WO2012043533A1 - 抗ヒトccr7抗体、ハイブリドーマ、核酸、ベクター、細胞、医薬組成物、並びに、抗体固定化担体 - Google Patents
抗ヒトccr7抗体、ハイブリドーマ、核酸、ベクター、細胞、医薬組成物、並びに、抗体固定化担体 Download PDFInfo
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Definitions
- the present invention relates to an anti-human CCR7 antibody, hybridoma, nucleic acid, vector, cell, pharmaceutical composition, and antibody-immobilized carrier, and more specifically, an anti-human CCR7 antibody that specifically binds to the extracellular domain of human CCR7.
- a hybridoma producing the antibody, a nucleic acid encoding a heavy chain variable region or a light chain variable region of the antibody, a vector having the nucleic acid, a cell having the vector, a pharmaceutical composition containing the antibody, and the antibody The present invention relates to an immobilized antibody-immobilized carrier.
- Chemokines are cytokines that regulate the migration and cell function of various cells.
- the dysfunction of chemokines and their receptors is responsible for various diseases such as autoimmune diseases, acute and chronic inflammation, and cancer. So far, drugs that control the activity of chemokines and their receptors have been developed and clinically applied, but it is difficult to say that the problem has been sufficiently solved.
- chemokine-selective cell membrane receptor In order for a specific chemokine to express activities such as cell migration and regulation of cell function, it is necessary to bind to a chemokine-selective cell membrane receptor. About 20 chemokine receptors have already been discovered, and all chemokine receptors are 7-transmembrane proteins (GPCRs) that bind to trimeric G protein. When the chemokine binds to the receptor, it releases the G ⁇ unit of the trimer G protein. As a result, the intracellular Ca concentration is increased, and the function is expressed by activating phosphatidylinositol 3-kinase (PI3K), small Rho GTPases pathway and other pathways.
- PI3K phosphatidylinositol 3-kinase
- Non-patent Document 1 chemokine receptors are activated by relatively selective chemokines, the primary structure of proteins and the intracellular activation mechanism are very similar. Therefore, it is not easy to selectively block the function of a specific chemokine receptor. Functional expression of each chemokine and chemokine receptor in physiological and disease states is controlled by the expression of each protein in a specific cell or tissue at a specific time (during inflammation) (Non-patent Document 1).
- CCR7 Human CC motif receptor 7
- CC MOTIF RECEPTOR 7
- EBI1, CMKBR7 CC MOTIF
- CCR7 Human CC motif receptor 7
- ELC selective chemokine receptor for CCL19
- CCL21 also known as SLC, EXODUS 2
- CCR7 is relatively selectively expressed in cells such as CD4-positive T cells (Th1, Th2, Treg cells), mature dendritic cells, and B cells.
- CCR7 chronic myeloma
- autoimmune diseases fibrosis after acute and chronic inflammation
- cancer metastasis a lesion such as an inflammatory site via CCR7
- amino acid sequence of human CCR7 and the base sequence of the gene are already known (for example, GenBank: EAW60669.1).
- Inflammation is a protective reaction against tissue damage and infection. In response to various inflammatory stimuli, it promotes the infiltration of neutrophils and monocytes following the upregulation of inflammatory molecules (chemokines and cytokines). When the inflammatory reaction is further increased, T and B lymphocytes flow and the disease state becomes chronic. On the other hand, at the stage of inflammation convergence, excessive apoptosis of leukocytes or phagocytosis by tissue macrophages occurs, and repair of damaged tissues by stromal cells (for example, fibroblasts) is observed.
- stromal cells for example, fibroblasts
- fibrosis live fibrosis, renal fibrosis, pulmonary fibrosis, dermal fibrosis, cardiovascular fibrosis, gastrointestinal fibrosis and other fibrotic diseases
- Chronic pulmonary fibrosis is a disease with a poor prognosis due to scar formation throughout the lungs.
- Pirfenidone with anti-fibrotic effects, corticosteroids (eg prednisone) and / or other drugs that suppress the body's immune system are prescribed to reduce the process leading to fibrosis.
- corticosteroids eg prednisone
- the patient has a satisfactory therapeutic result.
- CD14 + peripheral blood monocyte precursor cells infiltrate the inflamed site and locally differentiate into stromal cell-like (collagen types I and III and fibronectin) (Non-patent Document 3). These cells secrete inflammatory cytokines as well as extracellular matrix proteins, other cytokines, which can lead to fibrosis. If excessive infiltration of blood fibroblast precursor cells into the inflamed site can be selectively suppressed, it can be expected to stop fibrosis to a minimum, but clinical application has not yet been achieved.
- Non-patent Document 1 a chemokine receptor expressed in fibroblast precursor cells. It has been shown that fibrosis does not develop even when a stimulus that causes pulmonary fibrosis or renal fibrosis is added to a gene-converted animal deficient in CCR7 (Non-patent Document 4). Therefore, if the function of CCR7 can be inhibited by a low molecular weight compound, a monoclonal antibody, RNAi, etc., it can be expected to suppress various fibrosis. However, since the primary structure of the chemokine receptor protein and the intracellular activation mechanism are very similar, a substance that can selectively inhibit CCR7 is desired. So far, studies on substances (monoclonal antibodies or RNAi) that inhibit the function of CCR7 have been conducted, but there has been no case of reaching clinical application.
- MMP inhibitors extracellular matrix proteinase inhibitors
- CCR7 is expressed in various tumor cells such as B-cell chronic lymphocytic leukemia, non-Hodgkin lymphoma, breast cancer cells, malignant breast tumors and the like. Furthermore, it has been revealed that CCR7 plays a role in lymph node metastasis of various cancers such as gastric cancer, melanoma, non-small cell lung cancer, and T cell leukemia cells (Non-patent Document 1). Since CCL19 and CCL21, which are ligands for CCR7, are highly expressed in lymph nodes, selective inhibition of CCR7 function can be expected to suppress lymphatic metastasis of cancer cells.
- the main mechanism of action of an antibody drug against a membrane protein (receptor) is that the antibody recognizes cells expressing the protein, and then complement-dependent cytolytic action (CDC) and antibody-dependent cell-mediated cytotoxicity. What was removed based on (ADCC) was common.
- CDC and ADCC are accompanied by activation of inflammatory cells such as macrophages and are not necessarily appropriate for treating fibrosis. Therefore, when a monoclonal antibody capable of selectively inhibiting CCR7 is applied to the treatment of fibrosis, a functional antibody not based on CDC or ADCC is desirable. That is, an antibody that selectively blocks CCR19- or CCL21-dependent intracellular signal transduction of CCR7 is desirable.
- An object of the present invention is to provide a novel anti-human CCR7 antibody useful as a therapeutic agent for fibrosis and cancer, a pharmaceutical composition containing the anti-human CCR7 antibody, and the like.
- an anti-human CCR7 antibody that specifically binds to the extracellular domain of human CCR7, comprising SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, an anti-human CCR7 antibody comprising a heavy chain complementarity determining region 3 (heavy chain CDR3) comprising the amino acid sequence represented by SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, or SEQ ID NO: 77 is there.
- Another aspect of the present invention for solving the same problem is an anti-human CCR7 antibody that specifically binds to the extracellular domain of human CCR7, wherein the complementarity determining regions 1 to 3 (CDR1 to 3)
- (A1) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 5, Having a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 6 and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 7,
- (A2) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 15, Having a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 16 and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 17,
- (A3) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 25, Having a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 26 and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 27,
- (A4) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 35, Having a heavy chain CDR2 comprising the amino acid sequence represented
- Another aspect of the present invention for solving the same problem is an anti-human CCR7 antibody that specifically binds to the extracellular domain of human CCR7, wherein the complementarity determining regions 1 to 3 (CDR1 to 3)
- (B1) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 5, Heavy chain CDR2, comprising the amino acid sequence represented by SEQ ID NO: 6, A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 7, A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 8, Having a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 9 and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 10.
- (B2) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 15, Heavy chain CDR2, comprising the amino acid sequence represented by SEQ ID NO: 16, Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 17, A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 18, Having a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 19 and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 20, (B3) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 25, A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 26; Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 27, A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 28, A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 29 and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 30;
- Another aspect of the present invention for solving the same problem is an anti-human CCR7 antibody that specifically binds to the extracellular domain of human CCR7, the amino acid sequences of the heavy chain variable region and the light chain variable region.
- An anti-human CCR7 antibody satisfying any of the following (C1) to (C8).
- C2 having a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 14.
- it has an activity of blocking a CCR7-dependent intracellular signal transduction mechanism by CCR7 ligand stimulation.
- it is a humanized antibody or a chimeric antibody.
- it is an antibody fragment, a single chain antibody, or a diabody.
- Another aspect of the present invention is an anti-human CCR7 antibody that specifically binds to the extracellular domain of human CCR7, wherein R7-01 (FERM BP-11369), R7-02 (FERM BP-11404), R7- 05 (FERM BP-11371), R7-09 (FERM BP-11372), R7-11 (FERM BP-11373), R7-18 (FERM BP-11374), R7-25 (FERM BP-11375), or R7 It is an anti-human CCR7 antibody produced by -47 (FERM BP-11376).
- Another aspect of the present invention is an anti-human CCR7 antibody characterized by binding to the same epitope as the above-mentioned anti-human CCR7 antibody.
- Still other aspects of the present invention include R7-01 (FERM BP-11369), R7-02 (FERM BP-11404), R7-05 (FERM BP-11371), R7-09 (FERM BP-11372), R7 -11 (FERM BP-11373), R7-18 (FERM BP-11374), R7-25 (FERM BP-11375), or R7-47 (FERM BP-11376).
- Still another aspect of the present invention is a nucleic acid encoding the heavy chain variable region or the light chain variable region of the anti-human CCR7 antibody of the present invention.
- SEQ ID NO: 1 SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 51, SEQ ID NO: 53 , SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 71, or SEQ ID NO: 73.
- Still another aspect of the present invention is a vector containing the nucleic acid.
- Still another aspect of the present invention is a cell into which the above vector has been introduced.
- Still another aspect of the present invention is a pharmaceutical composition
- a pharmaceutical composition comprising the above-described anti-human CCR7 antibody of the present invention and a pharmaceutically acceptable carrier.
- the CCR7-dependent intracellular information transmission mechanism by the CCR7 ligand is blocked.
- it is used to treat tissue fibrosis.
- the fibrosis of the tissue is selected from the group consisting of liver fibrosis, renal fibrosis, pulmonary fibrosis, dermal fibrosis, cardiovascular fibrosis, gastrointestinal fibrosis and other fibrotic diseases It is.
- the liver fibrosis is from the group consisting of cirrhosis, ischemia reperfusion, injury after liver transplantation, necrotic hepatitis, hepatitis B, hepatitis C, primary biliary cirrhosis, and primary sclerosing cholangitis Selected.
- the cirrhosis is caused by at least one selected from the group consisting of alcohol induction, drug induction, and chemical induction.
- the renal fibrosis is proliferative glomerulonephritis, sclerosing glomerulonephritis, renal fibrosis dermatosis, diabetic nephropathy, renal tubulointerstitial fibrosis, and focal segmental glomeruli It is selected from the group consisting of sclerosis.
- the pulmonary fibrosis is pulmonary interstitial fibrosis, drug-induced sarcoidosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, diffuse alveolar injury disease, pulmonary hypertension, and It is selected from the group consisting of neonatal bronchopulmonary malformations.
- the dermatofibrosis is selected from the group consisting of scleroderma, keloid scarring, psoriasis, hypertrophic scarring, and pseudoscleroderma.
- the cardiovascular fibrosis is selected from the group consisting of atherosclerosis, coronary restenosis, congestive cardiomyopathy, heart failure, heart transplantation, and myocardial fibrosis.
- the gastrointestinal fibrosis is selected from the group consisting of collagenous colitis, villous atrophy, crypt hyperplasia, polyp formation, Crohn's disease fibrosis, gastric ulcer healing, and scar after abdominal adhesion surgery. is there.
- the fibrosis has a state resulting from a fibrotic disease associated with bone, and is rheumatoid pannus formation.
- it is used to treat cancer metastasis.
- the cancer is selected from the group consisting of pharyngeal cancer, chondrosarcoma, colon cancer, pancreatic cancer, leukemia, and breast cancer.
- Still another aspect of the present invention is an antibody-immobilized carrier in which the above-described anti-human CCR7 antibody of the present invention is immobilized on a carrier.
- it is used for removing CCR7-expressing cells from the body fluid by contacting blood containing CCR7-expressing cells.
- the anti-human CCR7 antibody of the present invention it is possible to provide a novel drug for fibrosis that is difficult to treat, lymphoid cancer metastasis, and the like.
- an anti-human CCR7 antibody can be produced as an active ingredient of a novel pharmaceutical agent for fibrosis and lymphoid cancer metastasis which are difficult to treat.
- the antibody of the present invention can be produced by recombinant technology. It can also be applied to gene therapy.
- the vector of the present invention and the antibody of the present invention can be produced by recombinant technology. It can also be applied to gene therapy.
- composition of the present invention it is possible to provide a novel pharmaceutical for fibrosis that is difficult to treat, lymphoid cancer metastasis, and the like.
- CCR7-expressing cells can be selectively removed from the blood of a patient suffering from fibrosis or cancer.
- FIG. 5 is a two-dimensional dot display diagram and a histogram showing the analysis results of the interaction with R7-02-derived recombinant anti-human CCR7 antibody.
- FIG. 6 is a two-dimensional dot display diagram and a histogram showing the analysis results of the interaction with the recombinant anti-human CCR7 antibody derived from R7-11.
- FIG. 2 is a two-dimensional dot display diagram and a histogram showing the analysis results of the interaction with R7-18-derived recombinant anti-human CCR7 antibody.
- CCR7 that is specifically recognized by the antibody of the present invention will be described focusing on its structure.
- CCR7 is a kind of G protein-coupled receptor (GPCR), and is present seven times through the cell membrane, with its N-terminus directed outside the cell and the C-terminus directed into the cell.
- GPCR G protein-coupled receptor
- a gene (cDNA) encoding human CCR7 has already been isolated, and the amino acid sequence of human CCR7 is also known.
- the sequence information can be obtained from a database such as GenBank (for example, GenBank: EAW60669.1).
- GenBank for example, GenBank: EAW60669.1
- SEQ ID NO: 81 shows the base sequence of the human CCR7 gene and the amino acid sequence corresponding to the base sequence
- SEQ ID NO: 82 shows only the amino acid sequence.
- Each domain of human CCR7 is considered to correspond to the following part of the amino acid sequence shown in SEQ ID NO: 82.
- the left side is the amino acid number
- the right side is each domain.
- some front and back may arise.
- 1 to 24 membrane translocation signal peptide sequence (cleaved and removed after expression) 25 to 59: N-terminal domain 87 to 95: intracellular first loop domain 117 to 130: extracellular first loop domain 153 to 170: intracellular second loop domain 192 to 219: extracellular second loop domain 248 to 263 : Intracellular third loop domain 290 to 313: extracellular third loop domain 332 to 378: C-terminal domain
- Human CCR7 various variants such as amino acid substitutions other than the one shown in SEQ ID NO: 82 are known. “Human CCR7” in the present invention includes the variants as long as it has an extracellular domain and functions as CCR7.
- the anti-human CCR7 antibody of the present invention specifically binds to the extracellular domain of human CCR7.
- the antibody of the present invention is represented by SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, or SEQ ID NO: 77. It has a heavy chain complementarity determining region 3 (heavy chain CDR3) containing the amino acid sequence represented.
- the antibody of the present invention satisfies any of the following (A1) to (A8) with respect to the amino acid sequence of its complementarity determining regions 1 to 3 (CDR1 to 3).
- A1 a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 5, Having a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 6 and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 7,
- A2 a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 15, Having a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 16 and a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 17,
- A3 a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 25, Having a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 26 and a heavy chain CDR3 comprising the amino acid sequence represented by
- the antibody of the present invention satisfies any of the following (B1) to (B8) with respect to the amino acid sequence of its complementarity determining regions 1 to 3 (CDR1 to 3).
- B1 a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 5, Heavy chain CDR2, comprising the amino acid sequence represented by SEQ ID NO: 6,
- a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 7
- a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 8
- (B2) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 15, Heavy chain CDR2, comprising the amino acid sequence represented by SEQ ID NO: 16, Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 17, A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 18, Having a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 19 and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 20, (B3) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 25, A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 26; Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 27, A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 28, A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 29 and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 30;
- the antibody of the present invention has its heavy chain variable region (hereinafter abbreviated as “VH”) and light chain variable region (hereinafter abbreviated as “VL”).
- the amino acid sequence satisfies any of the following (C1) to (C8).
- C2 having a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 12, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 14.
- SEQ ID NO: 5 (heavy chain CDR1), SEQ ID NO: 6 (heavy chain CDR2), SEQ ID NO: 7 (heavy chain CDR3) are amino acid numbers 27 to 35, 50 to 66, 97 to 112 of SEQ ID NO: 2 (VH), respectively.
- FIG. 1B ).
- SEQ ID NO: 15 (heavy chain CDR1), SEQ ID NO: 16 (heavy chain CDR2), SEQ ID NO: 17 (heavy chain CDR3) are amino acid numbers 43 to 51, 66 to 82, 113 to 124 of SEQ ID NO: 12 (VH), respectively.
- FIG. 2B ).
- SEQ ID NO: 25 (heavy chain CDR1), SEQ ID NO: 26 (heavy chain CDR2), SEQ ID NO: 27 (heavy chain CDR3) are amino acid numbers 30 to 38, 53 to 68, 100 to 111 of SEQ ID NO: 22 (VH), respectively.
- FIG. 3B ).
- SEQ ID NO: 35 (heavy chain CDR1), SEQ ID NO: 36 (heavy chain CDR2), SEQ ID NO: 37 (heavy chain CDR3) are amino acid numbers 28 to 36, 51 to 67, 98 to 109 of SEQ ID NO: 32 (VH), respectively.
- FIG. 4B ).
- SEQ ID NO: 45 (heavy chain CDR1), SEQ ID NO: 46 (heavy chain CDR2), SEQ ID NO: 47 (heavy chain CDR3) are amino acid numbers 29 to 37, 52 to 68, 98 to 110 of SEQ ID NO: 42 (VH), respectively.
- SEQ ID NO: 55 (heavy chain CDR1)
- SEQ ID NO: 56 (heavy chain CDR2)
- SEQ ID NO: 57 (heavy chain CDR3) are amino acid numbers 30 to 37, 53 to 69, 99 to 111 of SEQ ID NO: 52 (VH), respectively.
- SEQ ID NO: 58 (light chain CDR1)
- SEQ ID NO: 59 (light chain CDR2)
- SEQ ID NO: 60 are amino acid numbers 24-39, 55-61, 94-102 of SEQ ID NO: 54 (VL), respectively.
- FIG. 6B ).
- SEQ ID NO: 65 (heavy chain CDR1), SEQ ID NO: 66 (heavy chain CDR2), SEQ ID NO: 67 (heavy chain CDR3) are amino acid numbers 30 to 41, 54 to 69, and 100 to 111 of SEQ ID NO: 62 (VH), respectively. This corresponds to the part (FIG. 7A).
- FIG. 7B ).
- SEQ ID NO: 75 (heavy chain CDR1), SEQ ID NO: 76 (heavy chain CDR2), SEQ ID NO: 77 (heavy chain CDR3) are amino acid numbers 27 to 35, 50 to 66, 96 to 109 of SEQ ID NO: 72 (VH), respectively.
- SEQ ID NO: 78 (light chain CDR1), SEQ ID NO: 79 (light chain CDR2), SEQ ID NO: 80 (light chain CDR3) are amino acid numbers 24 to 39, 55 to 61, 94 to 102 of SEQ ID NO: 74 (VL), respectively. This corresponds to the part (FIG. 8B).
- antibody can be replaced with “immunoglobulin”.
- the antibody in the present invention includes a functional fragment thereof.
- the “functional fragment of an antibody” refers to a partial fragment of an antibody (ie, an immunoglobulin) that retains at least one action on an antigen.
- the partial fragments include F (ab ′) 2 , Fab, Fv, disulfide bond Fv, single chain antibody (scFv, VH-VL), VH, and polymers thereof, and heavy chain CH 3 Examples include fusions with regions.
- each CDR such as CDR1, CDR2, and CDR3, and a conjugate of these CDRs, and a fusion of these CDRs or CDR conjugates with a heavy chain CH3 region can be mentioned. That is, the antibody of the present invention includes partial fragments of the antibody as described above as long as it specifically binds to the extracellular domain of human CCR7.
- a partial fragment of an antibody is sometimes referred to as an “antibody fragment”.
- the antibody of the present invention may be a multispecific antibody.
- Diabody International Publication No. 93/11161 pamphlet
- bispecific antibody can be mentioned.
- the antibody of the present invention is a functional fragment
- the following effects are obtained. That is, when the anti-human CCR7 antibody of the present invention is applied to a medicine as described later, if a full-length antibody such as IgG type is used, in addition to signal inhibition of the target receptor, damage to the target tissue occurs. It may lead to side effects. In such a case, if the “functional fragment of an antibody” using only the variable region is employed, the above-mentioned side effects can be easily avoided.
- the class (isotype) of the antibody of the present invention is not particularly limited.
- any class such as IgG, IgM, IgA, IgD, and IgE may be used.
- the subclass of the antibody of the present invention is not particularly limited.
- any subclass such as IgG1, IgG2, and IgG3 may be used as long as it is IgG.
- it has an activity of blocking a CCR7-dependent intracellular signal transduction mechanism by CCR7 ligand stimulation.
- Still other aspects (fifth aspect) of the antibodies of the present invention are R7-01 (FERM BP-11369), R7-02 (FERM BP-11404), R7-05 (FERM BP-11371), R7-09. (FERM BP-11372), R7-11 (FERM BP-11373), R7-18 (FERM BP-11374), R7-25 (FERM BP-11375), or R7-47 (FERM BP-11376) Anti-human CCR7 antibody.
- the antibodies produced by these 8 hybridomas specifically bind to the extracellular domain of human CCR7.
- VH heavy chain variable regions
- VL light chain variable regions
- Table 1 summarizes the relationship between the sequence numbers of amino acid sequences of VH, VL, and CDRs and the corresponding clones.
- the extracellular domain of human CCR7 to which the antibody of the present invention specifically binds may be any of an N-terminal domain, an extracellular first loop domain, an extracellular second loop domain, and an extracellular third loop domain.
- the antibody of the present invention may bind to only one of these extracellular domains, or may bind to two or more.
- the antibody of the present invention also includes an anti-human CCR7 antibody that binds to the same epitope as the anti-human CCR7 antibody according to the first to fifth aspects described above.
- an anti-human CCR7 antibody having a CDR “functionally equivalent” to the CDR of the anti-human CCR7 antibody according to the first to fifth aspects described above is also included.
- the epitopes of 8 types of anti-human CCR7 antibodies specifically shown in the examples described later are analyzed by an epitope mapping method using a partial peptide of human CCR7.
- an anti-human CCR7 antibody that binds to the same epitope as the above-mentioned eight types of anti-human CCR7 antibodies can be obtained. Furthermore, the amino acid sequences of the heavy chain variable region and the light chain variable region of the obtained anti-human CCR7 antibody can be determined, and the amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 can be identified. Examples of “functionally equivalent” CDR amino acid sequences in the anti-human CCR7 antibody include the original amino acid sequences (SEQ ID NOs: 5 to 10, 15 to 20, 25 to 30, 35 to 40, and 45 to 50).
- one or several, preferably 1 to 5, more preferably 1 to 3, more preferably 1 amino acid has been deleted, substituted or added.
- examples thereof include amino acid sequences and CDRs having an equivalent function.
- Another example includes an amino acid sequence having a homology with the original amino acid sequence of 80% or more, preferably 90% or more, more preferably 95% or more, and having an equivalent function as a CDR.
- a method for examining whether or not two antibodies have the same epitope a method based on a competition experiment can be mentioned. For example, when the binding between the eight anti-human CCR7 antibodies as the first antibody and the receptor is competitively inhibited by the second antibody to be tested, the second antibody is the same as the first antibody. It can be said that it binds to an epitope.
- the nucleic acid of the present invention encodes the heavy chain variable region (VH) or the light chain variable region (VL) in the antibody of the present invention. That is, according to the first aspect, the sequence includes the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, or SEQ ID NO: 77.
- a nucleic acid encoding VH or VL of an anti-human CCR7 antibody having chain CDR3 is included in the nucleic acid of the present invention.
- nucleic acid encoding VH or VL that satisfies any of the above (A1) to (A8) according to the second aspect is included in the nucleic acid of the present invention.
- a nucleic acid encoding VH or VL that satisfies any of the above (B1) to (B8) according to the third aspect is included in the nucleic acid of the present invention.
- a nucleic acid encoding VH or VL that satisfies any of the above (C1) to (C8) according to the fourth aspect is included in the nucleic acid of the present invention.
- nucleic acids include SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: And a nucleic acid having the base sequence represented by SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 71, or SEQ ID NO: 73.
- the nucleic acid of the present invention can also be obtained from the above eight hybridomas using PCR or the like.
- the vector of the present invention contains the nucleic acid of the present invention.
- the type of vector is not particularly limited, and may be appropriately selected depending on the type of host cell introduced thereafter.
- the vector of the present invention includes a gene therapy vector. In this case, the vector itself can be administered directly into the living body.
- the cell of the present invention is a cell into which the vector of the present invention has been introduced.
- the type of cell is not particularly limited as long as the introduced vector functions. Examples include animal cells (COS cells, CHO cells, etc.), yeasts, bacteria (E. coli etc.), plant cells, insect cells, and the like.
- the antibody of the present invention can be produced, for example, as follows.
- any of the above eight hybridomas can be cultured, and the antibody of the present invention can be produced from the culture.
- a culture method a method generally employed as a hybridoma culture method can be applied as it is.
- the hybridoma is cultured using a medium for animal cells such as DMEM and RPMI 1640, and the antibody of the present invention can be obtained from the culture supernatant.
- ascites can be collected and the antibody of the present invention can be obtained from the ascites.
- the antibody of the present invention can also be produced using a gene recombination technique.
- a gene recombination technique In particular, when producing a chimeric antibody, a humanized antibody, a functional fragment of an antibody, etc., it is generally produced by a gene recombination technique.
- a method for producing an antibody having a heavy chain variable region and a light chain variable region satisfying any of the above (C1) to (C8) according to the fourth aspect will be described taking production of a chimeric antibody as an example.
- the term “chimeric antibody” as used herein refers to a heavy chain variable region (VH) and a light chain variable region (VL) derived from animals other than humans, and includes a heavy chain constant region (CH) and a light chain constant region (CL). Refers to an antibody in which other regions are of human origin.
- DNA encoding the amino acid sequence (VH) shown in SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: 62, or SEQ ID NO: 72 is prepared.
- DNA encoding the amino acid sequence (VL) shown in SEQ ID NO: 4, SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 34, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 64, or SEQ ID NO: 74 is prepared.
- DNAs include SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 51, Examples are shown in SEQ ID NO: 53, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 71, or SEQ ID NO: 73, but other base sequences may be used.
- the DNA can be prepared using a known method such as PCR.
- the DNA can also be prepared by chemical synthesis.
- the obtained DNA encoding VH or VL is inserted into a vector having a sequence encoding human antibody CH or CL, respectively, to construct a chimeric antibody expression vector.
- a vector having a sequence encoding CH or CL of a human antibody can be obtained from the market.
- a recombinant cell expressing the chimeric antibody is obtained.
- this recombinant cell is cultured, and a desired chimeric antibody is obtained from the culture.
- the host cell is not particularly limited as long as the expression vector can function.
- Animal cells such as COS cells and CHO cells
- yeasts such as E. coli
- plant cells such as E. coli
- insect cells as described above can be appropriately employed.
- the humanized antibody refers to an antibody in which CDR is derived from an animal other than human and other regions (such as framework region and constant region) are derived from human.
- DNAs encoding heavy chain CDR1-3 and light chain CDR1-3 SEQ ID NO: 5-10, SEQ ID NO: 15-20, SEQ ID NO: 25-30, SEQ ID NO: 35-40, SEQ ID NO: 45-50, DNAs encoding the amino acid sequences shown in Nos. 55 to 60, SEQ ID Nos. 65 to 70, or SEQ ID Nos. 75 to 80 are prepared.
- the DNA includes SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 51, SEQ ID NO: Examples of the sequence corresponding to each CDR in the nucleotide sequence shown in No. 53, SEQ ID No. 61, SEQ ID No. 63, SEQ ID No. 71, or SEQ ID No. 73 are exemplified, but other nucleotide sequences may be used.
- the DNA can be prepared using a known method such as PCR.
- the DNA can also be prepared by chemical synthesis.
- DNA encoding a variable region in which heavy chain CDRs 1 to 3 are grafted on the framework region (FR) of VH in any human antibody is prepared.
- DNA encoding a variable region in which light chain CDRs 1 to 3 are grafted on FR of VL in any human antibody is prepared.
- Each prepared DNA is inserted into a vector having a sequence encoding human antibody CH or CL to construct a humanized antibody expression vector.
- a recombinant cell expressing a humanized antibody is obtained.
- this recombinant cell is cultured, and a desired humanized antibody is obtained from the culture.
- An antibody having a specific CDR that satisfies any of the above (A1) to (A8) according to the second aspect can also be produced by the same procedure.
- Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67, or SEQ ID NO: 77 according to the first aspect
- An anti-human CCR7 antibody having a can also be produced by the same procedure.
- the method for purifying the antibody of the present invention is not particularly limited, and a known method can be employed.
- the culture supernatant of the hybridoma or the recombinant cell can be collected, and the antibody of the present invention can be purified by combining various known techniques such as chromatography, salting out, dialysis, membrane separation and the like.
- the antibody isotype is IgG, it can be easily purified by affinity chromatography using protein A.
- the hybridoma of the present invention has been selected and obtained using a known hybridoma production technique, as will be described in detail in Examples below.
- a gene immunization technique may be used.
- a fusion gene in which the CCR7 gene is linked to the GroEL gene, which is an E. coli chaperonin, as an immunogen antibody production may be facilitated. Details of the gene immunization technique are described in International Publication No. 2006/041157.
- the antibody of the present invention is useful as an active ingredient of a pharmaceutical composition (therapeutic agent).
- the pharmaceutical composition of the present invention comprises the anti-human CCR7 antibody of the present invention and a pharmaceutically acceptable carrier.
- a pharmaceutically acceptable carrier Preferably, the CCR7-dependent intracellular signal transduction mechanism by the CCR7 ligand is blocked.
- the pharmaceutical composition of the invention is used to treat tissue fibrosis.
- tissue fibrosis include fibrosis selected from the group consisting of liver fibrosis, renal fibrosis, pulmonary fibrosis, dermal fibrosis, cardiovascular fibrosis, gastrointestinal fibrosis and other fibrotic diseases. Can be mentioned.
- liver fibrosis are from the group consisting of cirrhosis, ischemia reperfusion, injury after liver transplantation, necrotic hepatitis, hepatitis B, hepatitis C, primary biliary cirrhosis, and primary sclerosing cholangitis
- liver fibrosis include selected liver fibrosis.
- Cirrhosis may be caused by at least one selected from the group consisting of alcohol induction, drug induction, and chemical induction.
- renal fibrosis include proliferative glomerulonephritis, sclerosing glomerulonephritis, renal fibrosis dermatosis, diabetic nephropathy, renal tubular interstitial fibrosis, and focal segmental glomeruli
- renal fibrosis selected from the group consisting of sclerosis.
- pulmonary fibrosis examples include pulmonary interstitial fibrosis, drug-induced sarcoidosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, asthma, chronic obstructive pulmonary disease, diffuse alveolar injury disease, pulmonary hypertension, and Examples include pulmonary fibrosis selected from the group consisting of neonatal bronchopulmonary malformations.
- dermatofibrosis examples include dermatofibrosis selected from the group consisting of scleroderma, keloid scarring, psoriasis, hypertrophic scarring, and pseudoscleroderma.
- cardiovascular fibrosis examples include cardiovascular fibrosis selected from the group consisting of atherosclerosis, coronary restenosis, congestive cardiomyopathy, heart failure, heart transplantation, and myocardial fibrosis.
- cardiovascular fibrosis include a gastrointestinal tract selected from the group consisting of collagen accumulation colitis, villous atrophy, crypt hyperplasia, polyp formation, Crohn's disease fibrosis, gastric ulcer healing, and scar after abdominal adhesion surgery Fibrosis can be mentioned.
- the fibrosis has a state resulting from a fibrotic disease related to bone, and may be rheumatoid pannus formation.
- the pharmaceutical composition of the present invention can also be used to treat cancer metastasis.
- cancer include cancer selected from the group consisting of pharyngeal cancer, chondrosarcoma, colon cancer, pancreatic cancer, leukemia, and breast cancer.
- the pharmaceutical composition of the present invention can be administered orally or parenterally, systemically or locally.
- administration forms include injection, nasal administration, pulmonary administration, and transdermal administration.
- an injection form it can be administered systemically or locally by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection and the like.
- an administration method can be appropriately selected depending on the age and symptoms of the patient.
- the dose of the antibody of the present invention can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight per time. Alternatively, for example, the dose can be selected within the range of 0.001 to 100,000 mg / body of antibody per patient. However, the dose of the antibody of the present invention is not limited to these ranges.
- a pharmaceutical composition containing the antibody of the present invention can be formulated according to a conventional method (for example, Remington's Pharmaceutical Science, Latest Edition, Mark Publishing Company, Easton, USA).
- the pharmaceutical composition of the present invention contains pharmaceutically acceptable carriers and additives.
- the carrier or the additive include surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), coloring agents, flavoring agents, preservatives, stabilizers, buffering agents (phosphorus). Acid, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonic agents, binders, disintegrating agents, lubricants, fluidity promoters, corrigents and the like.
- the present invention is not limited to these, and other commonly used carriers can be used as appropriate.
- other low molecular weight polypeptides, serum albumin, proteins such as gelatin and immunoglobulin, and amino acids such as glycine, glutamine, asparagine, arginine and lysine may be contained.
- aqueous solution for injection for example, isotonic solutions containing physiological saline, glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, etc.
- Adjuvants such as alcohols (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), nonionic surfactants (polysorbate 80, HCO-50), etc. may be used in combination.
- the antibody of the present invention can be encapsulated in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid]) or colloid drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles). And nanocapsules, etc. (see “Remingto's Pharmaceutical Science 16th edition", Oslo Ed. (1980), etc.).
- a technique for enhancing the therapeutic effect by directly fusing an antibody with another drug such as an antifibrotic agent, a low-molecular anticancer agent, or a cytokine
- another drug such as an antifibrotic agent, a low-molecular anticancer agent, or a cytokine
- a gene encoding the antibody of the present invention into a gene therapy vector to make a gene therapy drug.
- a method of packaging and administering to liposomes, retrovirus vector, adenovirus vector, vaccinia virus vector, poxvirus vector Method of administration by incorporating into various virus vectors such as adeno-associated virus vector and HVJ vector (see Adolph “Virus Genome Method”, CRC Press, Florida (1996)), colloidal gold particles and other coated beads (International Publication No. 93) / 17706 pamphlet etc.) and the like.
- the gene therapy drug may be administered by any method as long as the antibody of the present invention is expressed in vivo and can exert its action.
- suitable parenteral routes intravenous, intraperitoneal, subcutaneous, intradermal, adipose tissue, intramammary tissue, inhalation or intramuscular route, injection, infusion, or gas-induced particle bombardment (electronic A sufficient amount is administered by a method such as a gun), a method via a mucosal route such as an nasal drug, and the like.
- the gene therapy drug is administered ex vivo to cells using liposome transfection, particle bombardment (US Pat. No. 4,945,050), or viral infection, and the cells are reintroduced into the animal. May be administered.
- the present invention also includes a therapeutic method and a therapeutic agent relating to a disease caused by abnormal increase in CCR7 signal or a diseased mammal.
- treatment means to prevent or alleviate the progression and worsening of the disease state in a mammal that is likely to suffer from or suffers from the disease. Used to mean therapeutic treatment aimed at preventing or alleviating progression and worsening.
- the term “disease” means any disease that develops due to abnormal increase in CCR7 signal, and is not particularly limited.
- liver fibrosis, renal fibrosis, pulmonary fibrosis, skin It is a concept that includes fibrosis, cardiovascular fibrosis, gastrointestinal fibrosis and other fibrotic diseases.
- the concept also includes cancer metastasis originating in pharyngeal cancer, chondrosarcoma, colon cancer, pancreatic cancer, leukemia, and breast cancer.
- the “mammal” to be treated means any animal classified as a mammal, and is not particularly limited.
- pet animals such as dogs, cats, rabbits, cows, pigs, sheep , Livestock animals such as horses. Particularly preferred “mammals” are humans.
- the antibody-immobilized carrier of the present invention is obtained by immobilizing the anti-human CCR7 antibody of the present invention on a carrier.
- the antibody-immobilized carrier of the present invention is used for removing CCR7-expressing cells from the body fluid by contacting blood containing CCR7-expressing cells.
- the anti-human CCR7 antibody immobilized on the carrier may be only one type or two or more types.
- the antibody-immobilized carrier of the present invention include those in which the antibody of the present invention is immobilized on a water-insoluble carrier and filled in a container.
- any material can be used as the water-insoluble carrier, but polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, and polycarbonate are listed as preferable in view of low moldability, sterilization, and cytotoxicity.
- Synthetic polymers such as polyacrylamide and polyurethane, natural polymers such as agarose, cellulose, cellulose acetate, chitin, chitosan and alginate, inorganic materials such as hydroxyapatite, glass, alumina and titania, metal materials such as stainless steel and titanium Is mentioned.
- Examples of the shape of the carrier include granular, cotton-like, woven fabric, non-woven fabric, sponge-like porous body, flat plate shape, etc., but in terms of large surface area per volume, granular, cotton-like, woven fabric, non-woven fabric, A sponge-like porous body is preferred.
- CCR7-expressing cells associated with a disease can be efficiently removed by allowing peripheral blood to pass through a porous filter previously filled in a container with a water-insoluble carrier to which an antibody is immobilized.
- a CCR7-expressing cell removal kit can be prepared by combining the antibody-immobilized carrier of the present invention and other components.
- the other components include an anticoagulant and an extracorporeal circuit.
- the present invention includes the following anti-human CCR7 antibodies (1) to (5).
- An anti-human CCR7 antibody that specifically binds to the extracellular domain of human CCR7, comprising SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 57, SEQ ID NO: 67
- an anti-human CCR7 antibody comprising a heavy chain complementarity determining region 3 (heavy chain CDR3) comprising the amino acid sequence represented by SEQ ID NO: 77.
- the amino acid sequence of the complementarity determining regions 1 to 3 (CDR1 to 3) satisfies any of the following (B1) to (B8), and is described in (1) or (2) above Anti-human CCR7 antibody.
- B1 a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 5, Heavy chain CDR2, comprising the amino acid sequence represented by SEQ ID NO: 6, A heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 7, A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 8, Having a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 9 and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 10.
- (B2) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 15, Heavy chain CDR2, comprising the amino acid sequence represented by SEQ ID NO: 16, Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 17, A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 18, Having a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 19 and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 20, (B3) a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 25, A heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 26; Heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 27, A light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 28, A light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 29 and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 30;
- the amino acid sequences of the heavy chain variable region and the light chain variable region satisfy any of the following (C1) to (C8), according to any one of (1) to (3) above: Anti-human CCR7 antibody.
- the present invention includes a method for treating tissue fibrosis or cancer metastasis, to which an effective amount of the anti-human CCR7 antibody is administered.
- tissue fibrosis and cancer are as described above.
- the present invention includes the use of the anti-human CCR7 antibody for producing a therapeutic agent for tissue fibrosis or cancer metastasis.
- the present invention also includes the anti-human CCR7 antibody for the treatment of tissue fibrosis or cancer metastasis. Specific examples of tissue fibrosis and cancer are as described above.
- DNA fragment A the gene fragment prepared by cleaving the obtained vector at the NheI and SalI sites
- DNA fragment B the gene fragment prepared by cleaving at the NheI and EcoRI sites
- Genomic DNA was extracted and purified from Escherichia coli HMS174 (DE3) strain (Novagen). Next, PCR was performed using the purified genomic DNA as a template and the oligonucleotide having the nucleotide sequences shown in SEQ ID NOs: 84 and 85 as a primer pair, and a DNA fragment containing the GroEL subunit gene having the nucleotide sequence shown in SEQ ID NO: 86 ( Hereinafter, it is referred to as “DNA fragment C”). In DNA fragment C, a sequence encoding a SalI site at the 5 ′ end and two stop codons (TAATAG) at the 3 ′ end and a NotI site were introduced from the primer.
- TATAG two stop codons
- pCIneo-hCCR7 37.5 ⁇ L of a Lipofectamine solution, 625 ⁇ L of OPTI-MEMI medium, and 625 ⁇ L of OPTI-MEMI medium containing 20 ⁇ g of pCIneo-hCCR7 were mixed. Using this mixed solution, pCIneo-hCCR7 was introduced into 2 ⁇ 10 5 CHO-K1 cells (Dainippon Pharmaceutical Co., Ltd.). As a control, only pCIneo was introduced into CHO-K1 cells. Each CHO-K1 cell into which the gene was introduced was cultured in Ham's F12K + 10% FBS medium (ICN) for 30 hours.
- ICN FBS medium
- each cell was detached, suspended, seeded with 5 ⁇ 10 5 cells in a 100 mm dish, and treated with Ham's F12K + 10% FBS medium containing antibiotic G418 (Promega) at a concentration of 0.8 mg / mL for 2 weeks. Was done. After drug treatment, G418 resistant cells were cloned by limiting dilution. Furthermore, in order to increase the Ca signal response, pCEP-G ⁇ 16 (Molecular Devices) was introduced into the cloned cells, and the antibiotic hygromycin was similarly treated with a drug at a concentration of 0.2 mg / mL. Thereafter, hygromycin resistant cells were further cloned.
- G418 Promega
- each cell was washed with PBS, phycoerythrin-labeled anti-mouse IgG antibody (Beckman Coulter) was added as a secondary antibody, and each cell and serum were then used using a flow cytometer FACSCalibur (Becton Dickinson). The interaction with anti-human CCR7 antibody was analyzed.
- Booster immunization was performed on 6 mice genetically immunized by the same procedure as in (5) above. Three days after the booster, the spleen was removed and spleen cells were prepared. By the PEG method, 1 ⁇ 10 8 spleen cells and 1 ⁇ 10 7 BALB / C mouse-derived HAT-selective myeloma SP2 / 0 cells were fused (cell fusion). A population of fused cells (hybridoma) was suspended in RPMI medium and seeded in each well of 14 96-well microplates. At this stage, about 990 hybridomas were obtained.
- the medium in the microplate was replaced with RPMI medium supplemented with HAT Media Supplement (50 ⁇ ) (Sigma, product number: H0262) at a frequency of once every 3 days for 2 weeks from the day after cell fusion.
- Antibody binding was evaluated by the same flow cytometry as in (6) above, and the binding between the human CCR7 gene-introduced cells and the antibody in the hybridoma culture supernatant in each well was examined. As a result, the binding to the antibody was confirmed in the 8 holes. Eight types of hybridomas that were confirmed to be bound to antibodies were cloned by limiting dilution.
- hybridomas were seeded in a 96-well microplate so that there were 1 cell / hole or less and cultured. Two weeks later, the same flow cytometry was performed, and the binding of the antibody (anti-human CCR7 monoclonal antibody) in the cloned culture supernatant was confirmed. As a result, 8 hybridomas producing anti-human CCR7 monoclonal antibody were cloned.
- Each hybridoma was cultured in 100 mL of RPMI medium for 2 weeks. Each culture supernatant was subjected to protein G column (Amersham Bioscience) and purified and concentrated to obtain about 20 mg of each of the 8 types of purified anti-human CCR7 monoclonal antibodies.
- Each hybridoma was deposited with IPOD.
- the display of each hybridoma and the accession number are as described above.
- anti-human CCR7 antibody Each anti-human CCR7 monoclonal antibody (hereinafter simply referred to as “anti-human CCR7 antibody”) was subjected to the following tests.
- hybridoma display is also used as the antibody name.
- FIGS. 9 is a histogram showing the analysis results of the interaction between human CCR7 gene-introduced cells and mouse control IgG.
- FIG. 10 is a histogram showing the analysis results of the interaction between human CCR7 gene-introduced cells and anti-human CCR7 antibody R7-47.
- Table 2 shows the ratio of specific binding cells calculated from the analysis results of the interaction between human CCR7 gene-introduced cells and each anti-human CCR7 antibody. 9 and 10, the vertical axis represents the number of cells, and the horizontal axis represents the fluorescence intensity derived from phycoerythrin (PE). Moreover, the cell which belongs to M2 (right area
- FIG. 11 is a graph showing the relationship between the intracellular Ca 2+ concentration and the concentration of each additive. As shown in FIG. 11, a decrease in intracellular Ca 2+ concentration was observed depending on the concentration of R7-47. This indicated that R7-47 competitively inhibited the binding between CCL21 and human CCR7, resulting in inhibition of signal transduction into the cell. When the 50% inhibitory concentration (IC50) was calculated, it was 7.4 nM for R7-47.
- IC50 50% inhibitory concentration
- Table 3 shows the IC50 of anti-human CCR7 antibodies other than R7-47. From the above, it was shown that all of the obtained anti-human CCR7 antibodies can block the CCR7-dependent intracellular signal transduction mechanism by the human CCR7 ligand.
- cDNA cloning of antibody variable region and determination of complementarity determining region From the above 8 hybridomas, DNAs encoding the variable regions of the L chain and H chain of each antibody were cloned, and the nucleotide sequence was determined. Were determined. Cloning was performed as follows. First, RNA was isolated from the hybridoma using RNeasyMini Kit (QIAGEN). Next, using “SMARTer-RACE cDNA Amplification Kit” (Takara Bio Inc.), cDNA synthesis by 5′-RACE method and PCR products were obtained according to the manufacturer's manual.
- the sequence of SEQ ID NO: 87 was used for the ⁇ chain, the sequence of SEQ ID NOs: 88 to 90 for the ⁇ chain, and the sequence of SEQ ID NO: 91 for the ⁇ chain.
- the obtained DNA fragment was inserted into pT7 Blue Vector (Novagen) into DNA Ligation ver.2 (Takara Bio).
- XL10 Gold (Stratagene) was transformed with this vector, seeded on a plate containing X-gal, ampicillin, and IPTG, and white colonies were picked up.
- the DNA sequence was determined using an ABI PRISM 3130 type automatic sequencer. Since the determined sequence showed the same sequence in all three clones except that some mutations that were thought to be due to PCR errors were observed, the sequence was used as the target DNA sequence.
- the amino acid sequence corresponding to the obtained base sequence is represented by SEQ ID NO: 1 (VH of R7-01), SEQ ID NO: 3 (VL of R7-01), SEQ ID NO: 11 (VH of R7-02), SEQ ID NO: 13 (R7 -02), SEQ ID NO: 21 (V7 of R7-05), SEQ ID NO: 23 (VL of R7-05), SEQ ID NO: 31 (VH of R7-09), SEQ ID NO: 33 (VL of R7-09), SEQ ID NOs: 41 (VH of R7-11) and 43 (VL of R7-11), SEQ ID NO: 51 (VH of R7-18), SEQ ID NO: 53 (VL of R7-18), SEQ ID NO: 61 (V7 of R7-25) VH), SEQ ID NO: 63 (VL of R7-25), SEQ ID NO: 71 (VH of R7-47), SEQ ID NO: 73 (VL of R7-47).
- SEQ ID NO: 2 (VH of R7-01), SEQ ID NO: 4 (VL of R7-01), SEQ ID NO: 12 (VH of R7-02), SEQ ID NO: 14 (R7- 02, VL), SEQ ID NO: 22 (VH of R7-05), SEQ ID NO: 24 (VL of R7-05), SEQ ID NO: 32 (VH of R7-09), SEQ ID NO: 34 (VL of R7-09), SEQ ID NO: Nos.
- CDRs 1 to 3 were identified for each VH and VL (SEQ ID NOs: 5 to 10, 15 to 20, 25 to 30, 35 to 40, 45 to 50, 55 to 60, 65 to 70, 75 to 80). See Figures 1-9 and Table 1.
- PBMC Mononuclear cells
- PBMC peripheral blood mononuclear cells
- a washing buffer PBS solution containing 0.1% fetal bovine serum
- fluorescein isothiocyanate (FITC) labeled anti-mouse IgG antibody (Beckman Coulter, Inc.) was added as a secondary antibody and incubated at 4 ° C. for 1 hour.
- the PBMC was washed again with a washing buffer three times, and then analyzed using a flow cytometer (manufactured by Beckman Coulter).
- FIG. 12 shows the results of flow cytometry when using R7-47.
- FIG. 12 (a) shows the result of staining with an isotype control antibody and FITC-labeled anti-mouse IgG antibody
- FIG. 12 (b) shows the result of staining with R7-47 and FITC-labeled anti-mouse IgG antibody.
- the parts indicated by double arrows in FIGS. 12 (a) and 12 (b) indicate cell populations in which the isotype control antibody and R7-47 are specifically bound, respectively.
- the numbers in the figure indicate the proportion of the cell population when the number of analyzed PBMC cells is 100%.
- Table 5 shows the percentage of cells to which each anti-human CCR7 antibody specifically bound in PBMC.
- the anti-human CCR7 antibody recognizes human natural CCR7 and can bind to human natural CCR7.
- Effectiveness of anti-human CCR7 antibody in pulmonary fibrosis is that immunodeficient mice (for example, SCID mice) are administered with a stimulating substance (for example, bleomycin) that induces pulmonary fibrosis, and then transplanted with CD14 positive cells expressing human-derived CCR7. This can be shown by examining the migration of the cells to lung tissue.
- a stimulating substance for example, bleomycin
- PBMC peripheral blood of a healthy person according to a conventional method.
- the isolated PBMC was further contacted with human CD14-labeled magnetic beads (Miltenyi Biotech) to separate CD14-positive cells and used for cell transfer to mice.
- bleomycin Nippon Kayaku Co., Ltd., trade name: bleo
- physiological saline is administered via the respiratory tract to a dose of 5 mg / kg.
- Anti-human CCR7 antibody R7-11, R7-18, or R7- dissolved in physiological saline 4 days before bleomycin administration and 1, 4, 8, 11 days after administration 47 were administered intraperitoneally at a dose of 20 mg / kg each.
- isotype control IgG was administered on the same schedule.
- human CD14-positive cells containing cells expressing CCR7 were isolated by the method described above, and fluorescently labeled with PKH26PCL red fluorescent cell linker kit (Sigma). 1 ⁇ 10 6 fluorescently labeled cells per mouse were transferred from the tail vein.
- the mice were necropsied and the lung tissue was fixed with neutral formalin according to a conventional method to prepare pathological sections. The pathological tissue was observed with a fluorescence microscope, and the number of cells derived from fluorescently labeled human cells was counted in the entire right lung visual field. As a result, the number of fluorescently labeled cells in the lungs decreased in all mice administered with the anti-human CCR7 antibody compared to mice administered with isotype control IgG.
- RNA of the tissue was extracted from the left lung obtained by necropsy using TRIzol® reagent (Invitrogen), and the amount of RNA extracted using Nano® Drop® 1000® (Thermo® Fisher® Scientific) was quantified.
- a 2 ⁇ g RNA sample obtained from each mouse lung was subjected to a reverse transcription reaction using High®Capacity®cDNA®Reverse®transcription kit (Applied Biosystems), and the obtained cDNA sample was subjected to human CCR7 in lung tissue by real-time PCR. The amount of mRNA was measured.
- the analysis by real-time PCR was performed by TaqMan Gene Expression ⁇ ⁇ ⁇ ⁇ Assays system (Applied Biosystems).
- the mouse antibody light chain full-length gene containing the DNA represented by SEQ ID NO: 13 was cloned and introduced into the secretory expression vector pSecTag2.
- This vector was named pSecTag2-R702LC.
- a mouse antibody light chain full-length gene containing the DNA represented by SEQ ID NO: 43 was cloned and introduced into the secretion expression vector pSecTag2.
- This vector was named pSecTag2-R711LC.
- a mouse antibody light chain full-length gene containing the DNA represented by SEQ ID NO: 53 was cloned and introduced into a secretory expression vector pSecTag2.
- This vector was named pSecTag2-R718LC.
- PSecTag2-R702HC and pSecTag2-R702LC were introduced into HEK293 cells using Lipofectamine 2000 (Invitrogen). The cells were cultured for 2 days. The culture supernatant was applied to a Protein-G column to prepare a recombinant anti-human CCR7 antibody derived from hybridoma R7-02. Similarly, pSecTag2-R711HC and pSecTag2-R711LC were introduced into HEK293 cells using Lipofectamine 2000. The cells were cultured for 2 days. The culture supernatant was applied to a Protein-G column to prepare a recombinant anti-human CCR7 antibody derived from hybridoma R7-11.
- pSecTag2-R718HC and pSecTag2-R718LC were introduced into HEK293 cells using Lipofectamine 2000. The cells were cultured for 2 days. The culture supernatant was applied to a Protein-G column to prepare a recombinant anti-human CCR7 antibody derived from hybridoma R7-18.
- FIG. 14 shows the results using a recombinant anti-human CCR7 antibody derived from hybridoma R7-02.
- FIG. 15 shows the results using a recombinant anti-human CCR7 antibody derived from hybridoma R7-11.
- FIG. 16 shows the results using a recombinant anti-human CCR7 antibody derived from hybridoma R7-18. 14 to 16, (a) is a two-dimensional dot display diagram (left) and histogram (right) when CHO-K1 cells are used, and (b) is a two-dimensional dot when human CCR7 gene-introduced cells are used. A display diagram (left) and a histogram (right).
- the vertical axis represents the fluorescence intensity derived from phycoerythrin (PE), and the horizontal axis represents the fluorescence intensity derived from fluorescein isothiocyanate (FITC). That is, it was shown that any recombinant anti-human CCR7 antibody binds to human CCR7 gene-introduced cells but does not bind to CHO-K1 cells. From these results, it is shown that the antibody of the present invention can be produced in the form of a mouse antibody, a chimeric antibody, a humanized antibody, a functional fragment of an antibody, etc. using a gene recombination technique. It was.
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Abstract
Description
ヒトCCR7のアミノ酸配列と遺伝子の塩基配列は、すでに知られている(例えば、GenBank: EAW60669.1)。
(A1)配列番号5で表されるアミノ酸配列を含む重鎖CDR1、
配列番号6で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号7で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A2)配列番号15で表されるアミノ酸配列を含む重鎖CDR1、
配列番号16で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号17で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A3)配列番号25で表されるアミノ酸配列を含む重鎖CDR1、
配列番号26で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号27で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A4)配列番号35で表されるアミノ酸配列を含む重鎖CDR1、
配列番号36で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号37で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A5)配列番号45で表されるアミノ酸配列を含む重鎖CDR1、
配列番号46で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号47で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A6)配列番号55で表されるアミノ酸配列を含む重鎖CDR1、
配列番号56で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号57で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A7)配列番号65で表されるアミノ酸配列を含む重鎖CDR1、
配列番号66で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号67で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A8)配列番号75で表されるアミノ酸配列を含む重鎖CDR1、
配列番号76で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号77で表されるアミノ酸配列を含む重鎖CDR3を有する。
(B1)配列番号5で表されるアミノ酸配列を含む重鎖CDR1、
配列番号6で表されるアミノ酸配列を含む重鎖CDR2、
配列番号7で表されるアミノ酸配列を含む重鎖CDR3、
配列番号8で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号9で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号10で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B2)配列番号15で表されるアミノ酸配列を含む重鎖CDR1、
配列番号16で表されるアミノ酸配列を含む重鎖CDR2、
配列番号17で表されるアミノ酸配列を含む重鎖CDR3、
配列番号18で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号19で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号20で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B3)配列番号25で表されるアミノ酸配列を含む重鎖CDR1、
配列番号26で表されるアミノ酸配列を含む重鎖CDR2、
配列番号27で表されるアミノ酸配列を含む重鎖CDR3、
配列番号28で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号29で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号30で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B4)配列番号35で表されるアミノ酸配列を含む重鎖CDR1、
配列番号36で表されるアミノ酸配列を含む重鎖CDR2、
配列番号37で表されるアミノ酸配列を含む重鎖CDR3、
配列番号38で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号39で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号40で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B5)配列番号45で表されるアミノ酸配列を含む重鎖CDR1、
配列番号46で表されるアミノ酸配列を含む重鎖CDR2、
配列番号47で表されるアミノ酸配列を含む重鎖CDR3、
配列番号48で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号49で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号50で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B6)配列番号55で表されるアミノ酸配列を含む重鎖CDR1、
配列番号56で表されるアミノ酸配列を含む重鎖CDR2、
配列番号57で表されるアミノ酸配列を含む重鎖CDR3、
配列番号58で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号59で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号60で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B7)配列番号65で表されるアミノ酸配列を含む重鎖CDR1、
配列番号66で表されるアミノ酸配列を含む重鎖CDR2、
配列番号67で表されるアミノ酸配列を含む重鎖CDR3、
配列番号68で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号69で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号70で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B8)配列番号75で表されるアミノ酸配列を含む重鎖CDR1、
配列番号76で表されるアミノ酸配列を含む重鎖CDR2、
配列番号77で表されるアミノ酸配列を含む重鎖CDR3、
配列番号78で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号79で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号80で表されるアミノ酸配列を含む軽鎖CDR3を有する。
(C1)配列番号2で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号4で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C2)配列番号12で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号14で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C3)配列番号22で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号24で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C4)配列番号32で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号34で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C5)配列番号42で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号44で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C6)配列番号52で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号54で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C7)配列番号62で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号64で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C8)配列番号72で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号74で表されるアミノ酸配列を含む軽鎖可変領域を有する。
25~ 59:N末端ドメイン
87~ 95:細胞内第1ループドメイン
117~130:細胞外第1ループドメイン
153~170:細胞内第2ループドメイン
192~219:細胞外第2ループドメイン
248~263:細胞内第3ループドメイン
290~313:細胞外第3ループドメイン
332~378:C末端ドメイン
(A1)配列番号5で表されるアミノ酸配列を含む重鎖CDR1、
配列番号6で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号7で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A2)配列番号15で表されるアミノ酸配列を含む重鎖CDR1、
配列番号16で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号17で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A3)配列番号25で表されるアミノ酸配列を含む重鎖CDR1、
配列番号26で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号27で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A4)配列番号35で表されるアミノ酸配列を含む重鎖CDR1、
配列番号36で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号37で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A5)配列番号45で表されるアミノ酸配列を含む重鎖CDR1、
配列番号46で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号47で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A6)配列番号55で表されるアミノ酸配列を含む重鎖CDR1、
配列番号56で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号57で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A7)配列番号65で表されるアミノ酸配列を含む重鎖CDR1、
配列番号66で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号67で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A8)配列番号75で表されるアミノ酸配列を含む重鎖CDR1、
配列番号76で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号77で表されるアミノ酸配列を含む重鎖CDR3を有する。
(B1)配列番号5で表されるアミノ酸配列を含む重鎖CDR1、
配列番号6で表されるアミノ酸配列を含む重鎖CDR2、
配列番号7で表されるアミノ酸配列を含む重鎖CDR3、
配列番号8で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号9で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号10で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B2)配列番号15で表されるアミノ酸配列を含む重鎖CDR1、
配列番号16で表されるアミノ酸配列を含む重鎖CDR2、
配列番号17で表されるアミノ酸配列を含む重鎖CDR3、
配列番号18で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号19で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号20で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B3)配列番号25で表されるアミノ酸配列を含む重鎖CDR1、
配列番号26で表されるアミノ酸配列を含む重鎖CDR2、
配列番号27で表されるアミノ酸配列を含む重鎖CDR3、
配列番号28で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号29で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号30で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B4)配列番号35で表されるアミノ酸配列を含む重鎖CDR1、
配列番号36で表されるアミノ酸配列を含む重鎖CDR2、
配列番号37で表されるアミノ酸配列を含む重鎖CDR3、
配列番号38で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号39で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号40で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B5)配列番号45で表されるアミノ酸配列を含む重鎖CDR1、
配列番号46で表されるアミノ酸配列を含む重鎖CDR2、
配列番号47で表されるアミノ酸配列を含む重鎖CDR3、
配列番号48で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号49で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号50で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B6)配列番号55で表されるアミノ酸配列を含む重鎖CDR1、
配列番号56で表されるアミノ酸配列を含む重鎖CDR2、
配列番号57で表されるアミノ酸配列を含む重鎖CDR3、
配列番号58で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号59で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号60で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B7)配列番号65で表されるアミノ酸配列を含む重鎖CDR1、
配列番号66で表されるアミノ酸配列を含む重鎖CDR2、
配列番号67で表されるアミノ酸配列を含む重鎖CDR3、
配列番号68で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号69で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号70で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B8)配列番号75で表されるアミノ酸配列を含む重鎖CDR1、
配列番号76で表されるアミノ酸配列を含む重鎖CDR2、
配列番号77で表されるアミノ酸配列を含む重鎖CDR3、
配列番号78で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号79で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号80で表されるアミノ酸配列を含む軽鎖CDR3を有する。
(C1)配列番号2で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号4で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C2)配列番号12で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号14で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C3)配列番号22で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号24で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C4)配列番号32で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号34で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C5)配列番号42で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号44で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C6)配列番号52で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号54で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C7)配列番号62で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号64で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C8)配列番号72で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号74で表されるアミノ酸配列を含む軽鎖可変領域を有する。
配列番号8(軽鎖CDR1),配列番号9(軽鎖CDR2),配列番号10(軽鎖CDR3)は、それぞれ、配列番号4(VL)のアミノ酸番号24~39,55~68,94~102の部分に相当する(図1(b))。
配列番号18(軽鎖CDR1),配列番号19(軽鎖CDR2),配列番号20(軽鎖CDR3)は、それぞれ、配列番号14(VL)のアミノ酸番号24~37,53~59,87~99の部分に相当する(図2(b))。
配列番号28(軽鎖CDR1),配列番号29(軽鎖CDR2),配列番号30(軽鎖CDR3)は、それぞれ、配列番号24(VL)のアミノ酸番号23~36,52~58,91~99の部分に相当する(図3(b))。
配列番号38(軽鎖CDR1),配列番号39(軽鎖CDR2),配列番号40(軽鎖CDR3)は、それぞれ、配列番号34(VL)のアミノ酸番号23~36,52~58,91~99の部分に相当する(図4(b))。
配列番号48(軽鎖CDR1),配列番号49(軽鎖CDR2),配列番号50(軽鎖CDR3)は、それぞれ、配列番号44(VL)のアミノ酸番号24~39,55~61,94~102の部分に相当する(図5(b))。
配列番号58(軽鎖CDR1),配列番号59(軽鎖CDR2),配列番号60(軽鎖CDR3)は、それぞれ、配列番号54(VL)のアミノ酸番号24~39,55~61,94~102の部分に相当する(図6(b))。
配列番号68(軽鎖CDR1),配列番号69(軽鎖CDR2),配列番号70(軽鎖CDR3)は、それぞれ、配列番号64(VL)のアミノ酸番号24~39,56~62,95~102の部分に相当する(図7(b))。
配列番号78(軽鎖CDR1),配列番号79(軽鎖CDR2),配列番号80(軽鎖CDR3)は、それぞれ、配列番号74(VL)のアミノ酸番号24~39,55~61,94~102の部分に相当する(図8(b))。
後述の実施例で詳細に説明されるとおり、これらのハイブリドーマによって産生される8種の抗体は、それぞれ、上記した特定のアミノ酸配列からなる重鎖可変領域(VH)、軽鎖可変領域(VL)、及び各CDRを有する。表1に、VH、VL、各CDRのアミノ酸配列の配列番号と、対応するクローンの関係をまとめる。
受託番号:FERM BP-11369
受託日:平成22年(2010年)7月28日
(2010年7月28日に寄託されたFERM-21988より移管)
受託番号:FERM BP-11404
受託日:平成22年(2010年)7月28日
(2010年7月28日に寄託されたFERM-21989より移管)
受託番号:FERM BP-11371
受託日:平成22年(2010年)7月28日
(2010年7月28日に寄託されたFERM-21990より移管)
受託番号:FERM BP-11372
受託日:平成22年(2010年)7月28日
(2010年7月28日に寄託されたFERM-21991より移管)
受託番号:FERM BP-11373
受託日:平成22年(2010年)7月28日
(2010年7月28日に寄託されたFERM-21992より移管)
受託番号:FERM BP-11374
受託日:平成22年(2010年)7月28日
(2010年7月28日に寄託されたFERM-21993より移管)
受託番号:FERM BP-11375
受託日:平成22年(2010年)7月28日
(2010年7月28日に寄託されたFERM-21994より移管)
受託番号:FERM BP-11376
受託日:平成22年(2010年)7月28日
(2010年7月28日に寄託されたFERM-21995より移管)
なお、当該抗ヒトCCR7抗体における「機能的に同等」のCDRのアミノ酸配列の例としては、元のアミノ酸配列(配列番号5~10、15~20、25~30、35~40、45~50、55~60、65~70、75~80)において1若しくは数個、好ましくは1~5個、より好ましくは1~3個、さらに好ましくは1個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列で且つCDRとして同等の機能を有するものが挙げられる。別の例としては、前記元のアミノ酸配列との相同性が80%以上、好ましくは90%以上、より好ましくは95%以上のアミノ酸配列で且つCDRとして同等の機能を有するものが挙げられる。
2つの抗体についてエピトープが同一か否かを調べる方法としては、競合実験による方法が挙げられる。例えば、第一抗体たる前記8種の抗ヒトCCR7抗体と受容体との結合が、試験対象である第二抗体によって競合阻害を受ける場合には、当該第二抗体は、前記第一抗体と同じエピトープに結合するものであるといえる。
本発明の核酸は、上記8種のハイブリドーマからPCR等を用いて取得することもできる。
上記8種のいずれかのハイブリドーマを培養し、該培養物から本発明の抗体を生産することができる。培養の方法としては、ハイブリドーマの培養方法として一般に採用されている方法をそのまま適用することができる。例えば、DMEMやRPMI1640のような動物細胞用の培地を用いてハイブリドーマを培養し、その培養上清から本発明の抗体を得ることができる。動物の腹腔内でハイブリドーマを培養する場合には、腹水を採取し、その腹水から本発明の抗体を得ることができる。
本発明の抗体は、遺伝子組換えの手法を用いて生産することもできる。特に、キメラ型抗体、ヒト化抗体、抗体の機能的断片などを生産する場合には、遺伝子組換えの手法により生産することが一般的である。
まず、配列番号2、配列番号12、配列番号22、配列番号32、配列番号42、配列番号52、配列番号62、又は配列番号72に示すアミノ酸配列(VH)をコードするDNAを調製する。同様に、配列番号4、配列番号14、配列番号24、配列番号34、配列番号44、配列番号54、配列番号64、又は配列番号74に示すアミノ酸配列(VL)をコードするDNAを調製する。これらのDNAとしては、配列番号1、配列番号3、配列番号11、配列番号13、配列番号21、配列番号23、配列番号31、配列番号33、配列番号41、配列番号43、配列番号51、配列番号53、配列番号61、配列番号63、配列番号71、又は配列番号73に示すものが例示されるが、他の塩基配列のものでもよい。DNAの調製は、PCR等の公知の方法を用いて行うことができる。化学合成により当該DNAを調製することもできる。
得られたVH又はVLをコードするDNAを、ヒト抗体のCH又はCLをコードする配列を有するベクターにそれぞれ挿入し、キメラ型抗体発現ベクターを構築する。なお、ヒト抗体のCH又はCLをコードする配列を有するベクターは、市場から入手できる。構築した発現ベクターを宿主細胞に導入することにより、キメラ型抗体を発現する組換え細胞を得る。そして、この組換え細胞を培養し、該培養物から所望のキメラ型抗体を取得する。
次に、これらのDNAを用いて、任意のヒト抗体におけるVHのフレームワーク領域(FR)に重鎖CDR1~3が移植された可変領域をコードするDNAを作製する。同様に、任意のヒト抗体におけるVLのFRに軽鎖CDR1~3が移植された可変領域をコードするDNAを作製する。作製した各DNAを、ヒト抗体のCH又はCLをコードする配列を有するベクターに挿入し、ヒト化抗体発現ベクターを構築する。構築した発現ベクターを宿主細胞に導入することにより、ヒト化抗体を発現する組換え細胞を得る。そして、この組換え細胞を培養し、該培養物から所望のヒト化抗体を取得する。
第二の様相に係る、上記(A1)~(A8)のいずれかを満たす特定のCDRを有する抗体についても、同様の手順で生産することができる。
第一の様相に係る、配列番号7、配列番号17、配列番号27、配列番号37、配列番号47、配列番号57、配列番号67、又は配列番号77で表されるアミノ酸配列を含む重鎖CDR3を有する抗ヒトCCR7抗体についても、同様の手順で生産することができる。
前記肝線維症の例としては、肝硬変、虚血再灌流、肝移植後傷害、壊死性肝炎、B型肝炎、C型肝炎、原発性胆汁性肝硬変、及び原発性硬化性胆管炎からなる群から選択される肝線維症が挙げられる。肝硬変については、アルコールによる誘発、薬物による誘発、及び化学的な誘発からなる群から選択された少なくとも1つによるものが挙げられる。前記腎線維症の例としては、増殖性糸球体腎炎、硬化性糸球体腎炎、腎性線維化性皮膚症、糖尿病性腎症、腎尿細管間質性線維症、及び巣状分節性糸球体硬化症からなる群から選択される腎線維症が挙げられる。前記肺線維症の例としては、肺間質性線維症、薬物誘発サルコイドーシス、肺線維症、特発性肺線維症、喘息、慢性閉塞性肺疾患、びまん性肺胞損傷疾患、肺高血圧症、及び新生児気管支肺形成異常からなる群から選択される肺線維症が挙げられる。
ここで「治療」とは、疾患に罹患するおそれがあるか又は罹患した哺乳動物において、該疾患の病態の進行及び悪化を阻止又は緩和することを意味し、これによって該疾患の諸症状等の進行及び悪化を阻止又は緩和することを目的とする治療的処置の意味として使用される。
(A1)配列番号5で表されるアミノ酸配列を含む重鎖CDR1、
配列番号6で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号7で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A2)配列番号15で表されるアミノ酸配列を含む重鎖CDR1、
配列番号16で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号17で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A3)配列番号25で表されるアミノ酸配列を含む重鎖CDR1、
配列番号26で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号27で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A4)配列番号35で表されるアミノ酸配列を含む重鎖CDR1、
配列番号36で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号37で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A5)配列番号45で表されるアミノ酸配列を含む重鎖CDR1、
配列番号46で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号47で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A6)配列番号55で表されるアミノ酸配列を含む重鎖CDR1、
配列番号56で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号57で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A7)配列番号65で表されるアミノ酸配列を含む重鎖CDR1、
配列番号66で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号67で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A8)配列番号75で表されるアミノ酸配列を含む重鎖CDR1、
配列番号76で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号77で表されるアミノ酸配列を含む重鎖CDR3を有する。
(B1)配列番号5で表されるアミノ酸配列を含む重鎖CDR1、
配列番号6で表されるアミノ酸配列を含む重鎖CDR2、
配列番号7で表されるアミノ酸配列を含む重鎖CDR3、
配列番号8で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号9で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号10で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B2)配列番号15で表されるアミノ酸配列を含む重鎖CDR1、
配列番号16で表されるアミノ酸配列を含む重鎖CDR2、
配列番号17で表されるアミノ酸配列を含む重鎖CDR3、
配列番号18で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号19で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号20で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B3)配列番号25で表されるアミノ酸配列を含む重鎖CDR1、
配列番号26で表されるアミノ酸配列を含む重鎖CDR2、
配列番号27で表されるアミノ酸配列を含む重鎖CDR3、
配列番号28で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号29で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号30で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B4)配列番号35で表されるアミノ酸配列を含む重鎖CDR1、
配列番号36で表されるアミノ酸配列を含む重鎖CDR2、
配列番号37で表されるアミノ酸配列を含む重鎖CDR3、
配列番号38で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号39で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号40で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B5)配列番号45で表されるアミノ酸配列を含む重鎖CDR1、
配列番号46で表されるアミノ酸配列を含む重鎖CDR2、
配列番号47で表されるアミノ酸配列を含む重鎖CDR3、
配列番号48で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号49で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号50で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B6)配列番号55で表されるアミノ酸配列を含む重鎖CDR1、
配列番号56で表されるアミノ酸配列を含む重鎖CDR2、
配列番号57で表されるアミノ酸配列を含む重鎖CDR3、
配列番号58で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号59で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号60で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B7)配列番号65で表されるアミノ酸配列を含む重鎖CDR1、
配列番号66で表されるアミノ酸配列を含む重鎖CDR2、
配列番号67で表されるアミノ酸配列を含む重鎖CDR3、
配列番号68で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号69で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号70で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B8)配列番号75で表されるアミノ酸配列を含む重鎖CDR1、
配列番号76で表されるアミノ酸配列を含む重鎖CDR2、
配列番号77で表されるアミノ酸配列を含む重鎖CDR3、
配列番号78で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号79で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号80で表されるアミノ酸配列を含む軽鎖CDR3を有する。
(C1)配列番号2で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号4で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C2)配列番号12で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号14で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C3)配列番号22で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号24で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C4)配列番号32で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号34で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C5)配列番号42で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号44で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C6)配列番号52で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号54で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C7)配列番号62で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号64で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C8)配列番号72で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号74で表されるアミノ酸配列を含む軽鎖可変領域を有する。
ジーンバンク登録済みのヒトCCR7遺伝子配列(NM_001838、配列番号81)の5'末端にGCTAGC配列、3'末端にGTCGACTAGGAATTC配列(配列番号83)を付加した人工合成遺伝子をDNA合成装置により作製した。その後、この遺伝子をpUC57クローニングベクターに導入し、ヒトCCR7遺伝子クローンを調製した。以下、得られたベクターをNheIとSalIサイトで切断して調製される遺伝子断片を「DNA断片A」と称し、NheIとEcoRIサイトで切断して調製される遺伝子断片を「DNA断片B」と称する。
大腸菌HMS174(DE3)株(ノバジェン社)からゲノムDNAを抽出・精製した。次に、精製したゲノムDNAを鋳型とし、配列番号84及び85に示す塩基配列を有するオリゴヌクレオチドをプライマー対としてPCRを行い、配列番号86に示す塩基配列を有するGroELサブユニット遺伝子を含むDNA断片(以下、「DNA断片C」と称する。)を増幅した。DNA断片Cには、プライマーに由来して、5'末端にSalIサイト、3'末端に2個の停止コドン(TAATAG)をコードする配列及びNotIサイトが導入された。
哺乳動物発現ベクターpCI Mammalian Expression Vector(プロメガ社)を制限酵素NheIとSalIで消化し、バクテリア由来アルカリフォスファターゼ(BAP)にて末端を脱リン酸化処理した後、上記(1)で調製したDNA断片Aを挿入した。さらに、この発現ベクターをSalIおよびNotIで消化し、BAPにて末端を脱リン酸化処理した後、上記(2)で調製したDNA断片Cを挿入し、ベクターpCI-hCCR7・GroELを構築した。すなわち、ベクターpCI-hCCR7・GroELは、ヒトCCR7をコードする遺伝子とGroELサブユニットをコードする遺伝子との融合遺伝子を有している。
上記(1)で調製したDNA断片BをpCIneo(プロメガ社)のNheI-EcoRIサイトに導入し、pCIneo-hCCR7を構築した。
生理食塩水にベクターpCI-hCCR7・GroELを250μg/mLの濃度になるよう溶解し、免疫用組成物を調製した。この免疫用組成物を、8週齢のマウスBALB/c(雌)の両足大腿筋に各0.12mLずつ注射を行い、免疫した(0日目)。これにより、pCI-hCCR7・GroELを両足に各30μgずつ、すなわち、1匹につき1回あたり60μg投与した。その後、7日目、21日目、及び28日目にも同様して繰り返し免疫した。そして、0、7、14、21、28、35、42日目に採血を行い、血清を調製した。対照として、hCCR7を単独で発現するベクターpCI-hCCR7を用いてマウスを免疫した。
pCIneo-hCCR7が導入され安定発現を確認したCHO-K1細胞(以下、「hCCR7遺伝子導入細胞」と称する。)及び、pCIneoが導入されたCHO-K1細胞(対照の細胞)をPBSで洗浄した。免疫後56日目の血清を500倍希釈し、各細胞と一緒にインキュベートした。さらに、各細胞をPBSで洗浄し、2次抗体としてフィコエリスリン標識抗マウスIgG抗体(ベックマンコールター社)を添加した後、フローサイトメーターFACScalibur(ベクトンディッキンソン社)を用いて、各細胞と血清中の抗ヒトCCR7抗体との相互作用を解析した。
以上のことから、ベクターpCI-hCCR7・GroELによる遺伝子免疫によって、マウス血清中に活性型ヒトCCR7の細胞外ドメインを特異的に認識する抗体の産生を誘導することができた。
上記(5)と同様の手順で遺伝子免疫したマウス6匹に対して追加免疫を行った。追加免疫の3日後に脾臓を摘出し、脾細胞を調製した。PEG法により、1×108個の脾細胞と、1×107個のBALB/Cマウス由来のHAT選択性のミエローマSP2/0細胞とを融合した(細胞融合)。融合した細胞(ハイブリドーマ)の集団をRPMI培地に懸濁し、96穴マイクロプレート14枚の各穴に播種した。この段階で、約990種のハイブリドーマが得られた。
上記(6)と同様のフローサイトメトリーによって抗体結合評価を行い、ヒトCCR7遺伝子導入細胞と各穴のハイブリドーマ培養上清中の抗体との結合性を調べた。その結果、8穴において抗体との結合性が確認された。
抗体との結合性が確認された8種のハイブリドーマについて、限界希釈法によるクローニングを行った。すなわち、8種のハイブリドーマについて96穴マイクロプレートに細胞1個以下/穴になるように播種し、培養した。2週間後、同様のフローサイトメトリーを行い、クローニングされた培養上清中の抗体(抗ヒトCCR7モノクローナル抗体)の結合性を確認した。その結果、抗ヒトCCR7モノクローナル抗体を産生する8種のハイブリドーマがクローニングされた。
各ハイブリドーマについてRPMI培地100mL内でフラスコ培養を2週間行った。各培養上清を、プロテインGカラム(アマシャムバイオサイエンス社)に供して精製・濃縮し、精製された8種の抗ヒトCCR7モノクローナル抗体を各々約20mg得た。
10μg/mL濃度の抗ヒトCCR7モノクローナル抗体及びマウスコントロールIgG(Thermo、陰性対照)のPBS溶液を調製した(以下、「抗体溶液」と称する。)。一方、ヒトCCR7遺伝子導入細胞をPBSで洗浄した後、抗体溶液を細胞と共にインキュベートした。さらに、細胞をPBSで洗浄し、2次抗体としてフィコエリスリン標識抗マウスIgG抗体(ベックマンコールター社)を添加した後、フローサイトメーターFACScalibur(ベクトンディッキンソン社)を用いて、細胞と抗ヒトCCR7抗体及びマウスコントロールIgGとの相互作用を解析した。結果を図9、10及び表2に示す。図9はヒトCCR7遺伝子導入細胞と、マウスコントロールIgGとの相互作用の解析結果を表すヒストグラムである。図10はヒトCCR7遺伝子導入細胞と、抗ヒトCCR7抗体R7-47との相互作用の解析結果を表すヒストグラムである。表2は、ヒトCCR7遺伝子導入細胞と、各抗ヒトCCR7抗体との相互作用の解析結果より、算出された特異的結合細胞の割合を示す。図9、10において、縦軸は細胞数、横軸はフィコエリスリン(PE)由来の蛍光強度を表す。また、2つのエリア(M1、M2)のうち、M2(右領域)に属する細胞が抗体と結合した細胞を表す。表2において、特異的に結合した細胞の割合は、M2/(M1+M2)で表される。
ヒトCCR7遺伝子導入細胞を、2×104個/100μLの初期細胞濃度にて、96穴マイクロタイタープレートを用いて一昼夜培養した。培養終了後、各穴に10-6~10-11Mの濃度範囲内の抗ヒトCCR7抗体を添加した。また対照として、マウスコントロールIgG(Thermo社、陰性対照)を同様に10-6~10-11Mの濃度範囲内で添加したものも調製した。1時間後、1×10-7MのCCL21(R&D systems社)によって各細胞を刺激した際の細胞内Ca2+濃度の一過的上昇の抗体濃度依存的減少度を測定した。Ca2+濃度の測定は、Ca2+シグナル解析装置(FLIPR;Molecular Devices社)及び細胞内Ca染色キット(Ca3kit;Molecular Devices社)を用いて行った。抗ヒトCCR7抗体R7-47を添加した結果を図11に示す。図11は細胞内Ca2+濃度と各添加物の濃度との関係を表すグラフである。図11に示すように、R7-47の濃度依存的に、細胞内Ca2+濃度の低下がみられた。これは、R7-47がCCL21とヒトCCR7との結合を競合的に阻害した結果、細胞内へのシグナル伝達が阻害されたことを示していた。50%阻害濃度(IC50)を算出すると、R7-47では7.4nMであった。表3は、R7-47以外の抗ヒトCCR7抗体のIC50を示す。
以上より、得られた抗ヒトCCR7抗体は、いずれもヒトCCR7リガンドによるCCR7依存的な細胞内情報伝達機構を遮断できることが示された。
マウスモノクローナル抗体アイソタイピングキット(GEヘルスケア社)を用いて、抗ヒトCCR7抗体のアイソタイプを決定した。検出はホースラディッシュペルオキシダーゼ標識マウスIgG抗体を用いたサンドイッチELISAによった。結果を表4に示す。
上記8種のハイブリドーマより、各抗体のL鎖及びH鎖の可変領域をコードするDNAをクローニングし、その塩基配列を決定した。クローニングは以下のように行った。まずハイブリドーマからRNeasyMini Kit(QIAGEN社)を用いてRNAを単離した。次に、「SMARTer-RACE cDNA Amplification Kit」(タカラバイオ社)を用いて、メーカーマニュアルに従い5’-RACE法によるcDNA合成、及びPCR産物を得た。PCRの3'側プライマーとして、γ鎖の場合は、配列番号87の配列を、κ鎖の場合は配列番号88~90の配列を、λ鎖の場合は配列番号91の配列をそれぞれ使用した。
健常人の末梢血から、常法に従い Lymphoprep (Axis-Shield社)を用いて単核球細胞(PBMC)を単離した。単離したPBMCを、1mg/mLのヒトガンマグロブリン(Jackson Immuno Research Laboratories社)を含む Phosphate Buffered Saline(PBS)溶液に懸濁し、室温で30分間インキュベートすることでブロッキングした。ブロッキング後のPBMC(3×105細胞)を、各抗ヒトCCR7抗体(0.5μg)もしくはアイソタイプコントロール抗体(0.5μg)と共に4℃で1時間インキュベートした。その後、PBMCを洗浄バッファー(0.1% ウシ胎児血清を含むPBS溶液)で3回洗浄した。次に2次抗体として、フルオレセインイソチオシアネート(FITC)標識抗マウスIgG抗体(ベックマンコールター社)を添加し、4℃で1時間インキュベートした。このPBMCを再度洗浄バッファーで3回洗浄した後、フローサイトメーター(ベックマンコールター社製)を用いて分析した。
PBMC(1.6×105細胞)を24ウェルセルカルチャーインサート(ベクトン・ディッキンソン社)のインサートに播種した。さらに、各抗ヒトCCR7抗体もしくはアイソタイプコントロールIgG抗体10μg/mLを添加し、室温にて5分間反応させた。セルカルチャーインサートの各ウェルにはヒトリコンビナントCCL21(150ng/mL)を添加した。インサートをプレート上に設置後、37℃にて1.5時間反応させた。反応後、インサートを除去し、各ウェル中に遊走した細胞数を測定した。表6と表7に、CCL21依存的細胞遊走に対する当該抗体による機能阻害の結果を示す。
ヒトPBMC中の、CCR7を発現するCD14陽性細胞は、肺線維における線維化形成に重要な細胞である(Abe R, Donnelly SC, Peng T, Bucala R, Metz CN, "Peripheral blood fibrocytes: differentiation pathway and migration to wound sites." J Immunol. 2001 Jun 15;166(12):7556-62;Curnow SJ, Fairclough M, Schmutz C, Kissane S, Denniston AK, Nash K, Buckley CD, Lord JM, Salmon M,"Distinct types of fibrocyte can differentiate from mononuclear cells in the presence and absence of serum." PLoS One. 2010 Mar 18;5(3):e9730)。抗ヒトCCR7抗体の肺線維症における有効性は、免疫不全マウス(例えばSCIDマウス)に肺線維症を誘発させる刺激物質(例えばブレオマイシン)を投与後、ヒト由来のCCR7を発現するCD14陽性細胞を移入した際の当該細胞の肺組織への移行を調べることで示すことができる。
配列番号11で表されるDNAを含むマウス抗体重鎖全長の遺伝子をクローニングし、分泌発現用ベクターpSecTag2(インビトロジェン社)に導入した。このベクターをpSecTag2-R702HCと命名した。同様に、配列番号41で表されるDNAを含むマウス抗体重鎖全長の遺伝子をクローニングし、分泌発現用ベクターpSecTag2に導入した。このベクターをpSecTag2-R711HCと命名した。同様に、配列番号51で表されるDNAを含むマウス抗体重鎖全長の遺伝子をクローニングし、分泌発現用ベクターpSecTag2に導入した。このベクターをpSecTag2-R718HCと命名した。
Claims (30)
- ヒトCCR7の細胞外ドメインに特異的に結合する抗ヒトCCR7抗体であって、配列番号7、配列番号17、配列番号27、配列番号37、配列番号47、配列番号57、配列番号67、又は配列番号77で表されるアミノ酸配列を含む重鎖相補性決定領域3(重鎖CDR3)を有することを特徴とする抗ヒトCCR7抗体。
- ヒトCCR7の細胞外ドメインに特異的に結合する抗ヒトCCR7抗体であって、その相補性決定領域1~3(CDR1~3)のアミノ酸配列について、下記(A1)~(A8)のいずれかを満たすことを特徴とする抗ヒトCCR7抗体。
(A1)配列番号5で表されるアミノ酸配列を含む重鎖CDR1、
配列番号6で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号7で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A2)配列番号15で表されるアミノ酸配列を含む重鎖CDR1、
配列番号16で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号17で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A3)配列番号25で表されるアミノ酸配列を含む重鎖CDR1、
配列番号26で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号27で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A4)配列番号35で表されるアミノ酸配列を含む重鎖CDR1、
配列番号36で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号37で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A5)配列番号45で表されるアミノ酸配列を含む重鎖CDR1、
配列番号46で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号47で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A6)配列番号55で表されるアミノ酸配列を含む重鎖CDR1、
配列番号56で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号57で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A7)配列番号65で表されるアミノ酸配列を含む重鎖CDR1、
配列番号66で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号67で表されるアミノ酸配列を含む重鎖CDR3を有する、
(A8)配列番号75で表されるアミノ酸配列を含む重鎖CDR1、
配列番号76で表されるアミノ酸配列を含む重鎖CDR2、及び
配列番号77で表されるアミノ酸配列を含む重鎖CDR3を有する。 - ヒトCCR7の細胞外ドメインに特異的に結合する抗ヒトCCR7抗体であって、その相補性決定領域1~3(CDR1~3)のアミノ酸配列について、下記(B1)~(B8)のいずれかを満たすことを特徴とする抗ヒトCCR7抗体。
(B1)配列番号5で表されるアミノ酸配列を含む重鎖CDR1、
配列番号6で表されるアミノ酸配列を含む重鎖CDR2、
配列番号7で表されるアミノ酸配列を含む重鎖CDR3、
配列番号8で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号9で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号10で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B2)配列番号15で表されるアミノ酸配列を含む重鎖CDR1、
配列番号16で表されるアミノ酸配列を含む重鎖CDR2、
配列番号17で表されるアミノ酸配列を含む重鎖CDR3、
配列番号18で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号19で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号20で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B3)配列番号25で表されるアミノ酸配列を含む重鎖CDR1、
配列番号26で表されるアミノ酸配列を含む重鎖CDR2、
配列番号27で表されるアミノ酸配列を含む重鎖CDR3、
配列番号28で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号29で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号30で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B4)配列番号35で表されるアミノ酸配列を含む重鎖CDR1、
配列番号36で表されるアミノ酸配列を含む重鎖CDR2、
配列番号37で表されるアミノ酸配列を含む重鎖CDR3、
配列番号38で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号39で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号40で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B5)配列番号45で表されるアミノ酸配列を含む重鎖CDR1、
配列番号46で表されるアミノ酸配列を含む重鎖CDR2、
配列番号47で表されるアミノ酸配列を含む重鎖CDR3、
配列番号48で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号49で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号50で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B6)配列番号55で表されるアミノ酸配列を含む重鎖CDR1、
配列番号56で表されるアミノ酸配列を含む重鎖CDR2、
配列番号57で表されるアミノ酸配列を含む重鎖CDR3、
配列番号58で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号59で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号60で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B7)配列番号65で表されるアミノ酸配列を含む重鎖CDR1、
配列番号66で表されるアミノ酸配列を含む重鎖CDR2、
配列番号67で表されるアミノ酸配列を含む重鎖CDR3、
配列番号68で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号69で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号70で表されるアミノ酸配列を含む軽鎖CDR3を有する、
(B8)配列番号75で表されるアミノ酸配列を含む重鎖CDR1、
配列番号76で表されるアミノ酸配列を含む重鎖CDR2、
配列番号77で表されるアミノ酸配列を含む重鎖CDR3、
配列番号78で表されるアミノ酸配列を含む軽鎖CDR1、
配列番号79で表されるアミノ酸配列を含む軽鎖CDR2、及び
配列番号80で表されるアミノ酸配列を含む軽鎖CDR3を有する。 - ヒトCCR7の細胞外ドメインに特異的に結合する抗ヒトCCR7抗体であって、その重鎖可変領域と軽鎖可変領域のアミノ酸配列について、下記(C1)~(C8)のいずれかを満たすことを特徴とする抗ヒトCCR7抗体。
(C1)配列番号2で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号4で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C2)配列番号12で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号14で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C3)配列番号22で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号24で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C4)配列番号32で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号34で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C5)配列番号42で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号44で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C6)配列番号52で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号54で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C7)配列番号62で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号64で表されるアミノ酸配列を含む軽鎖可変領域を有する、
(C8)配列番号72で表されるアミノ酸配列を含む重鎖可変領域、及び配列番号74で表されるアミノ酸配列を含む軽鎖可変領域を有する。 - CCR7リガンド刺激によるCCR7依存的な細胞内情報伝達機構を遮断する活性を有することを特徴とする請求項1~4のいずれかに記載の抗ヒトCCR7抗体。
- ヒト化抗体又はキメラ抗体であることを特徴とする請求項1~5のいずれかに記載の抗ヒトCCR7抗体。
- 抗体断片、一本鎖抗体、又はダイアボディであることを特徴とする請求項1~6のいずれかに記載の抗ヒトCCR7抗体。
- ヒトCCR7の細胞外ドメインに特異的に結合する抗ヒトCCR7抗体であって、R7-01(FERM BP-11369)、R7-02(FERM BP-11404)、R7-05(FERM BP-11371)、R7-09(FERM BP-11372)、R7-11(FERM BP-11373)、R7-18(FERM BP-11374)、R7-25(FERM BP-11375)、又はR7-47(FERM BP-11376)により産生されることを特徴とする抗ヒトCCR7抗体。
- 請求項1~8のいずれかに記載の抗ヒトCCR7抗体と同一のエピトープに結合することを特徴とする抗ヒトCCR7抗体。
- R7-01(FERM BP-11369)、R7-02(FERM BP-11404)、R7-05(FERM BP-11371)、R7-09(FERM BP-11372)、R7-11(FERM BP-11373)、R7-18(FERM BP-11374)、R7-25(FERM BP-11375)、又はR7-47(FERM BP-11376)であることを特徴とするハイブリドーマ。
- 請求項1~9のいずれかに記載の抗ヒトCCR7抗体の重鎖可変領域又は軽鎖可変領域をコードする核酸。
- 配列番号1、配列番号3、配列番号11、配列番号13、配列番号21、配列番号23、配列番号31、配列番号33、配列番号41、配列番号43、配列番号51、配列番号53、配列番号61、配列番号63、配列番号71、又は配列番号73で表される塩基配列を有する請求項11に記載の核酸。
- 請求項11又は12に記載の核酸を含むベクター。
- 請求項13に記載のベクターが導入された細胞。
- 請求項1~9のいずれかに記載の抗ヒトCCR7抗体と、薬学的に許容される担体とを含むことを特徴とする医薬組成物。
- CCR7リガンドによるCCR7依存的な細胞内情報伝達機構を遮断するものであることを特徴とする請求項15に記載の医薬組成物。
- 組織の線維化を治療するために用いられることを特徴とする請求項15又は16に記載の医薬組成物。
- 前記組織の線維化が、肝線維症、腎線維症、肺線維症、皮膚線維症、心血管線維症、消化管線維症及び他の線維性疾患からなる群から選択される線維症であることを特徴とする請求項17に記載の医薬組成物。
- 前記肝線維症が、肝硬変、虚血再灌流、肝移植後傷害、壊死性肝炎、B型肝炎、C型肝炎、原発性胆汁性肝硬変、及び原発性硬化性胆管炎からなる群から選択されることを特徴とする請求項18に記載の医薬組成物。
- 前記肝硬変が、アルコールによる誘発、薬物による誘発、及び化学的な誘発からなる群から選択された少なくとも1つによるものであることを特徴とする請求項19に記載の医薬組成物。
- 前記腎線維症が、増殖性糸球体腎炎、硬化性糸球体腎炎、腎性線維化性皮膚症、糖尿病性腎症、腎尿細管間質性線維症、及び巣状分節性糸球体硬化症からなる群から選択されることを特徴とする請求項18に記載の医薬組成物。
- 前記肺線維症が、肺間質性線維症、薬物誘発サルコイドーシス、肺線維症、特発性肺線維症、喘息、慢性閉塞性肺疾患、びまん性肺胞損傷疾患、肺高血圧症、及び新生児気管支肺形成異常からなる群から選択されることを特徴とする請求項18に記載の医薬組成物。
- 前記皮膚線維症が、強皮症、ケロイド瘢痕化、乾癬、肥厚性瘢痕化、及び偽性強皮症からなる群から選択されることを特徴とする請求項18に記載の医薬組成物。
- 前記心血管線維症が、アテローム性動脈硬化、冠動脈再狭窄、うっ血性心筋症、心不全、心移植、及び心筋線維症からなる群から選択されることを特徴とする請求項18に記載の医薬組成物。
- 前記消化管線維症が、コラーゲン蓄積大腸炎、絨毛萎縮、陰窩過形成、ポリープ形成、クローン病の線維症、胃潰瘍治癒、及び腹部癒着術後瘢痕からなる群から選択されることを特徴とする請求項18に記載の医薬組成物。
- 前記線維症が、骨に関連する線維化性疾患から生じる状態を有し、リウマチ様パンヌス形成であることを特徴とする請求項18に記載の医薬組成物。
- 癌の転移を治療するために用いられることを特徴とする請求項15に記載の医薬組成物。
- 前記癌が、咽頭癌、軟骨肉腫、大腸癌、膵臓癌、白血病、乳癌からなる群から選択されることを特徴とする請求項27に記載の医薬組成物。
- 請求項1~9のいずれかに記載の抗ヒトCCR7抗体が担体に固定化されてなる抗体固定化担体。
- CCR7発現細胞を含む血液を接触させて、前記体液からCCR7発現細胞を除去するために用いられることを特徴とする請求項29に記載の抗体固定化担体。
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CN201180056955.3A CN103261412B (zh) | 2010-09-28 | 2011-09-27 | 抗人ccr7抗体、杂交瘤、核酸、载体、细胞、医药组合物和抗体固定化担载体 |
US13/876,265 US8865170B2 (en) | 2010-09-28 | 2011-09-27 | Anti-human CCR7 antibody, hybridoma, nucleic acid, vector, cell, pharmaceutical composition, and antibody-immobilized carrier |
DK11829086.5T DK2623592T3 (da) | 2010-09-28 | 2011-09-27 | Anti-humane ccr7-antistoffer, hybridom, medicinsk sammensætning og antistof-immobiliseret bærer |
JP2012536465A JP5315495B2 (ja) | 2010-09-28 | 2011-09-27 | 抗ヒトccr7抗体、ハイブリドーマ、核酸、ベクター、細胞、医薬組成物、並びに、抗体固定化担体 |
EP11829086.5A EP2623592B1 (en) | 2010-09-28 | 2011-09-27 | Anti-human ccr7 antibodies, hybridoma, medicinal composition, and antibody-immobilized carrier |
AU2011309114A AU2011309114B2 (en) | 2010-09-28 | 2011-09-27 | Antihuman CCR7 antibodies, hybridoma, nucleic acid, vector, cell, medicinal composition, and antibody-immobilized carrier |
CA2813203A CA2813203C (en) | 2010-09-28 | 2011-09-27 | Anti-human ccr7 antibody, hybridoma, nucleic acid, vector, cell, pharmaceutical composition, and antibody-immobilized carrier |
KR1020137010575A KR101886890B1 (ko) | 2010-09-28 | 2011-09-27 | 항인간 ccr7 항체, 하이브리도마, 핵산, 벡터, 세포, 의약 조성물 및 항체 고정화 담체 |
NZ609154A NZ609154A (en) | 2010-09-28 | 2011-09-27 | Antihuman ccr7 antibodies, hybridoma, nucleic acid, vector, cell, medicinal composition, and antibody-immobilized carrier |
IL225461A IL225461B (en) | 2010-09-28 | 2013-03-24 | Human antibodies against ccr7, nucleic acids encoding them and preparations containing them |
US14/489,918 US20150017167A1 (en) | 2010-09-28 | 2014-09-18 | Anti-human ccr7 antibody, hybridoma, nucleic acid, vector, cell, pharmaceutical composition, and antibody-immobilized carrier |
US14/489,898 US9505844B2 (en) | 2010-09-28 | 2014-09-18 | Anti-human CCR7 antibody, hybridoma, nucleic acid, vector, cell, pharmaceutical composition, and antibody-immobilized carrier |
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US14/489,898 Division US9505844B2 (en) | 2010-09-28 | 2014-09-18 | Anti-human CCR7 antibody, hybridoma, nucleic acid, vector, cell, pharmaceutical composition, and antibody-immobilized carrier |
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WO2013184200A1 (en) * | 2012-06-05 | 2013-12-12 | Msm Protein Technologies | Human monoclonal antibodies against human chemokine receptor ccr7 |
JP2016519066A (ja) * | 2013-03-15 | 2016-06-30 | 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 |
US10526411B2 (en) | 2013-03-15 | 2020-01-07 | Amgen Inc. | Methods and compositions relating to anti-CCR7 antigen binding proteins |
US11254749B2 (en) | 2015-08-10 | 2022-02-22 | Pepmab B.V. | Humanized anti-CCR7 receptor antibodies |
JP2018522568A (ja) * | 2015-08-10 | 2018-08-16 | ペプマブ ビー.ヴィー.PepMab B.V. | ヒト化抗ccr7受容体抗体 |
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JP7312111B2 (ja) | 2017-02-03 | 2023-07-20 | ノバルティス アーゲー | 抗ccr7抗体薬物コンジュゲート |
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WO2020171020A1 (ja) | 2019-02-18 | 2020-08-27 | 株式会社エヌビィー健康研究所 | 細胞の選抜方法、核酸の製造方法、組換え細胞の製造方法、目的物質の製造方法、医薬組成物の製造方法、及び試薬 |
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JPWO2012043533A1 (ja) | 2014-02-24 |
CA2813203C (en) | 2022-10-18 |
EP2623592B1 (en) | 2019-05-15 |
JP2013059343A (ja) | 2013-04-04 |
KR101886890B1 (ko) | 2018-08-08 |
EP2623592A4 (en) | 2014-05-14 |
CN105884895A (zh) | 2016-08-24 |
US20150010998A1 (en) | 2015-01-08 |
CN105884895B (zh) | 2019-12-13 |
US20130195869A1 (en) | 2013-08-01 |
DK2623592T3 (da) | 2019-08-19 |
US8865170B2 (en) | 2014-10-21 |
EP2623592A1 (en) | 2013-08-07 |
US20150017167A1 (en) | 2015-01-15 |
CN103261412B (zh) | 2016-03-23 |
KR20130119923A (ko) | 2013-11-01 |
IL225461A0 (en) | 2013-06-27 |
CA2813203A1 (en) | 2012-04-05 |
AU2011309114A1 (en) | 2013-05-02 |
CN103261412A (zh) | 2013-08-21 |
IL225461B (en) | 2019-08-29 |
AU2011309114A8 (en) | 2013-09-05 |
JP5315495B2 (ja) | 2013-10-16 |
NZ609154A (en) | 2014-10-31 |
AU2011309114B2 (en) | 2015-03-19 |
US9505844B2 (en) | 2016-11-29 |
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