EP2285403A2 - Anticorps monoclonaux humains dirigés contre le récepteur de chimiokines humaines ccr7 - Google Patents

Anticorps monoclonaux humains dirigés contre le récepteur de chimiokines humaines ccr7

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Publication number
EP2285403A2
EP2285403A2 EP09746941A EP09746941A EP2285403A2 EP 2285403 A2 EP2285403 A2 EP 2285403A2 EP 09746941 A EP09746941 A EP 09746941A EP 09746941 A EP09746941 A EP 09746941A EP 2285403 A2 EP2285403 A2 EP 2285403A2
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Prior art keywords
ccr7
seq
human
antibody
msm
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German (de)
English (en)
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Tajib Mirzabekov
David Kreimer
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Msm Protein Technologies Inc
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Msm Protein Technologies Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • 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.
  • GPCRs G-protein coupled receptors
  • 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.
  • GPCRs G- protein coupled receptors
  • Fully human antibodies that can specifically bind to human chemokine receptor CCR7 on the surfaces of living cells.
  • 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..
  • 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.
  • 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.
  • 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.
  • 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)).
  • 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)).
  • 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.
  • 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.
  • 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.
  • CCR7 receptors can be isolated from membranes of cells expressing the protein and used as an immunogen to produce CCR7-specific antibodies.
  • PCT International Patent Application No: PCT/US2007/003169 filed 5 February 2007 (WO 2007/092457). This application is expressly incorporated herein fully by reference.
  • 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.
  • 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.
  • inflammatory diseases such as (1) Rheumatoid Arthritis; (2) Inflammatory Bowel Disease; (3) Psoriasis; ?(4). Lupus; (5) Multiple Sclerosis, and (6) Asthma.
  • Rheumatoid Arthritis such as (1) Rheumatoid Arthritis;
  • 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.
  • 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.
  • anti-hCCR7 human anti-human CCR7
  • MAbs Monoclonal Antibodies
  • fragments thereof which just bind to CCR7 but do not affect its natural ligand binding properties and signaling;
  • MAbs and fragments thereof which bind to CCR7 and activate the signaling by natural ligands (agonists);
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • anti-CCR7 antibodies raised against non-natively configured CCR7 may not detect mis-folded or mis-inserted CCR7 into cell membranes.
  • use of antibodies of this invention along with more routine analyses can shed light upon the functional state of a cell's CCR7 status.
  • fully human CCR7 antibodies can be useful in treating conditions involving defects in CCR7, include cancers.
  • CCR7 can play important roles in dysregulation of cell growth and tumor metastasis.
  • use of fully human anti-CCR7 antibodies of this invention can bind to the CCR7 receptor.
  • 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.
  • 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.
  • anti-CCR7 antibodies of this invention can act as agonists and thereby increase the functioning of CCR7-dependent processes.
  • antibodies and fragments thereof of this invention can find therapeutic use in a variety of pathological conditions, including cancer.
  • 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.
  • qPCR quantative PCR
  • 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.
  • ELISA enzyme-linked immunosorbent assays
  • Western blotting or other methods known in the art.
  • the therapeutic goal can include reducing function of the CCR7 pathways.
  • Antagonist antibodies of this invention can be particularly useful for these situations.
  • using antibodies that specifically bind to CCR7 can be used to reduce the numbers of CCR7 expressing cells.
  • compositions containing fully human anti-CCR7 antibodies are also included within the scope of this invention.
  • a suitable composition can include one or more anti-CCR7 antibodies, a physiologically compatible solution, and one or more pharmacological excipients.
  • 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.
  • cancers including chronic leukocytic leukemia, head and neck cancers, non-small cell lung cancers, gastric cancer, breast cancer, melanoma and colorectal cancer.
  • cancers including chronic leukocytic leukemia, head and neck cancers, non-small cell lung cancers, gastric cancer, breast cancer, melanoma and colorectal cancer.
  • CLL Chronic Leukocytic Leukemia
  • SCC squamous cell carcinomas
  • ACC adenoid cystic carcinomas
  • CCR7 mediates survival and invasiveness of metastatic squamous cell carcinoma of the head and neck (SCCHN) to regional lymph nodes.
  • SCCHN head and neck
  • EGFR epidermal growth factor receptor
  • 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.).
  • 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.
  • 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.
  • anti-CCR7 antibodies of this invention can be used for diagnosing or evaluating the CCR7 status of a cell or tissue.
  • CCR7 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mice 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.
  • Lu-99 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.”
  • Tumor cells can express various receptors that facilitate such metastatic spread to lymph nodes and other non-lymphoid organs.
  • Chemokine receptors CCR
  • CCR Chemokine receptors
  • CMOS complementary metal-oxide-semiconductor
  • 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.
  • 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.
  • 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.”
  • 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.
  • chemokine receptor 7 CCR7
  • CCR7 CC chemokine receptor 7
  • CCL21 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.
  • CCL Chemokine ligand
  • BM-MSCs marrow-derived mesenchymal stem cells
  • 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.
  • 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.”
  • GFP green fluorescent protein
  • 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.
  • CCR7 in efficient priming of allospecific cytotoxic CD8(+) T cells is poorly characterized.
  • CCR7(-/-) mice completely failed to reject subcutaneously injected MHC class I mismatched tumor cells and cytotoxic activity of allospecific T cells was severely compromised.
  • recipient CCR7(-/-) mice were capable of rejecting the allografts.
  • GenbankTM alanine
  • CGC arginine
  • AAC asparagine
  • GAC aspartic acid
  • TGC cysteine
  • GCG glutamic acid
  • GAG glutamine
  • GGC histidine
  • CAC histidine
  • ATC isoleucine
  • CCG leucine
  • CAG methionine
  • TTC proline
  • TTC threonine
  • ACC tryptophan
  • TGG tyrosine
  • TGG tyrosine
  • GTG valiine
  • 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.
  • 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).
  • 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
  • PND a gift from Dr. Rhodes, University of California, Davis
  • 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.
  • FACS fluorescence activated cell sorting
  • Cells were washed twice with PBS and lysed in 1 ml of solubilization medium composed of 100 mM (NH 4 ) 2 SO 4 , 20 mM Tris-HCl (pH 7.5), 10% glycerol, l%(w/v) detergent (see below), and Protease Inhibitor Mixture (one tablet of CompleteTM (Roche Molecular Biochemicals) per 25 ml.
  • solubilization medium composed of 100 mM (NH 4 ) 2 SO 4 , 20 mM Tris-HCl (pH 7.5), 10% glycerol, l%(w/v) detergent (see below), and Protease Inhibitor Mixture (one tablet of CompleteTM (Roche Molecular Biochemicals) per 25 ml.
  • the lysate was incubated at 4 0 C 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 ⁇ l 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 55 0 C. Samples were run on 11% SDS-polyacrylamide minigels and visualized.
  • Detergents were used as components of solubilization buffers.
  • the detergents were rc-octyl- ⁇ -D- glucopyranoside (23.4 mM), H-decyl- ⁇ -D-maltoside (1.8mM), w-dodecyl- ⁇ -D-maltoside (DDM; 0.17 mM), cyclohexyl-butyl- ⁇ -D-maltoside (CymalTM-4; 7.6 mM), cyclohexyl-pentyl- ⁇ -D- maltoside (CymalTM-6; 0.56 mM), cyclohexyl-heptyl- ⁇ -D-maltoside (CymalTM-7; 0.19 mM), cyclo-hexylpropanoyl-N-hydroxyethylglucamide (108 mM), cyclohexylbutanoyl-N- hydroxye
  • 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 CymalTM-5 and centrifuged for 30 min at 14,000 x g. The cell lysate as incubated with 50 ⁇ l of 1D4-Sepharose beads on a rocking platform at 4 0 C for 10 -12 h.
  • the SepharoseTM beads were washed about five times with the washing buffer (100 mM (NH 4 ) 2 SO 4 , 20 mM Tris-HCl (pH 7.5), 10% glycerol and 1% CymalTM-5) and once with washing buffer plus 500 mM MgCl 2 .
  • CCR7 was eluted from the beads by three successive washes with 50 ⁇ l of medium containing 200 mM C9 peptide (TETSQVAPA: SEQ ID NO: 2), 500 mM MgCl 2 , 100 mM (NH 4 ) 2 SO 4 , 20 mM Tris-HCl (pH 7.5), 10% glycerol, and 0.5% CymalTM-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.
  • 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 GenePORTERTM 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.
  • 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.
  • cells can be transfected with vectors that encode tagged monomers and other vectors that encode un-tagged monomers.
  • 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.
  • 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 (GeneticinTM; 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
  • 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.
  • antibodies of this invention can recognize the ectodomain of the CCR7, and thus, can bind to native CCR7 expressed in cells, including human cells.
  • anti-CCR7 antibodies can be used therapeutically or diagnostically, as explained further herein.
  • 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)).
  • 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 10 9 -10 13 ). 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.
  • 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.
  • CDRs complementarity-determining regions
  • Example 7 Human Antibodies Obtained By Immunization of Transgenic Mice
  • mice that are transgenic for human immunoglobulin genes and have disrupted mouse immunoglobulin heavy-chain and Ig ⁇ light-chain loci were 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.
  • GPCRs G-protein coupled receptors
  • ion channels and transporters 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.
  • GPCRs G-protein coupled receptors
  • preparation of the native immunogen 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.
  • the antibodies generated using this last method rarely appear with desirable neutralizing (antagonistic) properties.
  • 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 37 0 C, 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.
  • IPTG Upon reaching logarithmic phase of growth (OD ⁇ 0.6) at 37 0 C, 250 rpm, IPTG was added to bacterial cultures to a final concentration of 0.05 mM. Cultures were incubated overnight at 3O 0 C, 250 rpm.
  • Ni-agarose resin High-Density IDA-Agarose 6 BCL Nickel Charged Resin (ABT)
  • ABT Nickel Charged Resin
  • 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.
  • CHO-Kl Choinese Hamster Ovary cells, ATCC Cat # CCL-61;
  • CF2Th Canine Thymocytes, ATCC Cat # CRL- 1430
  • R1610 Choinese Hamster Lung Fibroblasts, ATCC Cat # CRL- 1657
  • HEK-293T Human Embryonic Kidney cells, Cat # CRC- 1573.
  • GPCRs G-Protein Coupled Receptors
  • CHO-Kl -hCCR7 Choinese Hamster Ovary cells CHO-Kl expressing human chemokine receptor CCR7;
  • CHO-Kl -hFPR Choinese Hamster Ovary cells CHO-Kl expressing human Formyl Peptide Receptor FPR-I
  • CHO-K 1 -hCCR5 Choinese Hamster Ovary cells CHO-K 1 expressing human CCR5;
  • BHK-21 -hCCR7 (Syrian Hamster Fibroblasts BHK-21 expressing human CCR7);
  • CF2Th-hCXCR2 Canine Thymocytes expressing human CXCR2
  • CF2Th-hCXCR3 Canine Thymocytes expressing human CXCR3
  • Rl 610-hCCR7 Choinese Hamster Lung Fibroblasts expressing human CCR7
  • HEK-293T -hFRR-1 Human Embryonic Kidney cells expressing human FPR-I
  • CHO-Kl -hCCR7 Choinese Hamster Ovary cells CHO-Kl expressing mouse chemokine receptor CCR7.
  • scFvs in each of 9 samples appeared as single bands and thus were present in a highly purified forms.
  • 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.
  • 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.
  • FIGs. 2-9 demonstrate that all eight shown CCR7 antibody clones selectively bind to CCR7 but not to other G-protein coupled receptors.
  • 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 c ⁇ th-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.
  • 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.
  • 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.
  • MSM-R7-015 RLYGDYNDAFDI SEQ ID NO: 14
  • MSM-R7-049 RALEGGWWDY SEQ ID NO: 16 15. MSM-R7-053 DYGDYESGADYMDV SEQ ID NO: 17
  • MSM-R7-246 GSSWSVMDV SEQIDNO:70 69.
  • MSM-R7-1035 DSVAAAGHFDY SEQ ID NO: 102
  • MSM-R7-1211 NRAGFDS SEQ ID NO: 122
  • 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 V
  • 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
  • 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 F ⁇ rster, 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.
  • FACS fluorescence activated cell sorting
  • 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).
  • FACS fluorescence activated cell sorting
  • FIGs. HA and HB depict results of these studies.
  • Binding of CCL 19 Fc to cells expressing CCR7 produces a fluorescence signal.
  • 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.

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Abstract

La présente invention concerne dans certains aspects des anticorps totalement humains ou des fragments de ceux-ci qui se lient spécifiquement au récepteur humain CCR7. De tels anticorps ou de tels fragments peuvent être utilisés pour traiter des pathologies impliquant une fonction excessive du récepteur CCR7, comprenant les cancers. D’autres utilisations comprennent la détection du récepteur humain CCR7 dans des échantillons biologiques à des fins de diagnostic ou d’évaluation.
EP09746941A 2008-05-14 2009-05-11 Anticorps monoclonaux humains dirigés contre le récepteur de chimiokines humaines ccr7 Withdrawn EP2285403A2 (fr)

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DK2623592T3 (da) * 2010-09-28 2019-08-19 Nb Health Laboratory Co Ltd Anti-humane ccr7-antistoffer, hybridom, medicinsk sammensætning og antistof-immobiliseret bærer
US9670277B2 (en) 2011-02-24 2017-06-06 The Schepens Eye Reasearch Institute, Inc. Compositions and methods for treating inflammatory conditions of the ocular surface
WO2013184200A1 (fr) * 2012-06-05 2013-12-12 Msm Protein Technologies Anticorps monoclonaux humains contre le récepteur de chimiokine ccr7 humain
WO2014093870A2 (fr) * 2012-12-13 2014-06-19 The Schepens Eye Research Institute, Inc. Utilisation d'inhibiteurs du récepteur de chimiokines c-c de type 7 (ccr7)
JP6469644B2 (ja) 2013-03-15 2019-02-13 アムジエン・インコーポレーテツド 抗ｃｃｒ７抗原結合タンパク質に関係する方法および組成物
EP3334457B1 (fr) * 2015-08-10 2020-08-26 PepMab B.V. Anticorps humanisés du récepteur anti-ccr7
JOP20190187A1 (ar) 2017-02-03 2019-08-01 Novartis Ag مترافقات عقار جسم مضاد لـ ccr7
KR20210132644A (ko) 2018-12-18 2021-11-04 캐터펄트 테라퓨틱스 비.브이. 이식편 대 숙주 질환(GvHD)의 예방 또는 치료를 위한 항-CCR7 mAb의 용도
US20230348571A1 (en) * 2020-04-06 2023-11-02 Vanderbilt University Cross-reactive coronavirus antibodies and uses thereof

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