EP2285403A2 - Human monoclonal antibodies against human chemokine receptor ccr7 - Google Patents

Abstract

Aspects of this invention include fully human antibodies of fragments thereof that bind specifically to human CCR7 receptor. Such antibodies or fragments thereof can be used to treat disorders involving over function of the CCR7 receptor, including cancers. Other uses include detection of human CCR7 receptor in biological samples for diagnostic or evaluative purposes.

Description

HUMAN MONOCLONAL ANTIBODIES AGAINST HUMAN CHEMOKINE RECEPTOR CCR7

Claim of Priority

This application claims priority to United States Provisional Patent Application No: 61/127,747, filed 14 May 2008, entitled "Human Monoclonal Antibodies Against Human Chemokine Receptor CCR7," Tajib Mirzabekov, David Kreimer and Eldar Kim, Inventors. This application is expressly incorporated herein fully by reference.

Field of the Invention

This invention relates to antibodies against human G-protein coupled receptors (GPCRs). Particularly, this invention relates to fully human antibodies and fragments thereof directed against GPCRs. More particularly, this invention relates to fully human antibodies and fragments thereof directed against the human chemokine receptor CCR7.

BACKGROUND

Chemokines are molecules having diverse function. They are extracellular molecules that can initiate and/or maintain numerous cell processes, including chemotaxis, cell growth and in some cases, tumor growth and metastasis. Chemokines can act by binding to, activating, or inhibiting receptors known as chemokine receptors. Chemokine receptors are in the class of G- protein coupled receptors (GPCRs) and include multispanning membrane proteins, in which the protein has one or more regions that span a cellular membrane.

SUMMARY

We disclose fully human antibodies that can specifically bind to human chemokine receptor CCR7 on the surfaces of living cells. We disclose 129 different antibodies with different variable domain (CDR3) sequences. These fully human antibodies can be used as therapeutics for the treatment of different types of cancer, inflammation, and other diseases. These fully human antibodies selectively bind to human CCR7, and include antibodies having agonist properties and antibodies having antagonist (neutralizing) properties. Cancers such as Chronic Lymphocytic Leukemia (CLL), Head and Neck Cancer (HNC), Non-Small Cell Lung Cancer (NSCLC), Breast Cancer, Gastric Cancer, Melanoma and other types of cancer that express chemokine receptor CCR7 are therapeutic targets for the fully human anti-CCR7 antibodies and fragments there of this invention..

BRIEF DESCRIPTION OF THE FIGURES

This invention is described with reference to specific embodiments thereof. Other features and aspects of this invention can be appreciated with reference to the Figures, in which:

FIG. 1 depicts an SDS gel showing banding of 9 monoclonal antibody fragments (svFv) of this invention directed against human CCR7. The antibody fragments (svFv) all have molecular weights of approximately 25 to 28 kDa.

FIG. 2 depicts a graph of fluorescence of cells expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-107 of this invention. Columns 1-11 show results for the cells: (1) CHO-Kl-hCCR7, (2) BHK-21-hCCR7, (3) R1610-hCCR7, (4) CHO- Kl-hFPR, (5) CHO-Kl-hCCR5, (6) BHK-21 -parental, (7) R1610-hCXCR2, (8) CF2TH- hCXCR2, (9) Cf2Th-hCXCR3, (10) 293Trex-hFPR and (11) CHO-Kl-mouse CCR7, respectively.

FIG. 3 depicts a graph of fluorescence of cells as in FIG. 2 expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-201 of this invention.

FIG. 4 depicts a graph of fluorescence of cells as in FIG. 2 expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-312 of this invention.

FIG. 5 depicts a graph of fluorescence of cells as in FIG. 2 expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-315 of this invention.

FIG. 6 depicts a graph of fluorescence of cells as in FIG. 2 expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-327 of this invention.

FIG. 7 depicts a graph of fluorescence of cells as in FIG. 2 expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-333 of this invention.

FIG. 8 depicts a graph of fluorescence of cells as in FIG. 2 expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-334 of this invention. FIG. 9 depicts a graph of fluorescence of cells as in FIG. 2 expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-337 of this invention.

FIG. 10 depicts a graph of fluorescence of cells as in FIG. 2 expressing CCR7 or other GPCRs, and labeled with mouse antibodies. Cells in lanes 1 to 11 are: (1) CHO-Kl-hCCCR7 + mouse anti-hCCR7-PE, (2) BHK-21-hCCR7 + mouse anti-hCCR7-PE, (3) R1610-hCCR7 + Mouse anti-hCCR7-PE, (4) CHO-Kl-FPR, (5) CHO-Kl-hCCR5, (6) BHK-21 -Parental, (7) F1610-hCXCR2, (8) CF2Th-hCXCR2, (9) CF2Th-hCXCR3, (10) 293Trex-hFPR, and (11) CHO-Kl -mouse CCR7 + rat anti-mouse CCR7-PE, respectively.

FIG. 11 depicts a graph of fluorescence of cells expressing CCR7 or control cells in the presence of the natural ligand CCLl 9-Fc labeled with mouse antibodies.

DETAILED DESCRIPTION

Aspects of this invention include fully human antibodies and fragments thereof directed against CCR7. CCR7 is involved in cancer, and antibodies and fragments there that bind to CCR7 can result in decreased cancer growth. In aspects of this invention, antibodies and fragments thereof are fully human. This provides therapeutic potential in treating human disease, because use of non-human antibodies or even humanized antibodies can produce unwanted side effects due to graft versus host immune responses to the antibodies. Thus, fully human antibodies can provide greater therapeutic index compared to other antibody-based approaches.

CCR7 is a GPCR that binds to CC chemokine ligands MIP-3beta (ELC/CCL19) and 6Ckine (CCL21) (Yoshida et al. Molecular cloning of a novel human CC chemokine EBIl- ligand chemokine that is a specific functional ligand for EBIl, CCRl . J Biol Chem. 272: 13803- 13809 (1997)). These ligands are expressed in the secondary lymphoid organs, and binding to CCR7 expressed in naive T cells, B cells and dendritic cells directs migration of these cells to sites of antigen presentation (Foster et al. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell, 99 :23-33 (1999)). Inhibition of CCR7/ligand interactions inhibits contact sensitivity, delayed type hypersensitivity, and graft vs host disease in experimental models (Foster et al. Id., Sasaki et al. Antagonist of secondary lymphoid-tissue chemokine (CCR ligand 21) prevents the development of chronic graft-versus-host disease in mice. J Immunol. 170: 588-596 (2003)). In addition, CCR7 expression by breast cancer, melanoma and other malignant cells are associated with lymph node metastasis (Muller et al., Involvement of chemokine receptors in breast cancer metastasis. Nature. 6824:50-56 (2001); Payne, A.S. and L. A. Cornelius. The role of chemokines in melanoma tumor growth and metastasis. J. Invest. Dermatol. 118: 915-922 (2002)).

Although the mechanisms of such action of the fully human antibodies of this invention are not completely known, there are several possible mechanisms. Ligands of CCR7, chemokine peptides CCL 19 and CCL21, are expressed in lymph nodes. A signal induced by interaction of such ligands with CCR7-expressing tumor cells can result in metastatic homing of tumor cells in the lymph nodes. Blockade of CCR7 with antagonistic antibodies can prevent or inhibit the signaling, and thus can prevent or inhibit homing of tumor cells in the lymph nodes.

Even in the absence of antagonistic effects, important therapeutic effects of antibodies can arise from binding of antibodies to CCR7. In such cases, activation of ADCC and CDC mechanisms can result to the elimination of CCR7 expressing cells. Part of the mechanism is similat to the mechanism of how Rituxan™ is thought to eliminate CD20-expressing cells.

Another mode of possible therapeutic efficacy of the antibodies of this invention includes the inhibition of chemotaxis of CCR7-expressing cells due to binding of these antibodies to CCR7 and thereby inhibiting chemoattractant signals induced by the chemokine ligands CCL 19 and CCL21.

CCR7 is highly important receptor with a role in trafficking of B and T lymphocytes and dendritic cells to and across high endothelial venules and positioning those cells correctly in T cell zones of secondary lymphoid organs. The natural ligands of CCR7 are chemokines CCLl 9 (also called MIP-3beta, ELC, or Exodus-3) and CCL21 (also called 6Ckine, SLC, and Exodus- T), both biding to T-cells and actT and mDC cell types.

Binding of chemokines to their corresponding GPCRs induce cell signaling. In case of CCR7, the ligand binding induced signaling can be involved in the progression of cancer and some inflammatory diseases. Therefore the blocking of this signaling can be therapeutically useful. We have found the some human antibodies binding to CCR7 and neutralize the binding of chemokines to this receptor, e.g. the signaling. We call these antibodies antagonists or neutralizing antibodies. It should be appreciated that the above mechanisms are for purposes of illustration only, and are not considered to be the only mechanisms possible. The proper scope of this invention includes all possible mechanisms of action of the fully human antibodies of this invention.

Methods for Producing CCR7 Antibodies

In certain embodiments of this invention, CCR7 receptors can be isolated from membranes of cells expressing the protein and used as an immunogen to produce CCR7-specific antibodies. In general, we used methods described in PCT International Patent Application No: PCT/US2007/003169, filed 5 February 2007 (WO 2007/092457). This application is expressly incorporated herein fully by reference.

Applications of Human Antibodies Against Human CCR7

Fully human anti-CCR7 antibodies and/or fragments thereof can be used as therapeutic agents for different types of cancer where CCR7 plays a role. The types of cancer include: (1) Chronic Leukocytic Leukemia; (2) Head and Neck cancer; (3) Non-Small Cell Lung Cancer; (4) Breast Cancer; (5) Gastric Cancer as well as other types of human cancers. In addition to cancer, anti-CCR7 antibodies and fragments thereof may be used as a therapeutics for treatment of inflammatory diseases such as (1) Rheumatoid Arthritis; (2) Inflammatory Bowel Disease; (3) Psoriasis; ?(4). Lupus; (5) Multiple Sclerosis, and (6) Asthma. However, it can be appreciated that other disorders involving CCR7 can be treated as well. Thus, the full scope of therapeutics involving antibodies and fragments thereof of this invention includes any disorder in which CCR7's actions are at least partially responsible for the disorder. Further descriptions of applications are included herein below. As can be appreciated from Examples 11-13 herein, anti CCR7 antibodies can inhibit binding of natural ligands of CCR7, thereby demonstrating that thereapeutic uses of fully human CCR7 antibodies can be a viable alternative to existing treatments for such disorders.

Description of Antibodies

We have identified three classes of human anti-human CCR7 ("anti-hCCR7") antibodies: 1. Monoclonal Antibodies (MAbs) and fragments thereof which just bind to CCR7 but do not affect its natural ligand binding properties and signaling;

2. MAbs and fragments thereof which bind to CCR7 and activate the signaling by natural ligands (agonists); and

3. Antibodies what bind to CCR7 and inhibit the binding of natural ligands CCL 19 and CCL21 to CCR7 (antagonists). Therefore they are called neutralizing MABs.

All the antibodies were identified from phage display human antibody libraries presenting broad repertoire (up to lθ") of different human antibody variable domains Vl and Vh as a fusion protein (scFvs libraries).

Fully human antibodies against human CCR7 of this invention that cross react with mouse CCR7 can be useful in further development of drugs affecting CCR7 in human beings for treatment of a variety of diseases and conditions. Numerous mouse models can be employed to demonstrate an efficacy of anti-CCR7 antibodies. Cross reactivity of human antibodies of this invention with mouse CCR7 can make the use of these well established models straightforward. Therefore, data obtained in mouse models using fully human antibodies human are reasonably predictive of effects observed in human beings. Thus, the antibodies of this invention can be useful for treatment of human diseases and conditions, such as asthma, arteriosclerosis, various types and stages of cancer, including metastasis, various inflammatory conditions and others in which CCR7 and its natural ligands CCLl 9 and CCL21 are involved.

Applications of Fully Human Anti-CCR7 Antibodies

Fully human antibodies and fragments thereof against CCR7 can be useful diagnostic and/or therapeutic agents in treatment of a variety of conditions in which CCR7 is overexpressed, or in which ligands for CCR7 are over-expressed or released in pathological situations.

Detection of Natively Configured CCR7

In certain embodiments of this invention, anti-CCR7 antibodies can be useful for detection of expressed CCR7 in native configuration. Prior methods of determining expression CCR7 inadequately identify non-natively configured CCR7, and as such, may misrepresent the true amount of such CCR7 in a particular state. For example, RNA arrays and PCR assays (including quantitative PCR or "qPCR") measure only the mRNA for CCR7 and do not reflect expression of the mature protein. Because CCR7 and other GPCRs are multispanning membrane proteins, misfolding of nascent protein chains may be important aspects of loss of CCR7 function and may lead to pathological conditions.

Additionally, anti-CCR7 antibodies raised against non-natively configured CCR7 may not detect mis-folded or mis-inserted CCR7 into cell membranes. Thus, using the antibodies of this invention, better understanding of the CCR7 status of patients can be achieved. In certain aspects, use of antibodies of this invention along with more routine analyses (qPCR, RNA arrays, prior art antibodies) can shed light upon the functional state of a cell's CCR7 status.

Therapeutic Applications

In certain aspects of this invention, fully human CCR7 antibodies can be useful in treating conditions involving defects in CCR7, include cancers. In several types of cancer CCR7 can play important roles in dysregulation of cell growth and tumor metastasis. Thus, according to certain embodiments, use of fully human anti-CCR7 antibodies of this invention can bind to the CCR7 receptor. In some of these embodiments, binding of an antibody to a receptor can lead to loss of cells expressing CCR7. Whether this is by cell death or other mechanism is not crucial to the use of antibodies of this invention. In other embodiments, an anti-CCR7 antibody can act as an antagonist of the function of the CCR7 receptor, and these embodiments are useful to treat disorders in which CCR7 function is too high for normal functioning of the cell. In still further embodiments, anti-CCR7 antibodies of this invention can act as agonists and thereby increase the functioning of CCR7-dependent processes. Thus, antibodies and fragments thereof of this invention can find therapeutic use in a variety of pathological conditions, including cancer.

In certain aspects of this invention, an anti-CCR7 antibody of this invention can be selected based upon diagnostic findings. For example, in many types of cancer, CCR7 is over- expressed. It can be useful in some cases to determine whether a particular patient's cancer involves CCR7 over-expression. To determine whether CCR7 is over-expressed, a sample of the patient's tumor can be obtained through biopsy or resection of mass tumors, or by sampling blood in cases of leukemias, and CCR7 expression measured using measurement of mRNA expression or the natively configured CCR7 protein itself. Methods for measuring mRNA expression include solid phase arrays for mRNA, quantative PCR (qPCR) or other methods known in the art. Methods for determining expression of CCR7 protein include enzyme-linked immunosorbent assays (ELISA), Western blotting or other methods known in the art. These methods need not be further described herein. Rather, persons of ordinary skill in the art can easily refer to published articles, textbooks, or laboratory manuals for details of these methods. However, with the use of the fully human antibodies against natively configured CCR7, diagnosis can be improved. As noted, using a combination of RNA expression and production of natively configured CCR7 can lead to an understanding of whether the particular defect is more related to RNA expression or rather, to misfolding, improper post-expression processing of the CCR7 or whether the CCR7 is improperly inserted into the cell membrane.

In cases in which CCR7 expression is undesirably high, the therapeutic goal can include reducing function of the CCR7 pathways. Antagonist antibodies of this invention can be particularly useful for these situations. In other situations, using antibodies that specifically bind to CCR7 can be used to reduce the numbers of CCR7 expressing cells.

Pharmaceutical compositions containing fully human anti-CCR7 antibodies are also included within the scope of this invention. Thus, a suitable composition can include one or more anti-CCR7 antibodies, a physiologically compatible solution, and one or more pharmacological excipients.

Treatment of Cancer

In certain embodiments, antibodies and/or fragments thereof of this invention can be used to treat cancers, including chronic leukocytic leukemia, head and neck cancers, non-small cell lung cancers, gastric cancer, breast cancer, melanoma and colorectal cancer. Each of these disorders, the roles of CCR7 and roles of anti-CCR7 antibodies are described further herein.

Chronic Leukocytic Leukemia

Chronic Leukocytic Leukemia (CLL) is one of the diseases where CCR7 molecules are overexpressed. Thus, use of binding or antagonist anti-CCR7 antibodies can be useful. Head and Neck Cancer

Head and neck carcinomas are histologically and clinically heterogeneous. While squamous cell carcinomas (SCC) are characterized by lymphogenous spread, adenoid cystic carcinomas (ACC) disseminate preferentially hematogenously. Analysis at the mRNA and protein level of human chemokine receptors showed that SCC and ACC cells exhibited distinct and nonrandom expression profiles for these receptors. SCC predominantly expressed receptors for chemokines homeostatically expressed in lymph nodes, including CC chemokine receptor CCR7.

CCR7 mediates survival and invasiveness of metastatic squamous cell carcinoma of the head and neck (SCCHN) to regional lymph nodes. According to Wang et al., Autocrine and paracrine chemokine receptor 7 activation in head and neck cancer: implications for therapy. J. Natl. Cancer Inst., 100: 502-512. (2008), constitutive pro-survival signaling by the phosphoinositide-3 kinase/Akt pathway has been observed in SCCHN cells independent of epidermal growth factor receptor (EGFR) signaling. Expression and secretion of chemokines by primary tumors, metastatic nodes, and benign nodes of patients with SCCHN were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively (Wang et al. Id.). The role of paracrine activation of CCR7 on tumor growth was analyzed by comparing the growth of orthotopic tumors derived from B7E3 murine oral carcinoma cells in wild-type BALB/c mice, in paucity of lymphoid T cell (pit, deficient in CCL 19 and CCL21 expression) mice, and in pit mice in which the implanted B7E3 cells overexpressed CCR7 (Id.). In the absence of exogenous ligand treatment, blockade of CCR7 signaling reduced levels of phosphorylated (activated) Akt and decreased SCCHN cell viability by up to 59%, enhancing the effect of EGFR inhibition (Id.). CCR7 stimulation protected metastatic SCCHN cells from cisplatin-induced apoptosis in an Akt-dependent manner (Id.). Metastatic nodes expressed and secreted higher levels of CCLl 9 than benign nodes or primary tumors. Secretion of CCLl 9 and CCL21 by SCCHN cells and by paracrine sources combine to promote activation of CCR7 pro-survival signaling associated with tumor progression and disease relapse. CCR7 and its cognate chemokines may be useful biomarkers of SCCHN progression, and blockade of CCR7-mediated signaling may enhance the efficacy of platinum- and EGFR-based therapies. (Id.). In treating patients with head and neck cancer, after a diagnosis is made, the patient is treated with the anti-CCR7 antibody or fragment of this invention until one or more characteristic signs and/or clinical findings indicate that therapy has been at least partially successful.

It can also be appreciated that anti-CCR7 antibodies of this invention can be used for diagnosing or evaluating the CCR7 status of a cell or tissue.

Non-Small Cell Lung Cancer

Tumor cell migration into the lymph nodes is an important aspect of cancer and CCR7 has been shown to play an important role in tumor cell migration and lymph node metastasis. Takanami, I. Overexpression of CCR7 mRNA in nonsmall cell lung cancer: correlation with lymph node metastasis. Int. J. Cancer 2003 Jun 10;105(2):186-189 investigated CCR7 expression in 71 patients with NSCLC who underwent curative tumor resection and found that CCR7 mRNA was expressed in 45 cases (63.3%; Takanami, Id.).. The CCR7 mRNA expression was significantly associated with lymph node metastasis, stage, lymphatic invasion. Twenty-six (57.8%) of 45 cases with CCR7 mRNA expression in their cancer tissues were node-positive, whereas only 3 (11.5%) of 26 cases without CCR7 mRNA expression were node-positive. Furthermore, expression of CCR7 mRNA was shown to be an independent predictor of lymph node metastasis by multivariate analysis (p = 0.0117). Our study demonstrates that CCR7 might be related to the development of lymph node metastasis in NSCLC. The expression of CCR7 mRNA could open up a new window for the diagnostic staging and treatment of NSCLC {Id.).

Expression of CCR7 in pulmonary tumor tissues and metastasized lymph nodes in NSCLC has been measured in specimens from 17 cases of adenocarcinoma, 17 cases of Squamous cell Carcinoma, 12 cases of Adenosquamous Carcinoma, 4 cases of large cell carcinoma and 28 cases of metastasized lymph nodes of lung cancer (Zeng, T., Wen, J. The value and association of CCRl expression in NSCLC with lymph node metastasis. Chinese Journal of Lung Cancer, 11 : No 2 (2008)). The expression of CCR7 in pulmonary tumor tissue was remarkably higher than normal lung tissue {Id.). In treating patients with non-small cell lung cancer, after a diagnosis is made, the patient is treated with the anti-CCR7 antibody or fragment of this invention until the characteristic signs and/or clinical findings indicate that therapy has been at least partially successful.

Gastric Cancer

Chemokine receptor CCR7 is a key molecule for migration of lymphocytes and dendritic cells into lymph nodes (Ishigami et al., Prognostic value ofCCR7 expression in gastric cancer. Hepatogastroenterology 54:1025-1028 (2007)). Expression of CCR7 in tumor cells has been reported in malignancies, and CCR7 expression in tumor cells has been investigated in vitro and in vivo. A total of 224 gastric cancer patients who underwent curative surgery were enrolled and CCR7 expression in the primary tumor was detected. Patients showing more than 10% positivity for CCR7 were defined as having high CCR7 expression, as previously reported. CCR7 expression was detected in tumor cells and inflammatory cells in the tumor nest. CCR7- positive patients exhibited deeper tumor invasion, more frequent lymph node metastasis, higher rates of lymphatic invasion and more venous invasion than CCR7-negative patients. Most significant clinical factor for CCR7 was lymph node metastasis followed by lymphatic invasion. CCR7-positive gastric cancer patients had significantly poorer surgical outcomes than CCR7- negative patients. Our results suggest that CCR7 expression in gastric cancer is related to the onset of preferential conditions for lymphatic spread, such as lymph node metastasis. CCR7 expression of preoperative biopsy specimen can predict lymph node metastasis.

In treating patients with gastric cancer, after a diagnosis is made, the patient is treated with the anti-CCR7 antibody or fragment of this invention until the characteristic signs and/or clinical findings indicate that therapy has been at least partially successful.

Breast Cancer

Recent studies have indicated that expression of chemokine receptors CXCR4 and CCR7 could be an indicator of the metastatic potential of breast cancer (Cabioglu N. et al., Expression of growth factor and chemokine receptors: new insights in the biology of inflammatory breast cancer. Ann Oncol. 2007 Jun; 18(6): 1021-9). Expression of CXCR4 and CCR7 along with the biomarkers HER2-neu and epidermal growth factor receptor (EGFR) was investigated in inflammatory breast cancer (IBC) to evaluate their prognostic implications (Cabioglu N. et al. Id.). CXCR4, CCR7, and EGFR were evaluated by immunohistochemical staining (IHC) of paraffin-embedded tissue sections. HER2-neu amplification was assessed by FISH and/or IHC. All patients received chemotherapy, surgery, and radiation (Id.). Forty-four cases diagnosed with IBC from 1994 to 2002 were included in the study. In all, 18 (40.9%) patients had positive CXCR4, 10 (22.7%) had positive CCR7, 21 (47.7%) had positive HER2- neu, and EGFR was positive in 12 of 40 patients (30%). The 5-year overall survival (OS) was 24.8% for CXCR4-positive disease versus 42.3% for CXCR4-negative patients (P = 0.53) and 20.0% for CCR7-positive disease versus 41.9% for CCR7-negative patients (P = 0.24). EGFR- positive disease had significantly worse OS compared with EGFR-negative disease (P = 0.01). These data demonstrate the roles of expression of growth factor and chemokine in pathogenesis ofthis diease.

Animal Studies are Predictive of Human Efficacy

Among mouse models that can be used to demonstrate the efficacy of anti-CCR7 antibodies are, for example, a murine transplantation model of atherosclerosis regression as described in Feig JE, Quick JS, and Fisher EA, The role of a murine transplantation model of atherosclerosis regression in drug discovery. Curr Opin Investig Drugs. 2009 Mar;10(3):232-8. incorporated herein fully by reference. According to the authors, "a transplantation-based mouse model of atherosclerosis regression has been developed by allowing plaques to form in a model of human atherosclerosis, the apoE-deficient mouse, and then placing these plaques into recipient mice with a normolipidemic plasma environment. Under these conditions, the depletion of foam cells occurs. Interestingly, the disappearance of foam cells was primarily due to migration in a CCR7-dependent manner to regional and systemic lymph nodes after 3 days in the normolipidemic (regression) environment." Thus, this model can be useful in determining the effect on anti-CCR7 antibodies on the disease progression.

Several other well-established models can be used for development of anti-cancer drugs based upon anti-CCR7 antibodies of this invention. For example, such as experimental mouse model described in Kochetkova M, Kumar S, McCoIl SR, Chemokine receptors CXCR4 and CCR7 promote metastasis by preventing anoikis in cancer cells. Cell Death Differ. 2009 May;16(5):664-73. Epub 2009 Jan 9 incorporated herein fully by reference. This model was used to uncover "a novel property of the chemokine receptors CXCR4 and CCR7 in inhibiting detachment-induced cell death—anoikis, which is believed to be one of the major blocks in the metastatic spread of various neoplasms." The results obtained provide evidence for a previously unknown axis in malignant tumors, which connects chemokine receptors with deregulated apoptosis and relates to metastatic breast and potentially other tumors.

Another model that links CCR7 and cancer is described in Yu S, Duan J, Zhou Z, Pang Q, Wuyang J, Liu T, He X, Xinfa L, Chen Y, A critical role of CCR7 in invasiveness and metastasis of SW620 colon cancer cell in vitro and in vivo. Cancer Biol Ther. 2008 Jul;7(7): 1037-43. Epub 2008 Apr 7 that is incorporated herein fully by reference. The authors employed RNA interference to detect the in vitro effects of anti-CCR7 siRNAs on proliferation and invasiveness of SW620 cells and evaluated the ability of these siRNAs to inhibit the lymphogenesis and the lymph node metastasis in xenografted SW620 tumors in mice. In this animal model, blocking CCR7 expression at the mRNA level impaired invasion of colon cancer cells and inhibited lymph node metastasis of colon cancer and lymphogenesis.

Yet another cancer-related model that can be applied for the exploration of CCR7 antibodies of this invention is provided in Koizumi K, Kozawa Y, Ohashi Y, Nakamura ES, Aozuka Y, Sakurai H, Ichiki K, Doki Y, Misaki T, Saiki I., CCL21 promotes the migration and adhesion of highly lymph node metastatic human non-small cell lung cancer Lu-99 in vitro. Oncol Rep. 2007 Jun; 17(6): 151 1-6 (incorporated herein in full by reference). According to this article, "[t]o develop new therapy strategies for lung cancer, we established an animal model, which reflects the clinical features of mediastinal lymph node metastasis of lung cancer. This study was designed to determine whether CCL21 induced biological functions associated with the metastasis of highly lymph node metastatic human non-small cell lung cancer (NSCLC) selected by our model. Orthotopic intrapulmonary implantation of human NSCLC (Lu-99 and A549) was performed to analyze the metastatic characteristics of these cells. The expression of CCR7, which is a receptor of CCL21, was detected using CCL 19 [also called EBIl-ligand chemokine (ELQ]-Fc chimera by flow cytometric analysis. The effects of CCL21 on the migration, adhesion and growth of human NSCLC were investigated. After orthotopic implantation of human NSCLC cell lines, Lu-99, but not A549, metastasized to mediastinal lymph nodes, forming large size nodules, and expressed CCR7 on the surface. Accordingly, its ligand CCL21 induced chemotactic migration and alpha4betal -mediated adhesion to VCAM-I of Lu-99. The expression of CCR7 and vigorous responses to its ligand CCL21 potentially account for lymph node metastasis of a human NSCLC line Lu-99."

Another cancer-related model that can be applied for the exploration of fully human CCR7 antibodies of this invention is provided in Wang J, Xi L, Hunt JL, Gooding W, Whiteside TL, Chen Z, Godfrey TE, Ferris RL., Expression pattern of chemokine receptor 6 (CCR6) and CCRl in squamous cell carcinoma of the head and neck identifies a novel metastatic phenotype. Cancer Res. 2004 Mar l ;64(5):1861-6., incorporated herein fully by reference. According to this paper, "squamous cell carcinoma of the head and neck (SCCHN) metastasizes predictably to cervical lymph nodes, with low rates of distant metastases. Tumor cells can express various receptors that facilitate such metastatic spread to lymph nodes and other non-lymphoid organs. Chemokine receptors (CCR), normally expressed on lymphocytes, control immune and inflammatory cell migration, providing a link between innate and adaptive immunity. Chemokine receptor expression was evaluated in SCCHN, using paired primary and metastatic tumors cell lines, and paired primary and metastatic biopsies from the same patients. Quantitative reverse transcription-PCR showed a consistent pattern of CCR6 down-regulation and up-regulation of CCR7 in metastatic cells and tissues. Chemotaxis assays, ligand-induced receptor down-regulation, and specific antibody blocking experiments supported the quantitative reverse transcription-PCR results, indicating that these surface receptors were functional on metastatic tumor cells. Cells derived from a highly metastatic mouse model of SCCHN were used to confirm CCR7 up-regulation in tumor cells with higher metastatic potential. CCR6 down-regulation is consistent with its decreased expression in cells emigrating from peripheral mucosal sites, whereas CCR7, important for homing of immune cells to secondary lymphoid organs, was significantly up-regulated. Thus, CCR6, CCR7, and their ligands, normally important in controlling immune cell trafficking in response to inflammatory stimuli, may have an important role in determining the metastasis of SCCHN cells in vivo."

Yet another cancer-related use of the anti-CCR7 antibodies of this invention can be explored as described in Arenberg DA, Zlotnick A, Strom SR, Burdick MD, Strieter RM., The murine CC chemokine, 6C-kine, inhibits tumor growth and angiogenesis in a human lung cancer SCID mouse model. Cancer Immunol Immunother. 2001 Jan;49(l l):587-92, incorporated herein fully by reference. In this study tumor growth in severe combined immunodeficiency (SCID) mice was linked to interaction of CCR7 ligand with murine 6C-kine binding to one of the CXC chemokine receptors CXCR3, in addition to its other known receptor CCR7.

A further cancer model useful for studying anti-CCR7 antibodies can be applied as described in Saur D, Seidler B, Schneider G, Algiil H, Beck R, Senekowitsch-Schmidtke R, Schwaiger M, Schmid RM., CXCR4 expression increases liver and lung metastasis in a mouse model of pancreatic cancer. Gastroenterology. 2005 Oct; 129(4): 1237-50, incorporated herein fully by reference. That study utilized noninvasive imaging of targeted metastasis in a mouse model of pancreatic cancer; functional expression of the chemokine receptors CXCR4 and CCR7 was achieved by stable transfection of murine TD-2 pancreatic cancer cells and analyzed by flow cytometry, calcium flux, migration, and proliferation assays. The metastatic potential of the different stable TD-2 cell clones was assessed by tail vein metastatic assays in nude mice using in vivo bioluminescent imaging.

Another cancer-related model that can be used to obtain support for the use of anti-CCR7 antibodies of this invention of the treatment of cancer is described in Murakami T, Cardones AR, Hwang ST., Chemokine receptors and melanoma metastasis. J Dermatol Sci. 2004 Nov;36(2):71-8 (incorporated herein fully by reference). According to this article, "[c]ancer metastasis is the end result of a complex series of biologic events that leads to the formation of clinically significant secondary tumors at distant sites. The sites of distant metastasis are not random since certain tumors show a tendency to develop metastases in specific organs. Human melanoma, for example, demonstrates frequent metastasis to brain, lungs, lymph nodes, and skin. Herein, we review the evidence that suggests that a limited number of chemokine receptors may play critical roles in determining organ-selective metastasis in melanoma by regulating diverse processes such as chemoattraction, adhesion, and survival. In particular, we describe roles for CC chemokine receptor 7 (CCR7) in lymph node metastasis ... using a mouse model of melanoma. Preliminary evidence in this preclinical model suggests that inhibiting the function of these receptors may decrease the ability of cancer cells to disseminate to other sites and/or block their ability to survive and form tumors." Other models can be used by those skillful in the art to explore the use of anti-human CCR7 antibodies of this invention in inflammatory treatment, similarly to that described for example in Xia M, Hou C, DeMong DE, Pollack SR, Pan M, Brackley JA, Jain N, Gerchak C, Singer M, Malaviya R, Matheis M, Olini G, Cavender D, Wachter M., Synthesis, structure- activity relationship and in vivo antiinflammatory efficacy of substituted dipiperidines as CCR2 antagonists. J Med Chem. 2007 Nov 15;50(23):5561-3. Epub 2007 Oct 1 1 (incorporated by reference in full) for a series of substituted dipiperidine compounds synthesized and identified as selective CCR2 antagonists, some had outstanding selectivity over CCRl, CCR3, CCR4, CCR5, CCR6, CCR7, and CCR8 and showed excellent efficacy in adjuvant-induced arthritis model, collagen-induced arthritis model, and allergic asthma model.

Yet another model is as in Wengner AM, Hδpken UE, Petrow PK, Hartmann S, Schurigt U, Brauer R, Lipp M., CXCR5- and CCRl -dependent lymphoid neogenesis in a murine model of chronic antigen-induced arthritis. Arthritis Rheum. 2007 Oct;56(10):3271-83, by (incorporated fully by reference). This study has established a murine model of chronic arthritis in which the development of tertiary lymphoid tissue, a hallmark of human rheumatoid arthritis, is locally induced. The role of the homeostatic chemokine receptor CCR7 in this process can be explored using the model of chronic antigen-induced arthritis in mice with a strong bias toward inflammation, as described therein.

Yet another model for exploring the use of anti-human CCR7 antibodies of this invention is described in Pierce EM, Carpenter K, Jakubzick C, Kunkel SL, Flaherty KR, Martinez FJ, Hogaboam CM., Therapeutic targeting of CC ligand 21 or CC chemokine receptor 7 abrogates pulmonary fibrosis induced by the adoptive transfer of human pulmonary fibroblasts to immunodeficient mice. Am J Pathol. 2007 Apr; 170(4): 1152-64 (incorporated herein fully by reference). According to this article, "[idiopathic interstitial pneumonias (IIPs) are a collection of pulmonary fibrotic diseases of unknown etiopathogenesis. CC chemokine receptor 7 (CCR7) is expressed in IIP biopsies and primary fibroblast lines, but its role in pulmonary fibrosis was not previously examined. To study the in vivo role of CCR7 in a novel model of pulmonary fibrosis, 1.0 x 10(6) primary fibroblasts grown from idiopathic pulmonary fibrosis/usual interstitial pneumonia, nonspecific interstitial pneumonia, or histologically normal biopsies were injected intravenously into CB- 17 severe combined immunodeficiency (SCID)/beige (bg) mice. At days 35 and 63 after idiopathic pulmonary fibrosis/usual interstitial pneumonia fibroblast injection, patchy interstitial fibrosis and increased hydroxyproline were present in the lungs of immunodefϊcient mice. Adoptively transferred nonspecific interstitial pneumonia fibroblasts caused a more diffuse interstitial fibrosis and increased hydroxyproline levels at both times, but injected normal human fibroblasts did not induce interstitial remodeling changes in CB- 17SCID/bg mice. Systemic therapeutic immunoneutralization of either human CCR7 or CC ligand 21, its ligand, significantly attenuated the pulmonary fibrosis in groups of C.B-17SCID/bg mice that received either type of IIP fibroblasts. Thus, the present study demonstrates that pulmonary fibrosis is initiated by the intravenous introduction of primary human fibroblast lines into immunodeficient mice, and this fibrotic response is dependent on the interaction between CC ligand 21 and CCR7."

And yet another use of anti-CCR7 antibodies of this invention can be explored as described in Yamashita N, Tashimo H, Matsuo Y, Ishida H, Yoshiura K, Sato K, Yamashita N, Kakiuchi T, Ohta K., Role of CCL21 and CCL19 in allergic inflammation in the ovalbumin- specific murine asthmatic model. J Allergy Clin Immunol. 2006 May; 1 17(5): 1040-6 (incorporated herein fully by reference. According to this article, dendritic cells are the most powerful of the antigen-presenting cells and are known to play important roles in sensitization and inflammation in allergen-specific asthma. The role of CCL21 in airway inflammation in asthma was explored by using BALB/c-plt/plt (pit) mice, which possess genetic defects in expression of both CCL21 and CCLl 9. Chemokine ligand (CCL)21, a key chemokine in the entry of naive T cells and antigen-stimulated dendritic cells into the T-cell zones of secondary lymphoid organs, which is a critical process in antigen-specific T-cell activation. Pit and control BALB/c mice were immunized with ovalbumin and alum 4 times and thereafter were subjected to a 2-week regimen of ovalbumin inhalation. In pit mice, ovalbumin-specific IgE response was delayed compared with control BALB/c mice, but they had the same level of response after final immunization. Although airway inflammation and response to acetylcholine were significantly reduced compared with BALB/c mice, significant eosinophilic inflammation and hyperresponsiveness were also observed in pit mice after 2 weeks of inhalation. Four weeks after cessation of inhalation, airway inflammation and hyperresponsiveness in pit mice were greater than in BALB/c mice. At the time of resolution of airway inflammation, IL-10 production was enhanced in BALB/c mice but not in pit mice. The chemokines CCL21 and CCLl 9 were critical for resolution of airway inflammation. The findings about the chemokines for induction and resolution of inflammation are key to establishing a new strategy for asthma immunotherapy.

Another use of the antibodies of this invention can be in stem cell treatment. Sordi V, Malosio ML, Marchesi F, Mercalli A, Melzi R, Giordano T, Belmonte N, Ferrari G, Leone BE, Bertuzzi F, Zerbini G, Allavena P,Bonifacio E, Piemonti L., Bone marrow mesenchymal stem cells express a restricted set of functionally active chemokine receptors capable of promoting migration to pancreatic islets. Blood. 2005 JuI 15;106(2):419-27. Epub 2005 Mar 22 incorporated herein fully by reference. According to this article, "[b]one marrow-derived mesenchymal stem cells (BM-MSCs) are stromal cells with the ability to proliferate and differentiate into many tissues. Although they represent powerful tools for several therapeutic settings, mechanisms regulating their migration to peripheral tissues are still unknown. Here, we report chemokine receptor expression on human BM-MSCs and their role in mediating migration to tissues. A minority of BM-MSCs (2% to 25%) expressed a restricted set of chemokine receptors (CXC receptor 4 [CXCR4], CX3C receptor 1 [CX3CR1], CXCR6, CC chemokine receptor 1 [CCRl], CCR7) and, accordingly, showed appreciable chemo tactic migration in response to the chemokines CXC ligand 12 (CXCLl 2), CX3CL1, CXCLl 6, CC chemokine ligand 3 (CCL3), and CCLl 9. Using human pancreatic islets as an in vitro model of peripheral tissue, we showed that islet supernatants released factors able to attract BM-MSCs in vitro, and this attraction was principally mediated by CX3CL1 and CXCL12. Moreover, cells with features of BM-MSCs were detected within the pancreatic islets of mice injected with green fluorescent protein (GFP)-positive BM. A population of bona fide MSCs that also expressed CXCR4, CXCR6, CCRl, and CCR7 could be isolated from normal adult human pancreas. This study defines the chemokine receptor repertoire of human BM-MSCs that determines their migratory activity. Modulation of homing capacity may be instrumental for harnessing the therapeutic potential of BM-MSCs."

Yet another use of the anti-CCR7 antibodies of this invention can be developed by those skillful in the art using models and approaches described in Martin AP, Coronel EC, Sano G, Chen SC, Vassileva G, Canasto-Chibuque C, Sedgwick JD., Frenette PS, Lipp M, Furtado GC, Lira SA., A novel model for lymphocytic infiltration of the thyroid gland generated by transgenic expression of the CC chemokine CCL21, J Immunol. 2004 Oct 15;173(8):4791-8, incorporated herein fully by reference. According to this article, "[lymphocytic infiltrates and lymphoid follicles with germinal centers are often detected in autoimmune thyroid disease (AITD), but the mechanisms underlying lymphocyte entry and organization in the thyroid remain unknown. We tested the hypothesis that CCL21, a chemokine that regulates homeostatic lymphocyte trafficking, and whose expression has been detected in AITD, is involved in the migration of lymphocytes to the thyroid. We show that transgenic mice expressing CCL21 from the thyroglobulin promoter (TGCCL21 mice) have significant lymphocytic infiltrates, which are topologically segregated into B and T cell areas. Although high endothelial venules expressing peripheral lymph node addressin were frequently observed in the thyroid tissue, lymphocyte recruitment was independent of L-selectin or lymphotoxin-alpha but required CCR7 expression. Taken together, these results indicate that CCL21 is sufficient to drive lymphocyte recruitment to the thyroid, suggest that CCL21 is involved in AITD pathogenesis, and establish TGCCL21 transgenic mice as a novel model to study the formation and function of lymphoid follicles in the thyroid."

While those skilful in the art can suggest to apply the antibodies of this invention to many other treatments, we provide an addition model among these many, that can be used as described in Hόpken UE, Droese J, Li JP, Joergensen J, Breitfeld D, Zerwes HG, Lipp M., The chemokine receptor CCRl controls lymph node-dependent cytotoxic T cell priming in alloimmune responses. Eur J Immunol. 2004 Feb;34(2):461-70, incorporated herein fully by reference. According to this article, "[t]he chemokine receptor CCR7 and its ligands regulate migration and co-localization of T cells and mature dendritic cells to and within secondary lymphoid organs. The requirement of CCR7 in efficient priming of allospecific cytotoxic CD8(+) T cells is poorly characterized. Here, we demonstrate a role for CCR7 in the initiation of an alloimmune response and in the development of transplant rejection. Remarkably, in a model of acute allogeneic tumor rejection, CCR7(-/-) mice completely failed to reject subcutaneously injected MHC class I mismatched tumor cells and cytotoxic activity of allospecific T cells was severely compromised. When solid tumors derived from wild-type mice were transplanted, recipient CCR7(-/-) mice were capable of rejecting the allografts. In contrast, tumor allografts transplanted from CCR7(-/-) donors onto CCR7(-/-) recipients showed allograft survival up to 28 days, suggesting a critical function of CCR7 on donor-type passenger leukocytes in the initiation of cytotoxic CD8(+) T cell responses. In a heterotopic heart transplantation model CCR7 deficiency resulted in significantly prolonged but not indefinite allograft survival. Additional prolongation of graft survival was observed when hearts from CCR7(-/-) mice were used as donor organs. Our results define a key role for CCR7 in allogeneic T cell priming within the context of draining lymph nodes."

As can be appreciated from the above descriptions, there are animal models that can be used to develop treatments for disorders in humans characterized by CCR7-ligand interactions, that are reasonably predictive of their effects in treating human disease.

EXAMPLES

The following examples are presented to illustrate aspects and embodiments of this invention. As such, they are not intended to limit the scope of the invention. Rather, persons of skill in the art can use the descriptions and teachings herein to create, modify or produce other human antibodies and uses thereof without undue experimentation. All such embodiments are considered part of this invention.

Example 1: Construction and Expression of Codon-Opitimized CCR7 (synCCR7)

For certain embodiments, methods for construction and expression of codon-optimized CCR7 are described, in general, in Mirzabekov et al., Enhanced Expression, Native Purification, and Characterization of CCR5, a Principal HIV-I Coreceptor. J. Biol. Chem. 274(40):29745- 28750 (1999), expressly incorporated herein fully by reference.

In certain aspects, analysis of codon usage for 45 GPCRs representing different protein subfamilies was performed with Genbank™ data and software developed by the University of Wisconsin Genome Sequence Group. The sequence encoding human CCR7 was optimized for mammalian cell codon usage, utilizing the following codons: alanine (GCC), arginine (CGC), asparagine (AAC), aspartic acid (GAC), cysteine (TGC), glutamic acid (GAG), glutamine (CAG), glycine (GGC), histidine (CAC), isoleucine (ATC), leucine (CTG), lysine (AAG), methionine (ATG), phenylalanine (TTC), proline (CCC), serine (TCC), threonine (ACC), tryptophan (TGG), tyrosine (TAC) and valiine (GTG). The 5' and 3' sequences flanking the CCR7 coding sequence were modified. Following restriction sites for EcoRV, EcoRI and Hindlll, the Kozak consensus (GCCGCCACCATGG; SΕQ ID NO: 1) was placed immediately 5¹ to the CCR7 reading frame. A sequence encoding a single glycine residue followed by the bovine rhodopsin C9 peptide tag (TΕTSQVAPA; SΕQ ID NO:2) was introduced immediately 5' to the natural stop codon of CCR7. At the 3' end of the epitope-tagged CCR7 gene. Xhol, Sail, and Notl restriction sites were introduced. Analogous constructs were made for the wild-type human CCR7 gene and the bovine rhodopsin gene, except that the codons were not altered and, in the latter case, the C-terminal C9 sequence was naturally present.

Oligonucleotides, each approximately 70 nucleotides in length, corresponding to the complete sense and antisense strands of the synCCR7 gene and flanking sequences, were constructed so that approximately 50% of their sequences were complementary to those of each of the two complementary oligonucleotides from the opposite strand. Oligonucleotides were deprotected in pure ammonium hydroxide at 65° C for 4 h, after which the ammonium hydroxide was evaporated, and the oligonucleotides were dissolved in water at a final concentration of 2 nM. For gene synthesis, the oligonucleotides were separated into groups (about 6 to 8 oligonucleotides per group) and about 25 cycles of polymerase chain reaction (PCR) were performed using Pfu polymerase (Stratagene, La Jolla, CA) and a 3-fold molar excess of the 5' and 3' terminal oligonucleotides in each group. This step generated small segments of the sysCCR7 gene with complementary and overlapping ends. Equal amounts of each PCR product were combined with a 3-fold molar excess of the 5' and 3' terminal oligonucleotides of the complete synCCR7 sequence. A second round of about 25 cycles of PCR yielded the complete sysCCR7 sequence. The product was sequenced to ensure that the sequence was correct.

The synCCR7, wild-type CCR7 and bovine rhodopsin sequences were cloned into the following vectors: PMT4 (a gift from Dr. Reeves, Massachusetts Institute of Technology), PACH (a gift from Dr. Velan, Israel Institute for Biological Research), pcDNA 3.1(+) and pcDNA4/HisMax (Invitrogen), and PND (a gift from Dr. Rhodes, University of California, Davis). After cloning the synCCR7 gene into the pcDNA4/HisMax vector, the sequence encoding the N-terminal HisMax region was removed by QuikChange™ mutagenesis (Stratagene). Different cell lines were transfected with the synCCR7 gene and wild-type CCR7 genes using the GenePorter transfection reagent (San Diego, CA). Following transfection, cells expressing CCR7 were selected with 0.8mg/ml of neomycin (G418). Cells expressing the highest surface levels of CCR7 were selected by fluorescence activated cell sorting (FACS) after staining cells with R-phycoerythrin-conjugated anti-CCR7 antibody (Pharmagen, San Diego, CA). The highest synCCR7 expressing cells were selected by FACS.

Example 2: Radiolabeling and Immunoprecipitation of CCR7

Approximately 4 x 10⁸ CCR7-expressing Cf2Th or HEK-293T cells grown to full confluence in 100-mm dishes were washed twice in PBS and starved for 1 h at 37⁰C in Dulbecco's modified Eagle's medium without cysteine and methionine (Sigma) or in sulfate-free media (ICN, Costa Mesa, CA). The starvation medium was removed and 200 μCi each of [³⁵S]methionine and [³⁵S]cysteine or 500 μCi of [³⁵S]sulfate (NEN Life Science Products) in 4 ml of medium was added to the cells for various times for pulse-chase experiments or overnight (12 h) in other cases. Cells were washed twice with PBS and lysed in 1 ml of solubilization medium composed of 100 mM (NH₄)₂ SO₄, 20 mM Tris-HCl (pH 7.5), 10% glycerol, l%(w/v) detergent (see below), and Protease Inhibitor Mixture (one tablet of Complete™ (Roche Molecular Biochemicals) per 25 ml.

The lysate was incubated at 4⁰C for 30 minutes on a rocking platform, and cell debris was removed by centrifugation at 14,000 x g for 30 min. CCR7 was precipitated with 20 μl of 1D4-Sepharose beads overnight, after which the beads were washed six times in the solubilization medium and pelleted. An equal volume of 2 x SDS-sample buffer was added to the beads, followed by re-suspension and incubation for 1 h at 55⁰C. Samples were run on 11% SDS-polyacrylamide minigels and visualized.

Example 3: Solubilization Buffers

Detergents were used as components of solubilization buffers. The detergents, with abbreviations and critical micelle concentrations in parentheses, were rc-octyl-β-D- glucopyranoside (23.4 mM), H-decyl-β-D-maltoside (1.8mM), w-dodecyl-β-D-maltoside (DDM; 0.17 mM), cyclohexyl-butyl-β-D-maltoside (Cymal™-4; 7.6 mM), cyclohexyl-pentyl-β-D- maltoside (Cymal™-6; 0.56 mM), cyclohexyl-heptyl-β-D-maltoside (Cymal™-7; 0.19 mM), cyclo-hexylpropanoyl-N-hydroxyethylglucamide (108 mM), cyclohexylbutanoyl-N- hydroxyethylglucamide (35 mM), cyclohexylpentanoyl-N-hydroxyethylglucamide (11.5 mM), N- octylphosphocholine (Fos-Choline™ 8; 114 mM), N-decylphosphocholine (Fox-CholineTM 10; 1 mM), jV-dodecylphosphocholine (Fos-Choline™ 12; 1.5 mM), iV-tetradecylphosphocholine (Fos-Choline™ 14; 0.12 mM), Triton X-IOO (0.02 mM), CHAPS (8 mM), Nonidet P-40 (0.02 mM), and diheptanoyl-phosphocholine (DHPC; 1.4 mM). All detergents were purchased from Anairace (Maumee, OH) except DHPC, which was purchased from Avanti Polar Lipids (Alabaster, AL).

Example 4: Purification of CCR7

Stable Cf2Th/PACH/synCCR7 cells grown to full confluence in a 150 mm dish were incubated with medium containing 4 mM sodium butyrate for 40 h, washed in PBS, detached by treatment with 5 mM EDT A/PBS, pelleted, and again washed in PBS. Cells were solubilized for 30 min with 3 ml of the solubilization medium containing Cymal™-5 and centrifuged for 30 min at 14,000 x g. The cell lysate as incubated with 50 μl of 1D4-Sepharose beads on a rocking platform at 4⁰C for 10 -12 h. The Sepharose™ beads were washed about five times with the washing buffer (100 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 7.5), 10% glycerol and 1% Cymal™-5) and once with washing buffer plus 500 mM MgCl₂. CCR7 was eluted from the beads by three successive washes with 50 μl of medium containing 200 mM C9 peptide (TETSQVAPA: SEQ ID NO: 2), 500 mM MgCl₂, 100 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 7.5), 10% glycerol, and 0.5% Cymal™-5. The amount of CCR7 was estimated by Coomassie Blue staining of an SDS-polyacrylamide gel (SDS-PAGE) run with standard quantities of bovine serum albumin.

In other embodiments, CCR7 can be obtained using paramagnetic particles, chemically derivatized with a capture agent, using the protocol provided by the Dynal Biotech Inc. A capture reagent can be an antibody capable of selective binding a tag or streptavidin that can bind a know peptide tag; either of the tags can be attached at the C-terminus of CCR7.

CCR7 protein can be over-expressed in a mammalian cell by transfecting, using for example, a GenePORTER™ transmembrane reagent and protocol (Gelantis), a line of mammalian cells (which can be purchased from ATCC) with a vector (for example, pcDNA3.1, from Invitrogen) carrying the gene of the protein having an appropriate peptide tag at the C- terminus and genes that provide an antibiotic resistance to the cells. In some embodiments, CCR7 monomers can each have a C-terminal tag, and in other embodiments, some CCR7 monomers can have C-terminal tags and other CCR7 monomers can be untagged. For manufacture of hetero-multimeric proteins of this invention, cells can be transfected with vectors that encode tagged monomers and other vectors that encode un-tagged monomers. Alternatively, a single vector having two or more expression cassettes, one cassette having a sequence encoding a tagged monomer and another cassette encoding an untagged monomer) can be used. In such systems, a mixture of tagged and untagged monomers can be produced, that when associated with each other in a cell, can form a hetero-multimeric protein complex. Antibiotic resistance (for example, resistance to gentamycin (Geneticin™; G418), the feature acquired concomitantly with the capacity to over-express CCR7, can be used for selecting over- expressing cells that survive in the presence of added antibiotic.

Cells that over-express CCR7 can be harvested, and the membranes of the cells can be solubilized in a mixture of detergents. Solublilized CCR7 (and other solubilized proteins) can be clarified by centrifugation and the CCR7-containing protein-detergent complexes can be mixed with beads carrying a capture reagent capable of binding to the tag on the CCR7 protein.

Washing the beads can remove contaminants from the CCR7-detergent complexes. A magnet can be used to hold beads within a vessel (e.g., tube) and washing solutions can be added to carry away non-bound materials, including contaminants. Beads retaining CCR7 in the desirable orientation (i.e., the extracellular portion is exposed on the surface of the bead) can then be dialyzed to produce complexes having the desired detergents.

Example 5: Immunization

In other aspects, this invention includes immunization of mice having fully human immune systems. Such mice are known in the art and need not be described further herein. Mice are immunized with CCR7 protein and splenocytes isolated. The genetic components of splenocytes can be placed in phage display libraries constructed from splenocytes, and analyzed using phage display technology. Based on these methods, selection of clones that express antibodies against CCR7 can be obtained (see below). By immunizing such mice with isolated, purified synCCR7, antibodies can be produced against the CCR7 protein in its native configuration. In certain of these embodiments, antibodies of this invention can recognize the ectodomain of the CCR7, and thus, can bind to native CCR7 expressed in cells, including human cells. Thus, such anti-CCR7 antibodies can be used therapeutically or diagnostically, as explained further herein.

Example 6: Methods for Selection of Fully Human Antibodies

Phage-display libraries are among the most used technologies for generation and optimization of fully human antibodies (see Hoogenboom, H. R. Selecting and screening recombinant antibody libraries. Nature Biotechnol. 23, 1105—1116 (2005); Bradbury, A. R. & Marks, J. D. Antibodies from phage antibody libraries. J. Immunol. Methods 290, 29-49 (2004); and Fredericks, Z. L. et al. Identification of potent human anti-IL-lR I antagonist antibodies. Protein Eng. Des. SeI. 17, 95-106 (2004)). Other display technologies useful for the generation and affinity maturation (optimization) include yeast-, mRNA- and ribosome-display libraries — are gaining in popularity for selection and optimization of antibodies (see Hoogenboom, Id., Bradbury, Id., and Fredericks, Id.).

Display libraries display single-chain variable-domain antibody fragments (scFvs) or Fabs, and contain the encoding DNA or RNA. They have high genetic diversity or repertoire size (commonly 10⁹-10¹³). These technologies allow the selective recovery of clones that bind a target antigen from a library, and they provide the means to amplify the selected clones for further rounds of selection or analysis. The genetic diversity in these libraries is commonly created by cloning the repertoire of the immunoglobulin heavy-chain (Hl) and and light-chain (Vl) variable gene segments from naive or immunized individuals. Alternatively, this diversity can be achieved by using synthetic DNA to randomize the complementarity-determining regions ("CDRs", the antigen-binding loops) or by a combination of these two approaches. The binding step can be undertaken with the target in solution, immobilized on a surface or on cells. After extensive washing, specifically bound clones are recovered and amplified for the next round of selection. Example 7: Human Antibodies Obtained By Immunization of Transgenic Mice

The generation of human antibodies by immunization of mice that are transgenic for human immunoglobulin genes and have disrupted mouse immunoglobulin heavy-chain and Igκ light-chain loci was first described in 1994. Subsequent progress included the expression of more V gene segments by the transgenic mice, thereby expanding the potential repertoire of recovered antibodies. Mouse strains that encode human antibodies with different heavy-chain isotypes have also been created to tailor effector functions. One problem in the generation of human antibodies for multispanning membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels and transporters is that these proteins have a high rate of homology with the mouse protein, thus the animal immune system tolerance has to be broken. In addition, preparation of the native immunogen (better if purified) in the amounts required for the immunization may be a problem in the case of multispanning membrane proteins, although this problem may be overcome with the use of synthetic peptides and fusion proteins mimicking the fragments of the multispanning membrane proteins. However, the antibodies generated using this last method rarely appear with desirable neutralizing (antagonistic) properties.

Example 8: Purification of Anti-Human CCR7 Antibodies as scFv's

Antibodies (scFv's that have his-tag) were purified using his-tag affinity purification protocol as follows. Each of the E. coli clones carrying phagemid with an anti-hCCR7 scFv gene was grown in 2xTY medium supplemented with 100 microg/ml ampicillin and 2% glucose at 37⁰C, 250 rpm to saturation, and each of the cultures so produced was used to inoculate 6 vessels each containing 50 ml 2xTY media supplemented with 100 microg/ml ampicillin and 0.1% glucose. The total volume for each scFv culture thus was 300 ml. Upon reaching logarithmic phase of growth (OD ~ 0.6) at 37⁰C, 250 rpm, IPTG was added to bacterial cultures to a final concentration of 0.05 mM. Cultures were incubated overnight at 3O⁰C, 250 rpm.

In the morning, to recover csFv's accumulated in the periplasm, the cultures were centrifuged in 50 ml tubes at 2,500 rpm for 20 min using Beckman table-top centrifuge and the supernatant discarded. Each bacterial pellet (~500 microliter) was re-suspended in 1,200 miroliter ice-cold TES buffer containing 20% sucrose, 1 mM EDTA, 50 mM Tris-HCl, pH8.0, incubated on ice for 30 min, and then 800 μl of ice-cold 1OmM Tris-HCl, pH7.5 was added the mixture was then incubated on ice for another 30 min. All further purification procedures were performed at 4⁰C.

After centrifugation in 50 ml tubes at 2,500 rpm for 20 min in Beckman table-top centrifuge, the supernatants were collected into 2 ml Eppendorf tubes, clarified by centrifugation for 20 min at 14,000 rpm using Eppendorf table top refrigerated centrifuge, and the clarified supernatant was transferred into fresh 2 ml Eppendorf tubes (each tube contained thus ~l,900 microliter clarified periplasm material in approximately half-diluted TES buffer).

After pre-washing Ni-agarose resin (High-Density IDA-Agarose 6 BCL Nickel Charged Resin (ABT)) twice in the Bind/Wash Buffer (300 mM NaCl, 20 mM Imidazol, 50 mM Tris- HCl, pH7.5, 0.05% Tween-20), a 100 microliter aliquot of the Ni-resin was added to each tube. Then each tube was incubated on a rotator for 3 h, centrifuged for 20 min at 2,000 rpm using an Eppendorf table top refrigerated centrifuge, and supernatant removed. The resin pellets of each scFv was combined in a fresh 2 ml Eppendorf tube (thus, scFv samples were obtained, each in the form of ~600 micloliter Ni-resin carrying his-tag immobilized scFv).

Each sample was re-suspended in 1,400 microliter Bind/Wash buffer and incubated on a rotator for 40 min, centrifuged for 20 min at 2,000 rpm using an Eppendorf table top refrigerated centrifuge, and the supernatant was removed. To each of the resulting pellets (Ni-resin beads with scFv) a 600-microliter aliquot of the Elute Buffer (20 mM EDTA, 100 mM NaCl, 20 mM Tris-HCl, pH7.5, 0.05% Tween-20) was added, and after incubation with rotation for 40 min and centrifuged for 20 min at 14,000 rpm using an Eppendorf table top refrigerated centrifuge, the supernatants (500 microliters each) were collected and supplemented with MgSO₄ added to final concentration 21 mM.

Example 9: Binding of Anti-CCR7 Antibodies to Cell Lines

To determine whether fully human antibodies of this invention bind to CCR7 or other GPCRs, we carried out studies using a series of cell lines in culture. The cell lines used were:

1. CHO-Kl (Chinese Hamster Ovary cells, ATCC Cat # CCL-61);

2. BHK-21 (Syrian Hamster Fibroblasts, ATCC Cat # CCL-10);

3. CF2Th (Canine Thymocytes, ATCC Cat # CRL- 1430) ;

4. R1610 (Chinese Hamster Lung Fibroblasts, ATCC Cat # CRL- 1657); and 5. HEK-293T (Human Embryonic Kidney cells, Cat # CRC- 1573).

The same cell lines were also adapted to stable express six different G-Protein Coupled Receptors (GPCRs). Next GPCRs have been expressed and used in the work: human CCR7, human CCR5, Human CXCR2 (hCXCR2), human CXCR3, human FPR, and mouse CCR7. The mammalian cells adapted to stable expression of GPCRs included:

1. CHO-Kl -hCCR7 (Chinese Hamster Ovary cells CHO-Kl expressing human chemokine receptor CCR7);

2. CHO-Kl -hFPR (Chinese Hamster Ovary cells CHO-Kl expressing human Formyl Peptide Receptor FPR-I);

3. CHO-K 1 -hCCR5 (Chinese Hamster Ovary cells CHO-K 1 expressing human CCR5);

4. BHK-21 -hCCR7 (Syrian Hamster Fibroblasts BHK-21 expressing human CCR7);

5. CF2Th-hCXCR2 (Canine Thymocytes expressing human CXCR2);

6. CF2Th-hCXCR3 (Canine Thymocytes expressing human CXCR3);

7. Rl 610-hCCR7 (Chinese Hamster Lung Fibroblasts expressing human CCR7);

8. HEK-293T -hFRR-1 (Human Embryonic Kidney cells expressing human FPR-I); and

9. CHO-Kl -hCCR7 (Chinese Hamster Ovary cells CHO-Kl expressing mouse chemokine receptor CCR7).

Example 10: Isolation and Characterization of Anti-CCR7 Antibodies

According to the SDS-PAGE of the supernatants of 9 antibody preparations (FIG. 1), scFvs in each of 9 samples (lanes 1 -9) appeared as single bands and thus were present in a highly purified forms. The concentration of scFv's varied among these 9 samples— from as high as approximately 0.3 mg/ml to below the detection limit of 0.02 mg/ml. This variation in the concentration is typical and originated from differences in expression of scFv's in individual bacterial clones.

Eight histograms are shown demonstrating the signal collected from the cells expressing human CCR7 and different other GPCRs (used as controls). Cells have been incubated with selected CCR7 bidning scFv antibodies bearing the c-Myc tag. After washing out residual unbound antibodies the cells have been incubated with mouse anti c-Myc antibodies and secondary goat anti-mouse antibodies conjugated to the fluorescent dye phycoerythrin (IgG-PE). The fluorescent signal collecdet from individual cells were collected using fluorescence Activated Cell Sorter (FACS). The last histogram (Fig.9) shows the expression of CCR7 in generated cell lines, and is used as a control for CCR7 expression.

FIG. 2 depicts a graph of fluorescence of cells expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-107 of this invention.

FIG. 3 depicts a graph of fluorescence of cells expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-201 of this invention.

FIG. 4 depicts a graph of fluorescence of cells expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-312 of this invention.

FIG. 5 depicts a graph of fluorescence of cells expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-315 of this invention.

FIG. 6 depicts a graph of fluorescence of cells expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-327 of this invention.

FIG. 7 depicts a graph of fluorescence of cells expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-333 of this invention.

FIG. 8 depicts a graph of fluorescence of cells expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-334 of this invention.

FIG. 9 depicts a graph of fluorescence of cells expressing CCR7 or other GPCRs, and labeled with human antibody fragment MSM-R7-337 of this invention.

The data shown in FIGs. 2-9 demonstrate that all eight shown CCR7 antibody clones selectively bind to CCR7 but not to other G-protein coupled receptors.

The same holds true for many other clones listed in Table 1 below. Among the other 121 clones listed in the Table, MSM-R7-1051, MSM-R7-1052, MSM-R7-1124, and MSM-R7-1296 display particularly low background binding, whereas their signals upon binding to both human and mouse CCR7 are particularly strong and are comparable to or better than those of commercial mouse antibodies, as assayed using the following 17 types of cells: Seven types of human CCR7 expressing cell lines - BHK, two CHO, cell lines with differences in expression vector, HEK-293T, Rl 610, cGth, and HeLa, one cell line (CHO) expressing mouse CCR7, and as a control - HeLa parental, CHO parental, BHK parental, HEK-293T parental, R1610 parental, and four cell lines expressing unrelated GPCRs - CHO-FPR, R1610-CXCR4, cf2Th-CXCR4, and cΩth-CCR5.

FIG. 10 depicts a graph of fluorescence of cells expressing CCR7 or other GPCRs, and labeled with mouse antibodies. The data presented confirm that cells (correspondent to the legend) employed in the analysis of specificity of binding (FIGs. 2-9) indeed express, as expected, human and mouse CCR7, whereas other cells used in the assay, again as expected and correspondent to the legend, do not display any significant CCR7 expression. Along with the FIGs. 2-9, FIG. 10 shows that the fully human antibodies of this invention also cross react with the mouse CCR7 molecule. Mouse and human CCR7 molecules have a high degree of homology and the cross-reactivity has been both expected and desired, because the human- mouse cross-reactivity helps in the following animal based evaluation studies on the antibodies for selection of antibody candidates for therapeutics development.

Example 11: Sequencing of Antibody Fragments

Fully human antibodies depicted in FIGs. 2-9, and 121 (one hundred twenty one) other cloned, heavy chain variable CDR3 regions were sequenced. Table 1 shows sequence data for each of 8 clones for which cell binding data is presented above and for 121 other clones. Each of the clones has a unique HvCDR-3 sequence and thus the Table does not include duplications.

Table 1: Sequences of Anti-CCR7 Antibody Heavy Chain Variable CDR-3 Domains (Hv CDR-3)

Clone No: Clone Number Peptide sequence Sequence Id N

1. MSM-R7-107 GRRRTSIAGATIFDY SEQ ID NO:3

2. MSM-R7-281 RTRRTAIVTGAFDI SEQ ID NO:4

3. MSM-R7-312 RAGWSNGFDI SEQ ID NO:5

4. MSM-R7-315 RLSRWLGAFDI SEQ ID NO:6

5. MSM-R7-327 QRBPSLESYDNFDTSILDF SEQ ID NO:7

6. MSM-R7-333 GRALSF SEQ ID NO:8

7. MSM-R7-334 GRSMGV SEQ ID NO:9

8. MSM-R7-337 RGPTATTRAFDI SEQ ID NO: 10

9. MSM-R7-001 YDLSGNTDAFDY SEQ ID NO: 1 1

10. MSM-R7-002 YTSKTDAFDF SEQ ID NO: 12

1 1. MSM-R7-006 GLGRSSWYSGYYGLDV SEQ ID NO: 13

12. MSM-R7-015 RLYGDYNDAFDI SEQ ID NO: 14

13. MSM-R7-020 HFTRDDAFDI SEQ ID NO: 15

14. MSM-R7-049 RALEGGWWDY SEQ ID NO: 16 15. MSM-R7-053 DYGDYESGADYMDV SEQ ID NO: 17

16. MSM-R7-058 VRGVINGNNWFDP SEQ ID NO: 18

17. MSM-R7-060 GGGSYYPLTD SEQ ID NO: 19

18. MSM-R7-061 DGPPGTGNAFDl SEQ ID NO:20

19. MSM-R7-066 VARQVIGSGFGYMDV SEQIDNO:21

20. MSM-R7-067 ELVGAPGGFDP SEQ ID NO.22

21. MSM-R7-068 EEIDHTRSWYTRDRKDYCYMDV SEQIDNO:23

22. MSM-R7-069 DVEAYDFWTGYFAY SEQ ID NO:24

23. MSM-R7-070 LIPAPGNTYYYYGLDD SEQIDNO:25

24. MSM-R7-071 DMGQSGAAIGPIDF SEQ ID NO:26

25. MSM-R7-093 DFRGIDAFDI SEQIDNO:27

26. MSM-R7-096 GPEWMWSLFDF SEQIDNO:28

27. MSM-R7-113 RRVGHDY SEQIDNO:29

28. MSM-R7-122 DPRNSRDWLGFNWFDP SEQIDNO:30

29. MSM-R7-123 VSRITVGTVIAHPIDY SEQIDNO:31

30. MSM-R7-129 DSPYTRSLDY SEQIDNO:32

31. MSM-R7-132 GLAYVLRFPEWSHFDS SEQIDNO:33

32. MSM-R7-138 ERYDSSSGEEYLDY SEQIDNO:34

33. MSM-R7-140 GPNQGFCSDGPCYAGGYWFDP SEQIDNO:35

34. MSM-R7-141 VEVDYYDSSGYYLDD SEQIDNO:36

35. MSM-R7-142 ALFYDSRGYQNGLGY SEQIDNO:37

36. MSM-R7-144 DSPGGYFDY SEQIDNO:38

37. MSM-R7-145 EIHVWFGVDY SEQIDNO:39

38. MSM-R7-147 GSDSGWYEGPSPLLDY SEQIDNO:40

39. MSM-R7-148 DNSAKDDAFDL SEQIDNO:41

40. MSM-R7-149 DVSTVVTPDYFQH SEQ ID NO:42

41. MSM-R7-152 GHPIADPFHY SEQ ID NO:43

42. MSM-R7-153 VCSSTSCYDTY SEQIDNO:44

43. MSM-R7-154 DMLGLDYDALTGYLEY SEQIDNO:45

44. MSM-R7-155 DRWNSIGVPVYGMDV SEQIDNO:46

45. MSM-R7-156 APNGCPDY SEQIDNO:47

46. MSM-R7-157 GIEDYGDSKYLEY SEQIDNO:48

47. MSM-R7-158 APSNHYYYYYMDV SEQ ID NO:49

48. MSM-R7-159 VKRQLAPPDYFDS SEQIDNO:50

49. MSM-R7-160 GATLLNS SEQIDNO:51

50. MSM-R7-162 GSSSVYPY SEQIDNO:52

51. MSM-R7-164 AVLLTEIVVDEKWFAP SEQIDNO:53

52. MSM-R7-172 APNRYCRDGSCYGSYYYMDV SEQIDNO:54

53. MSM-R7-173 YEWNSWFDP SEQIDNO:55

54. MSM-R7-174 DHNRGSNWFDP SEQIDNO:56

55. MSM-R7-176 RREPGGWFDS SEQIDNO:57

56. MSM-R7-191 VGEYYYGSFEIGKAFDI SEQIDNO:58

57. MSM-R7-196 QVAHGDYDYGYFDL SEQIDNO:59

58. MSM-R7-197 HVIPMIVVIPYLYYFDY SEQIDNO:60

59. MSM-R7-206 DGPLGLDALDI SEQIDNO:61

60. MSM-R7-213 LSALRDAFDI SEQIDNO:62

61. MSM-R7-214 VFPGGNYYGSGSYLLDY SEQIDNO:63

62. MSM-R7-221 RHYYDTSGPTPYDGLDT SEQIDNO:64

63. MSM-R7-230 EYPPDYDFWSGYYHYYGMDV SEQIDNO:65

64. MSM-R7-232 IPTAFGVILSWYFDY SEQIDNO:66

65. MSM-R7-235 EISYYDSSGNYHYMDV SEQIDNO:67

66. MSM-R7-242 * VTSKSXPIHQNYYEMGL SEQIDNO:68

67. MSM-R7-245 SRSSNIGYWEYYYAMDV SEQIDNO:69

68. MSM-R7-246 GSSWSVMDV SEQIDNO:70 69. MSM-R7-249 VQWAYGSGSAIHYYYYMDV SEQIDNO:71

70. MSM-R7-254 DKEYCSSTSCHYYYGTDV SEQ ID NO:72

71. MSM-R7-255 GYDLWSGNDHYGMDV SEQ ID NO:73

72. MSM-R7-256 DPSLGSVFYYYMDV SEQ ID NO:74

73. MSM-R7-258 RPWLQGRAGWFDP SEQIDNO:75

74. MSM-R7-272 GQGAYPTELLVI SEQ ID NO:76

75. MSM-R7-274 DSEYHSGDYWYDAFDI SEQ ID NO:77

76. MSM-R7-275 QLNLLRALDV SEQ ID NO:78

77. MSM-R7-278 DTRLVLGRPPGPLLLLGAAKEDWPB SEQ ID NO:79

78. MSM-R7-280 GGGYYFDY SEQ ID NO: 80

79. MSM-R7-282 DRVMSWHDPPDLFAS SEQIDNO:81

80. MSM-R7-285 GQRFFRY SEQIDNO:82

81. MSM-R7-288 GGGRYYSSGSFDS SEQ ID NO:83

82. MSM-R7-289 DKDPVFGVVNHAYFDL SEQ ID NO:84

83. MSM-R7-294 DGKVGERGAFYHYGLDV SEQIDNO:85

84. MSM-R7-296 GRGRSGSYYKY SEQ ID NO: 86

85. MSM-R7-298 GGSGRPTYYYHGMDV SEQIDNO:87

86. MSM-R7-300 EGVAYSSNWYYFDY SEQ ID NO:88

87. MSM-R7-305 DFYNSGGYDDF SEQ ID NO:89

88. MSM-R7-307 DVNYYNAFDI SEQIDNO:90

89. MSM-R7-309 PPQIPFSYDDRGGYYFPHYYYYMDV SEQIDNO:91

90. MSM-R7-311 DGYCNSTSCYRALSL SEQIDNO:92

91. MSM-R7-315 RLSRWLGAFDI SEQ ID NO:93

92. MSM-R7-316 DPPMHGYNSFDY SEQ ID NO:94

93. MSM-R7-319 HGPDDLTGTAFFDY SEQIDNO:95

94. MSM-R7-320 DYYDGNGNYNYFDT SEQIDNO:96

95. MSM-R7-324 NYGSGFSD SEQ ID NO:97

96. MSM-R7-326 RLSKWLGAFDI SEQ ID NO:98

97. MSM-R7-335 GDSYRSGDYGADYYYGLDI SEQIDNO:99

98. MSM-R7-337 RGPTATTRAFDI SEQ ID NO: 100

99. MSM-R7-1016 GRIFDY SEQ ID NO: 101

100. MSM-R7-1035 DSVAAAGHFDY SEQ ID NO: 102

101. MSM-R7-1044 RFGGPAHFDH SEQ ID NO: 103

102. MSM-R7-1048 NYSSSWFLDY SEQ ID NO: 104

103. MSM-R7-1051 RVGTTLGAFDI SEQ ID NO: 105

104. MSM-R7-1052 RRGWDHGFDI SEQ ID NO: 106

105. MSM-R7-1064 RTRRTAIVTGAYDI SEQ ID NO: 107

106. MSM-R7-1092 NLHISGRAPGDY SEQ ID NO: 108

107 MSM-R7-1113 DSYANTLWY SEQ ID NO: 109

108. MSM-R7-1118 GAGGTMVRGNNWFDP SEQIDNO:110

109. MSM-R7-1119 GRGYSYGSGAFDI SEQIDNO:111

110. MSM-R7-1121 GLTSSWYIGY SEQ ID NO: 112

111. MSM-R7-1123 GRALSF SEQ ID NO: 113

112. MSM-R7-1124 RTRRTAIVSGAYDM SEQ ID NO: 114

113. MSM-R7-1141 VARLADF SEQIDNO:115

114. MSM-R7-1144 RFGGPAHFDH SEQIDNO:116

115. MSM-R7-1156 EAPVSINDYDFWTGGEYYYGMDV SEQ ID NO: 117

116. MSM-R7-1158 LTAGYSSSPIDY SEQ ID NO: 118

117. MSM-R7-1175 EWSGFDH SEQ ID NO: 119

118. MSM-R7-1183 ESGHGDYEGAFDI SEQ ID NO: 120

119. MSM-R7-1209 RLGYHDGFDI SEQIDNO:121

120. MSM-R7-1211 NRAGFDS SEQ ID NO: 122

121. MSM-R7-1215 DRGGIHNFYAMDV SEQ ID NO: 123

122. MSM-R7-1228 YRGSYEGYFDL SEQ ID NO: 124 123. MSM-R7-1223 AGWNYVLFDY SEQ ID NO: 125

124. MSM-R7-1231 DKVYDFWSGYPWDYYYYGTDV SEQ ID NO: 126

125. MSM-R7-1240 GRKVLSSRV A V AGPHFDF SEQ ID NO: 127

126. MSM-R7-1246 EGPGMQALDV SEQ ID NO: 128

127. MSM-R7-1269 RVVSV1GAFDY SEQ ID NO: 129

128. MSM-R7-1296 RLSRWLGAFDI SEQ ID NO: 130

129. MSM-R7-1303 DQRWEFDLPDALDV SEQ ID NO: 131 * For No: 66, X is not determined.

Example 12: Generation of R1610-hCCR7: Chinese Hamster Lung Fibroblasts expressing human CCR7

R1610-hCCR7 cells were obtained by transfecting the R1610 cells (Chinese Hamster Lung Fibroblasts; ATCC, catalog number CRLl 657) using Lipofectamin 2000 transfection reagent (Invitrogen, catalog number 1 1668019), according to the manufacturer's protocol, with the commercial pCMV-Script Vector (Catalog #212220, Stratagene) carrying a synthetic, mammalian cell expression optimized, human CCR7 gene (encodes the human CCR7 amino acid sequence of 378 amino acids; the Swissprot accession number P32248: MDLGKPMKSVLVV ALLVIFQVCLCQDEVTDDYIGDNTTVD YTLFESLCSKKDVRNFKA WFLPIMYSIICFVGLLGNGLVVLTYIYFKRLKTMTDTYLLNLAVADILFLLTLPFWAYSA AKSWVFGVHFCKLIF AIYKMSFFSGMLLLLCISIDRYV AIVQAVSAHRHRARVLLISKLSC VGIWILATVLSIPELL YSDLQRSSSEQAMRCSLITEHVEAFITIQV AQMVIGFLVPLLAMSF CYLVIIRTLLQARNFERNKAIKVIIAVVVVFIVFQLPYNGVVLAQTV ANFNITSSTCELSKQ LNIAYDVTYSLACVRCCVNPFLYAFIGVKFRNDLFKLFKDLGCLSQEQLRQWSSCRHIRR SSMSVEAETTTTFSP SEQ ID NO: 132

The CCR7 coding region had a C-terminal extension of nucleotides that encode a two amino acid (S and A) linker followed by the Streptavidin-tag

MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP SEQ ID NO: 133

followed by another two amino acid (GG) linker and the S-tag

KETAAAKFERQHMDS SEQ ID NO: 134

The Rl 610 cells so transfected were cloned and CCR7 expressing clones were selected. Example 13: The Human Natural Ligands of CCR7, CCL19 and CCL21

To determine whether the CCR7 is expressed in its native configuration, we performed a series of experiments in which we measured the binging of natural ligands of CCR7 to cells expressing CCR7 produced by our constructs. The natural ligands of CCR7, CCL19 and CCL21 are commercially available. The mouse CCLl 9-Fc fusion ligand with human Fc fragment (eBioscience, catalog number 14-1972) and the human CCL-21-stalk-His6 ligand (R&D Systems, catalog number 966-6C/CF) were employed. The commercial CCL- 19-Fc binds to both native mouse CCR7 according to the manufacturer's data; and human CCR7 on the cell surface, as described by Stefan Krautwald, Ekkehard Ziegler, Reinhold Fδrster, Lars OhI, Kerstin Amann, and Ulrich Kunzendorf, in: Ectopic expression of CCLl 9 impairs alloimmune response in mice. Immunology. 2004 June; 1 12(2): 301-309.

Binding of the ligand to CCR7 expressing cells was demonstrated by fluorescence activated cell sorting (FACS) obtained using a Guava FACS instrument. CCL 19-Fc binding to cells was detected using PE-conjugated Mouse Anti-Human Fc Monoclonal Antibody (1/50 diluted, eBioscience; catalog number 12-4998-82). Under the conditions of the experiment, if no Mouse Anti -Human Fc antibody bound to the cell, the ligand CCL 19-Fc was not bound to the cell. Conversely, detection of Mouse Anti-Human Fc antibody indicated the presence of CCL 19 to the cells.

To carry out these experiments, we produced cells expressing CCR7 using our expression vector. As a control, we used the same cells but not having the CCR7 expression vector.

Then, to each set of cells we added CCL- 19 Fc for 30 minutes on ice. Subsequently, we added Mouse Anti-Human Fc antibody for 30 minutes on ice, after which we washed the cells to remove unbound CCL- 19Fc and unbound Mouse Anti-Human Fc antibody. We fixed the cells using formalin-containing fixation buffer. We then measured fluorescence using a Guava-96 FACS device. Data is expressed as arbitrary units (PM 1 Fluorescence)

FIGs. HA and HB depict results of these studies. CCR7 expressing cells showed much higher fluorescence (MFL = 100; FIG. HA), compared to control, parental cells that showed only marginal binding of the ligand (MFL = 3.5; FIG. 11B).

We conclude that our expression system produced CCR7 in its native configuration and therefore capable of binding the natural ligands. We further conclude that such cells expressing CCR7 can be useful for determining whether human monoclonal antibodies against CCR7 can inhibit binding of the natural ligands for CCR7..

Example 14: Anti-CCR Antibodies Inhibit Binding of the Natural Ligand to CCR7

To determine if fully human antibodies against CCR7 can be useful in inhibiting CCR7 mediated disorders, we performed experiments to determine whether such antibodies compete with naturally occurring CCR7 ligands for natively configured CCR7.

Using cells expressing CCR7 as described above in Example 12, we performed direct competition assays. A 5 μL aliquot of a purified antibody against CCR7 or 5 μL buffer aliquot as a control was added to a 10 μL aliquot of cell suspension containing approximately 30,000 HEK-293T cells expressing human CCR7. Upon incubation on ice for 30 min, an aliquot of commercial CCL 19 Fc (80 nM) was then added, and cells were washed from unbound antibodies and the ligand by centrifugation at 1 ,200 rpm for 5 min using Sorvall Legend RT centrifuge (at 5⁰C). A 5 μL aliquot of phycoerythrin (PE)-conjugated Mouse anti-human Fc monoclonal antibody (1/50 or 1/20 dilution (eBioscience; catalog number 12-4998-82) was then added, and after 30 min-incubation on ice cells were washed twice by centrifugation. Fixed samples were analyzed by FACS using a Guava-96 instrument.

Binding of CCL 19 Fc to cells expressing CCR7 produces a fluorescence signal. Among 18 antibodies so analyzed, using MSM-R7-281 having the CDR3 sequence disclosed in Table 1 No: 2 (SEQ ID NO:4), we found a significant reduction of FACS signal by 8.4% ± 4.0% (mean ± SD, n=3) (dilution - 1 :20) compared to control signals obtained from cells expressing CCR7 but not pre-treated with antibody. The presence of MSM-R7-334 antibody, having the CDR3 sequence disclosed in Table 1 No:7 (SEQ ID NO:9) also decreased the FACS signal compared to control by 68% in one experiment and by 7.9% in another independent experiment performed more diluted Mouse anti-human Fc monoclonal antibody (dilution = 1:50).

We conclude from these studies that these two antibodies compete with natural ligands for CCR7. Without being bound by any particular theory of operation, we believe that the antibodies of this invention bound to natively configured CCR7 in such a way as to at least partially cover the binding site for the CCR7 ligand, therefore decreasing binding of the ligand for the binding site of CCR7. Regardless of the theory, we conclude that fully human antibodies of this invention can be used to decrease the effects of over-stimulation of CCR7 in a variety of conditions and disorders, including cancers.

All references cited herein are incorporated fully by reference, as if separately so incorported.

INDUSTRIAL APPLICATION

Fully human antibodies against human CCR7 can be used to detect the presence of CCR7 on cells, and therefore can be used to diagnose disorders involving CCR7. Further, antibodies of this invention can be useful for treating disorders involving CCR7 by inhibiting binding of native chemokines to the CCR7, and thereby decrease effects of those chemokines.

Claims

We claim:

1. A fully human antibody against human chemokine receptor CCR7.

2. The antibody of Claim 1 , wherein said antibody selectively binds to CCR7.

3. The antibody of Claim 1, wherein said antibody is essentially free of contaminants.

4. A fully human antibody fragment, said fragment capable of specifically binding to human CCR7.

5. A library of fully human anti-CCR7 antibodies.

6. A fully human anti-CCR7 antibody having a CDR3 sequence selected from Table 1.

7. The antibody of Claim 6, wherein said antibody has a heavy chain fragment having a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5., SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

8. A method for treating a disorder, comprising: providing a patient having said disorder involving over function of CCR7 receptor; and administering a pharmaceutically effective amount of a fully human antibody against

CCR7, said pharmaceutically effective amount being an amount that reduces the function of

CCR7 expressing cells in said patient.

9. The method of Claim 8, wherein said fully human antibody against CCR7 is an antibody fragment capable of specifically binding to human CCR7 receptor.

10. The method of Claim 8, wherein said antibody has a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5., SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20.

11. The method of Claim 8, wherein said antibody has the CDR3 sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO:9.

12. A kit, comprising: one or more fully human antibodies or fragments thereof against human CCR7 receptor, said antibodies or fragments thereof capable of selectively binding to said CCR7 receptor; a solution comprising a pharmaceutically acceptable excipient; and instructions for use.

13. A method for determining the presence of CCR7 receptor in a biological sample, comprising: providing a biological sample; exposing said sample to an antibody of Claim 1 ; and detecting the presence of said antibody bound to said biological sample.

14. Use of an fully human antibody against CCR7 in the manufacture of a medicament useful for treating a disorder in an animal caused by ligand-mediated overactivity of CCR7.

15. The use of Claim 14, wherein said antibody has a CDR3 region sequence selected from Table 1.