to a CCR7 receptor.

In other aspects, the invention relates to methods of attenuating migration of tumor cells expressing a CCR7 receptor to secondary lymphoid tissue and/or blocking dissemination of tumor cells into secondary lymphoid tissue in an individual in need of such treatment comprising administering to said individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to a CCR7 receptor.

Information on CCR7 receptor expressing tumor cells, antibodies, dosing regimens and dosages that are amenable to treatment by the methods of the invention are set forth above. In particular embodiments, the tumor cells expressing the CCR7 receptor susceptible to treatment with the methods described above are CLL or MCL cells.

In all cases, the phrase "therapeutically effective amount" refers to an amount effective for treating cancer as defined previously; the amount can be an amount sufficient to achieve a desired response or to ameliorate symptoms or indicators such as metastasis or primary tumor progression, size, or growth. The therapeutically effective amount for a particular individual may vary depending on factors such as the disease state being treated, the overall health of the individual, the method, route and dosage of administration, and the severity of the side effects. For example, the effect may result in a change in the basis weight of at least about 10%, preferably at least 20%, 30%, 50%, 70% or even 90% or more. When in combination, a therapeutically effective amount is the ratio of the combination of the relative components and the effect is not limited to a single component.

A therapeutically effective amount will generally modulate symptoms by at least about 10%; typically at least about 20%; preferably at least about 30%; or more preferably at least about 50%. Alternatively, modulation of migration refers to the migration or functioning of multiple cell types being affected. These will result in, for example, statistically significant and quantifiable changes in the number of affected cells. This may be a reduction in the number of target cells affected over a period of time or in a target area. The rate, size, spread or growth of progression of the primary tumor can also be monitored.

Any of the above methods of treatment may be used in any individual in need of such treatment, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably humans.

In other aspects, the invention relates to methods of identifying compounds for killing tumor cells expressing the CCR7 receptor or inducing apoptosis in tumor cells expressing the CCR7 receptor comprising

a) Contacting a cell expressing the CCR7 receptor with a candidate compound coupled to an anti-CCR 7 antibody or fragment thereof, and

b) determining whether the candidate compound kills the cells expressing the CCR7 receptor,

wherein the compound that kills the cells expressing the CCR7 receptor is a compound that is potentially useful for killing tumor cells expressing the CCR7 receptor or inducing apoptosis in tumor cells.

Virtually any cell, tumor cell or non-tumor cell, that expresses the CCR7 receptor can be used to perform the above methods. In particular embodiments, the cell expressing the CCR7 receptor is a CLL or MCL cell.

Death of cells expressing the CCR7 receptor can be determined by any conventional method, for example, according to the methods disclosed in the examples herein.

The following examples serve to illustrate the invention.

Examples

Antibodies to CCR7 as tools for treating CLL

I. Materials and methods

Sample, reagent and Flow Cytometry (FCM)

Peripheral blood samples from CLL (chronic lymphocytic leukemia) and MCL (mantle cell lymphoma) patients and healthy donors were obtained after informed consent. Initial immunophenotypic characterization of whole blood cells was performed by a standard four-color FCM using monoclonal antibodies (mAbs) against the following human surface antigens: CD45, CD19, kappa light chain, lambda light chain, CD20, CD23, CD5 and CD3 (all available from BD Biosciences, San Jose, CA). Less than 60% of the samples of CLL or MCL cells in the mononuclear subpopulation were discarded. CCR7 expression was then analyzed in electronically gated tumor B cells or normal T and B lymphocytes.

Phycoerythrin (PE) -conjugated mouse anti-human CCR7 was purchased from R & D Systems (McKinley Place, MN). Appropriate isotype controls were included in all cases. Immunofluorescent staining was analyzed on a FACScalibur flow cytometer using CellQuest software (BD Biosciences).

Peripheral Blood Mononuclear Cells (PBMC) were isolated by polysucrose (Ficoll) density gradient centrifugation (Histopaque-1077, Sigma-Aldrich, Madrid, Spain).

Purified murine anti-human CCR7mAbs (hereinafter "anti-CCR 7 mAbs") were obtained from BDBiosciences (clone 150503, IgG2a isotype) and R & D Systems (clone 2H4, IgM isotype). The DNA dye 7-amino-actinomycin D (7-AAD) used in cell cycle analysis and cell viability assays was obtained from BD Biosciences. Recombinant human chemokines CCL19 and CXCL12 were purchased from R & D Systems.

Receptor endocytosis assay

To investigate whether binding of anti-CCR 7mAbs resulted in internalization of chemokine receptor (CCR7), 2. mu.g/mL of anti-CCR 7mAbs were used at 37 ℃ with 5% CO₂Incubation under air 5X 10⁵CLL cells were cultured for various periods of time (from 30 seconds to 1 hour). CCL19, which is one of the physiological ligands of CCR7 and causes its endocytosis, was used as a positive control. After clearance of bound mAb or CCL19 using an acid wash (0.1M glycine, 0.15M nacl, pH 2.5), CCR7 expression on CLL cells was determined by FCM analysis as described above.

Complement Dependent Cytotoxicity (CDC)

Will contain 10⁵50 μ L of the cells in PBMC suspension and purified anti-CCR 7mAbs or Isotype Control (IC) at desired concentrations from 0.5mg/mL to 16mg/mL were plated in 96 round bottom well plates. After incubation at 37 ℃ for 30 min, the cells were centrifuged and washed. Then, young rabbit complement (Serotec, Oxford, UK) diluted to 25% in RPMI1640 medium was added. After 1-2 hours at 37 ℃, cells were stained with Fluorescein Isothiocyanate (FITC) -conjugated anti-CD 19, Allophycocyanin (APC) -conjugated anti-CD 5mAb, and 7-AAD. Both surface markers allow FCM to resolve CLL cells, MCL cells and T cell populations, and 7-AAD is used as a viability exclusion dye. The percentage of non-viable cells was measured separately in each population and the percentage of lysis using heat inactivated complement was according to the formula [1]Calculating characteristicsHeterolysis

Wherein

Antibody-dependent, cell-mediated cytotoxicity (ADCC)

Natural Killer (NK) cells were isolated from PBMCs obtained from buffy coats (buffy coats). Positive selection for NK cells was performed by PE-conjugated anti-CD 56mAb staining followed by magnetic cell sorting using anti-PE-conjugated microbeads (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). The purity of NK cells measured by FCM was 95% to 98%. anti-CCR 7 dependent, cell mediated cytotoxicity was measured with FCM after incubation of CLL cells (target cells) with NK cells (as cytolytic effectors) at different effect-to-target (E/T) ratios between 5 and 40, in the presence or absence of anti-CCR 7mAb or its isotype control for 4 hours. Then, cells were stained with FITC-conjugated anti-CD 19mAb, PE-conjugated anti-CD 56mAb and 7-AAD to distinguish CLL cells and effector NK cells, and viability of CLL cells was analyzed. CLL cells without NK cells used in the incubation were used as a control for spontaneous death and specific lysis was calculated as CDC assay.

Chemotaxis assay

CLL and MCL cells previously incubated for 30 minutes with 2 μ g/mL anti-CCR 7mAbs were assayed for chemotaxis in response to ligand CXCL12 of CCL19, CCL21 and CXCR4 in a Transwell cell culture chamber (6.5 mm diameter, 10 μm thickness, 5 μm pore diameter, Costar, Cambridge, MA). For chemotaxis assay, 5X 10⁵Tumor cells were suspended in 100. mu.l RPMl 1640 containing 0.5% BSA, added to the upper compartment of the culture chamber and chemokines added at the optimal concentration (100ng/mL for CXCL12 and 1. mu.g/mL for CCL19 and CCL 21) below600 μ l of the same medium in the partial chamber. Allowed to migrate at 37 ℃ and 5% CO₂Air was allowed to flow for 4 hours. Migrated cells were recovered from the lower compartment, stained with FITC-conjugated anti-CD 19mAb, and counted for 60 seconds with FCM after calibrating flow rate with a Trucount tube (BD Biosciences). Events were analyzed in gated B cell populations and compared to the number of cells in the initial suspension of cells to calculate the percentage of input (100 x number of migrated cells/number of cells counted in initial suspension). The measurement was repeated twice for each sample.

Apoptosis and cell cycle analysis

In some experiments, anti-CCR 7mAbs were diluted at 2. mu.g/mL in 50. mu.L medium containing 10⁵CLL cell culture suspension. In other experiments, anti-CCR 7mAbs were attached to 96 flat bottom well plates by incubation in RPMI1640 overnight. The plate was then washed and 10 added⁵CLL cells. In both cases, cells were incubated at 37 ℃ for up to 48 hours. Cells were washed with cold Phosphate Buffered Saline (PBS), stained with FITC-conjugated anti-CD 19mAb, resuspended in 0.5mL PBS and diluted in 5mL ice-cold 70% ethanol for fixation at-20 ℃ overnight. The following day, the fixed cells were centrifuged, washed and stained with 20. mu.g/mL of 7-AAD for DNA content analysis. A minimum of 10,000 total events were obtained and analyzed using Cell-Quest Pro software (BD biosciences). Briefly, the pulse area and pulse width of 7-AAD fluorescence was used to gate CD19 positive events and double lines were discarded. Analysis of sub-G as apoptotic cells in 7-AAD fluorescence histograms₁Percentage of peaks.

Proliferation assay

PBMCs were isolated from healthy donors and cultured for 72 hours in 96-well plates previously coated with anti-CCR 7mAbs and their corresponding isotype controls. Positive controls such as anti-CD 3 plus IL2 are also included. All measurements were repeated three times. Use of 1. mu. Ci per well of [ 2 ], [ 2 ] in the last 16 hours of culture³H]Thymidine (Amersham Biosciences GmbH, Freiburg, Germany) labels cells. Cells were then collected and scintillation counted.

Statistical analysis

All statistical tests were performed at SPSS version 8.0. Wilcoxon and Kendall's W nonparametric tests were used to compare the results of different treatments in a larger number of assays. For [ 2 ]³H]Thymidine proliferation assay, ANOVA for group comparison. Pearson correlation coefficients were calculated to investigate the relationship between CCR7 Mean Fluorescence Intensity (MFI) and percent solubility in CDC.

Results II

anti-CCR 7mAbs do not induce internalization of their targets

The use of unconjugated forms of therapeutic mAbs requires not only a high density of target antigens, but also the presence of antigen-antibody complexes on the cell surface to activate complement or bind Fc receptors of cytotoxic cells, i.e., it is important that binding of the mAb to the antigen does not induce its internalization, as this may reduce the therapeutic effect of the mAb. In this regard, it has been suggested that binding of antibodies to certain chemokine receptors can result in endocytosis of the receptor and subsequent proteolysis or re-expression of the receptor following degradation of the antibody. The inventors analyzed whether the binding of mAbs to CCR7 resulted in their endocytosis in CLL cells. CCR7 expression on CLL cell surfaces was 2-4 fold higher in CLL cells than in normal B or T cells (fig. 1A) and did not decrease at different times of incubation with anti-CCR 7mAbs for up to 60 min (fig. 1B). In contrast, it decreased by about 60% in the first 5 minutes when incubated with 1 μ g/mL CCL19 as a positive control.

Strong and specific CDC of CLL and MCL cells mediated by anti-CCR 7mAbs in vitro

Cytotoxicity mediated by the recruitment of complement proteins (CDC) or effector cells such as NK cells (ADCC) is the proposed primary mechanism for the in vivo clearance of tumor cells by unconjugated therapeutic mAbs. Therefore, CLL and MCL samples from different patients were tested for potency against CCR7mAbs in mediating CDC. A very high proportion of CLL cells pre-incubated with anti-CCR 7IgM mAb were killed after 1 hour of treatment with rabbit complement (fig. 2A). Similarly, anti-CCR 7mabs of IgG isotype also mediated significant CDC (fig. 2B).

Similar to CLL, MCL cells also expressed significant levels of CCR7 (fig. 3A), and CDC experiments showed that both anti-CCR 7mAbs were effective in killing CCR7 positive MCL cells (fig. 3B). No significant CDC was observed when using unrelated immunoglobulins of the same isotype (fig. 2A, 2B and 3B).

Although the potency of anti-CCR 7mAb of IgG isotype to cause CDC was relatively low, kinetic assays demonstrated that its complement activation capacity could be significantly increased by extending the time of incubation with complement source (data not shown). This is probably due to the slower activation of the complement cascade by mabs of the IgG2a isotype.

CCR7 is not limited to CLL or MCL cells, but is also expressed in certain normal lymphoid subpopulations, mainly naive B cells and naive T cells, which may be susceptible to cytotoxicity mediated by anti-CCR 7 mAbs. Interestingly, CDC mediated by anti-CCR 7mAbs was significantly higher in CLL cells than in T cells from the same patient (P ═ 0.001 for IgM mAbs and 0.016 for IgG mAbs). Similarly, B cells and T cells from healthy donors were resistant to complement activity when treated with the same experimental conditions as described above, even at higher doses of the two anti-CCR 7mAbs, as demonstrated by the dose-response assay (fig. 4A and 4B). This is especially true for T cells from CLL patients that suffer little lysis (fig. 4A and 4B), probably due to the high and fast depletion of complement by CLL cells present in these samples. On the other hand, both anti-CCR 7mAbs at concentrations as low as 1. mu.g/mL were sufficient to mediate CDC of CLL cells (FIGS. 4A and 4B).

One possible explanation is that there is a differential sensitivity to CDC activity, since CCR7 is expressed much less in normal T and B cells than in CLL cells (fig. 1A). Indeed, the expression level of CCR7 in CLL cells was significantly correlated with the sensitivity of different samples to CDC (fig. 5) (r ═ 0.602, P ═ 0.025, n ═ 11), confirming the importance of antigen density in selecting targets for immunotherapy.

Furthermore, it appears that differences in CDC activity are not associated with differential expression of complement regulatory proteins such as CD55 or CD59, as their expression levels are very similar between different CLL samples and between CLL and T cells from these patients (data not shown).

In vitro ADCC of CLL cells

The other major mechanism mediating the beneficial effects of therapeutic mAbs is cell-mediated cytotoxicity (ADCC). Thus, the inventors tested the ability of NK cells to cause lysis of CLL cells pre-treated with anti-CCR 7IgG mAb. In 6 experiments with E/T ratios of 5 to 40, no significant ADCC was observed in the presence of this anti-CCR 7mAb (mean solubility 13% ± 1, n ═ 6) when compared to an unrelated immunoglobulin of the same isotype (mean solubility 12% ± 5, n ═ 6) (figure 6). This may be associated with the low affinity of human Fc receptors for murine mAbs. In contrast, rituximab, a well-known chimeric anti-CD 20mAb, the IgG1 isotype, mediated ADCC against CLL cells with an average lysis of 34% ± 2(n ═ 6) (fig. 6). ADCC in the presence of anti-CCR 7IgM mAb was not tested because this isotype is not effective in mediating this type of cytotoxicity.

Apoptosis and proliferation in response to anti-CCR 7mAbs

Increasing evidence suggests that a significant portion of the beneficial responses of therapeutic mAbs are due to direct effects on the cell cycle or target cell survival. The effect of anti-CCR 7mAbs on apoptosis of CLL cells was studied by 7-AAD staining of cellular DNA and analysis of the sub-G1 peak. The apoptosis rate of CLL cells treated with soluble anti-CCR 7IgM mAb for 24 hours was not significantly different from that of CLL cells treated with IC (data not shown). Similarly, cross-linking of CCR7 generated by application of anti-CCR 7mAbs to 48 hours did not induce apoptosis (data not shown).

To study cell proliferation in response to anti-CCR 7mAbs, classical [ 2 ] was performed with PBMC from a healthy donor³H]Thymidine uptake proliferation assay, since CLL cells have a high rate of spontaneous apoptosis after 48-72 hours of incubation. These assays reveal that after incubation with an anti-CCR 7mAb of IgM isotype³H]Moderate increase of thymidine incorporation into cells, which is obtained with IgM ICThe results of (a) were not significantly different. In contrast, neither anti-CCR 7mAb of the IgG isotype nor its IC induced proliferation (fig. 7). As a positive control, PBMCs proliferated in response to incubation with anti-CD 3mAb plus IL 2.

anti-CCR 7mAbs inhibit migration of CLL and MCL cells in vitro

CCR7 plays a major role not only in the physiological homing of naive lymphocytes and mature dendritic cells, but also in the metastatic migration of tumor (cancer) cells expressing it, particularly CLL and MCL cells. To block the spread of these diseases to lymphoid tissues, neutralization of this chemokine receptor function may be of interest in CLL and MCL patients. In this regard, the inventors analyzed in vitro migration of anti-CCR 7mAbs neutralizing CLL and MCL cells in response to CCL19 or CCL 21. anti-CCR 7mAb of the IgG isotype very effectively blocked migration of these cells to CCL19 or CCL21, while anti-CCR 7mAb of the IgM isotype slightly reduced migration (fig. 8A-B).

In these assays, the migration of CLL cells in response to CXCL12 (a ligand for chemokine receptor CXCR 4) was not affected by the two mAbs used.

Discussion of

The foregoing data from the inventors show that expression levels of chemokine receptors CCR7, CXCR4, and CXCR5 in B cell lymphoproliferative disorders are very heterogeneous according to histological subtype and are significantly associated with Lymph Node (LN) involvement, as these molecules mediate lymphocyte entry and localization to secondary lymphoid tissues. Expression of CCR7 is particularly high in CLL and mantle cell lymphomas, explaining the high propensity of these diseases to invade LNs. Similarly, recent studies report expression of CCR7 on malignant cells from other hematological tumors such as hodgkin's disease, adult T-cell leukemia/lymphoma, and mycosis fungoides, or non-hematological solid tumors such as breast cancer, non-small cell lung cancer, melanoma, gastric cancer, or squamous cell carcinoma of the head and neck or colon cancer. Interestingly, in all cases, this expression correlated with a characteristic pattern of migration and metastasis to lymphoid tissues.

These data suggest the possibility of using CCR7 as a therapeutic target in immunotherapy, not only because of its high density in certain malignant cells and its limited expression in normal tissues, but also because of its critical role in disease progression and pathogenicity.

It has been accepted that CDC is the primary mechanism of action of unconjugated therapeutic mabs when the target antigen is highly expressed, as CDC is believed to require about 10-fold more surface antigen density than ADCC. Thus, the inventors analyzed the ability of anti-CCR 7mAbs to fix complement and mediate cytotoxicity against CLL cells, MCL cells and normal T cells from CLL patients. Their results show that the important lysis of CLL and MCL cells is due to complement fixation with little T cell destruction, even at saturating concentrations of anti-CCR 7mAbs and complement. This is probably due to the lower expression of CCR7 on T cells compared to CLL cells. In this regard, the inventors found that under the same experimental conditions, CCR7 density on CLL cell surface and the percentage of lysed cells were closely related (p 0.025, r 0.602). Importantly, these results suggest that treatment of CLL or MCL with anti-CCR 7mAbs, even at low concentrations, may result in efficient clearance of tumor cells without lysing normal lymphocytes expressing the molecule (CCR 7). It is possible to hypothesize that secondary immunodeficiency due to treatment with anti-CCR 7mAbs would not be very important, as CCR7 negative effector lymphocytes remain undisturbed. The inference of immunodeficiency as a characteristic of CLL or MCL patients from CCR7 deficient mice to treatment with anti-CCR 7mAbs is in some way difficult, as these individuals have developed an immunological memory that is deficient in CCR7 knockdown.

Unlike findings with respect to CDC, anti-CCR 7mabs of the IgG isotype are not activators of ACDD, possibly due to their murine origin. Nevertheless, it is well known that molecular engineering techniques make it possible to develop chimeric or humanized antibodies to improve clinical features such as the ability of the mAb to mediate cytotoxicity through Fc receptors of monocytes, neutrophils and NK cells. In any case, the importance of ADCC in the immunotherapy of CLL is currently under discussion, due to the strong expansion of malignant cells and the functional deficiency of T cells and NK cells in these patients. In this regard, several methods have recently been published to expand and activate cytotoxic lymphocytes and NK cells from healthy donors or CLL patients to take advantage of the mechanism of action of the therapeutic mAbs.

Blocking the function of the target antigen constitutes another mechanism of action for therapeutic mAbs. In the present invention, blocking anti-CCR 7mAbs may have additional advantages: inhibiting the function of possible major molecules involved in lymphoid tumors and other non-hematologic tumors expressing them. In this regard, neither rituximab (rituximab) nor alemtuzumab (alemtuzumab) are known to be very effective in reducing lymphadenopathy in CLL patients.

These results open new therapeutic opportunities for those pathologies where blocking anti-CCR 7mAbs can attenuate migration to LN and thus block tumor spread in addition to killing cells by CDC or ADCC.

Claims (11)

1. Use of an antibody or antigen-binding fragment thereof that binds to the CCR7 receptor for the preparation of a pharmaceutical composition for killing tumor cells expressing the CCR7 receptor or inducing apoptosis of tumor cells expressing the CCR7 receptor.

2. The use of claim 1 or 2, wherein the tumor cell is a Chronic Lymphocytic Leukemia (CLL) cell or a Mantle Cell Lymphoma (MCL) cell.

3. Use according to any of claims 1 or 2, wherein the antibody or antigen-binding fragment thereof that binds to the CCR7 receptor is used in combination with an additional therapeutically active compound.

4. A method for killing a tumor cell expressing a CCR7 receptor or inducing apoptosis of a tumor cell expressing a CCR7 receptor comprising contacting the cell with an antibody or antigen-binding fragment thereof that binds to the CCR7 receptor.

5. A method for killing tumor cells expressing the CCR7 receptor or inducing apoptosis of tumor cells expressing the CCR7 receptor in an individual in need of such treatment comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to the CCR7 receptor.

6. The method of claim 4 or 5, wherein the tumor cell is a Chronic Lymphocytic Leukemia (CLL) cell or a Mantle Cell Lymphoma (MCL) cell.

7. A method for attenuating migration of tumor cells expressing a CCR7 receptor to secondary lymphoid tissue and/or blocking dissemination of tumor cells into secondary lymphoid tissue, comprising contacting said tumor cells with an antibody or antigen-binding fragment thereof that binds to a CCR7 receptor.

8. A method for attenuating migration of tumor cells expressing a CCR7 receptor to secondary lymphoid tissue and/or blocking dissemination of tumor cells into secondary lymphoid tissue in an individual in need of such treatment comprising administering to said individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof that binds to a CCR7 receptor.

9. The method of claim 7 or 8, wherein the tumor cell is a Chronic Lymphocytic Leukemia (CLL) cell or a Mantle Cell Lymphoma (MCL) cell.

10. A method for identifying a compound for killing tumor cells expressing a CCR7 receptor or inducing apoptosis of tumor cells expressing a CCR7 receptor comprising

a) Contacting a cell expressing the CCR7 receptor with a candidate compound conjugated to an anti-CCR 7 antibody or fragment thereof, and

b) determining whether the candidate compound kills the cells expressing the CCR7 receptor,

wherein the compound that kills the CCR7 receptor-expressing cells is a compound that is potentially useful for killing tumor cells or inducing apoptosis of tumor cells.

11. The method of claim 10, wherein the cell is a Chronic Lymphocytic Leukemia (CLL) cell or a Mantle Cell Lymphoma (MCL) cell.