Antibody, nucleic acid, cell and drug

Abstract

Provided is an antibody that specifically binds to an extracellular domain of human CCR7, the antibody having the heavy chain CDR1 including the amino acid sequence represented by SEQ ID NO: 25, the heavy chain CDR2 including the amino acid sequence represented by SEQ ID NO: 27, the heavy chain CDR3 including the amino acid sequence represented by SEQ ID NO: 29, the light chain CDR1 including the amino acid sequence represented by SEQ ID NO: 35, the light chain CDR2 including the amino acid sequence represented by SEQ ID NO: 37, and the light chain CDR3 including the amino acid sequence represented by SEQ ID NO: 39.

Description

Antibodies, nucleic acids, cells, and drugs

[0001] The present disclosure relates to an antibody that specifically binds to the extracellular domain of human CCR7, a nucleic acid encoding the antibody, a cell containing the nucleic acid, and a pharmaceutical comprising the antibody as an active ingredient.

[0002] Chemokines are proteins that regulate the migration and function of various cells. Dysfunction of chemokines and their receptors is a cause of various diseases, such as fibrosis, autoimmune diseases, acute and chronic inflammation, and cancer. Although drugs that control the activity of chemokines and their receptors have been developed and are being used clinically, it is difficult to say that they have fully resolved the problems.

[0003] Specific chemokines must bind to specific cell membrane receptors to exert their activities, such as regulating cell migration and cell function. Approximately 20 types of chemokine receptors have been discovered, all of which are seven-transmembrane receptors (GPCRs) that couple to trimeric G proteins. Chemokine receptor binding releases the Gα unit of the trimeric G protein, which then elevates intracellular Ca concentration and activates phosphatidylinositol 3-kinase (PI3K), small Rho GTPases, and other pathways, resulting in their function. Although each chemokine receptor is activated by relatively selective chemokines, their primary protein structures and intracellular activation mechanisms are very similar. Therefore, it is not easy to selectively block the function of a specific chemokine receptor. The functional expression of each chemokine and chemokine receptor under physiological and pathological conditions is regulated by the expression of each protein in specific cells and tissues at specific times (during inflammation) (Non-Patent Document 1).

[0004] Human CC motif receptor 7 (CC MOTIF, RECEPTOR 7; also known as EBI1 or CMKBR7; hereafter referred to as "CCR7") was initially discovered as a GPCR selectively expressed in lymphocytes following Epstein-Barr virus infection (Non-Patent Document 2). CCR7 was subsequently found to be a selective chemokine receptor for CCL19 (also known as ELC) and CCL21 (also known as SLC or EXODUS 2). Under physiological conditions, CCR7 is relatively selectively expressed on cells such as CD4+ T cells (Th1, Th2, and Treg cells), mature dendritic cells, and B cells. It is known that these cells are attracted to inflammatory sites and other lesions via CCR7, enhancing inflammatory and immune responses. Abnormal CCR7 activation has also been implicated in various diseases, including autoimmune diseases, fibrosis following acute and chronic inflammation, and cancer metastasis.

[0005] In cancer treatment, it is important to suppress the growth of the primary cancer and prevent recurrence accompanied by distant metastasis. While treatment outcomes have improved with conventional surgical therapy and chemotherapy, as well as molecularly targeted drugs (e.g., kinase inhibitors), antibody drug therapy, and cancer immunotherapy, there is still a need for the development of therapeutic agents that can prevent recurrence accompanied by distant metastasis. Distant metastasis can occur via blood vessels from the primary cancer or via lymphatic tissue. Although matrix metalloproteinase (MMP) inhibitors have been developed to prevent metastasis, no clinical applications have been reported.

[0006] Various studies have shown that CCR7 is expressed in various tumor cells, such as B-cell chronic lymphocytic leukemia, non-Hodgkin's lymphoma, breast cancer cells, and malignant breast tumors. Furthermore, it has become clear that CCR7 plays a role in lymph node metastasis of various cancers, such as gastric cancer, melanoma, non-small cell lung cancer, lung adenocarcinoma, T-cell leukemia cells, cervical cancer, various squamous cell carcinomas, hepatocellular carcinoma, urothelial carcinoma, and renal cell carcinoma (Non-Patent Document 1). Since CCL19 and CCL21, which are CCR7 ligands, are highly expressed in lymph nodes, selective inhibition of CCR7 function is expected to suppress lymphatic metastasis of cancer cells.

[0007] As selective CCR7 function inhibitors, low molecular weight compounds and monoclonal antibodies that selectively bind to the extracellular domain of the receptor and block the CCR7 signal transduction mechanism stimulated by CCL19 / CCL21 are considered as candidates.

[0008] Patent Document 1 discloses eight monoclonal antibodies that specifically bind to the extracellular domain of human CCR7. It also discloses the amino acid sequences of the complementarity determining regions (CDRs), heavy chain variable regions, and light chain variable regions of each monoclonal antibody. These anti-human CCR7 antibodies are considered to be particularly effective in treating fibrosis.

[0009] Patent Document 2 discloses a humanized antibody of one type of monoclonal antibody that specifically binds to human CCR7, and discloses that the humanized antibody or an Fc variant with enhanced complement-dependent cytotoxicity kills multiple tumor cells of blood cancers through its effector function.

[0010] Patent Document 3 discloses an antibody-drug conjugate in which an anticancer drug is bound to a monoclonal antibody that specifically binds to human CCR7 and is a mutant that has no effector function.

[0011] However, the antibodies described in Patent Documents 1 to 3 have not yet been put to practical use as therapeutic agents. There is a need in the art for additional or improved anti-human CCR7 antibodies that have superior properties as pharmaceutical raw materials.

[0012] The properties of monoclonal antibodies required as raw materials for antibody drugs that inhibit the function of CCR7 include high selectivity for the receptor, strong functional inhibitory activity, high solubility, thermal stability, low aggregation, and low immunogenicity. All of these properties are necessary for the practical application of antibody drugs, but low immunogenicity in humans is particularly important in terms of the safety of antibody drugs and determining the administration period. The importance of low immunogenicity is also indicated in the guidelines for the development of biopharmaceuticals from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA).

[0013] A common method for achieving low immunogenicity of antibodies is antibody humanization. Specifically, in order to reduce immunogenicity while maintaining high receptor selectivity and strong functional inhibitory activity for antibodies obtained using rodents, the antibodies can be humanized by CDR grafting while maintaining 100% of the amino acid sequences of the heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3. However, there are only two types of antibodies against GPCRs on the market as pharmaceuticals, and it is not easy to reduce immunogenicity while maintaining high target molecule specificity and strong functional inhibitory activity.

[0014] In addition to directly blocking the intracellular signaling mechanism of receptors, the in vivo antitumor activity of antibody drugs is known to be induced by antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC). ADCC is a mechanism in which, when an antibody binds to an antigen on a cancer cell or target cell, immune cells such as macrophages and natural killer (NK) cells, which have Fc receptors that recognize the Fc region of the antibody, are attracted to the target cell and kill the antibody-bound cancer cell or target cell. In cancer therapy, antibodies that have ADCC activity in addition to neutralizing activity are considered useful because they exert antitumor activity at low doses. The strength of ADCC activity is determined by the amount of antigen expression on the target cell, the strength of antigen-antibody binding, the selectivity of the antibody, and the strength of the affinity between the antibody's Fc region and the Fc receptor. It is not clear whether all GPCR antibodies have ADCC activity, and the strength of ADCC activity varies from antibody to antibody.

[0015] Methods for artificially enhancing the ADCC activity of an antibody include modifying the amino acid sequence of the Fc region of the antibody and controlling the sugar chain structure of the antibody. A known method for controlling the sugar chain structure of an antibody is to remove fucose from the reducing end of the N-type complex sugar chain of the antibody (defucosylation).

[0016] International Publication No. 2012 / 043533 International Publication No. 2017 / 025569 International Publication No. 2021 / 220199

[0017] Viola A, Luster AD "Chemokines and their receptors: drug targets in immunity and inflammation", Annu Rev Pharmacol Toxicol. 2008;48:171-197Birkenbach, M., Josefsen, K., Yalamanchili, R., Lenoir, G., Kieff, E., "Epstein-Barr virus-induced genes: first lymphocyte-specific G protein-coupled peptide receptors", J. Virol. 67: 2209-2220, 1993.

[0018] As described above, antibodies that inhibit the function of CCR7 are expected to be useful as antibody pharmaceuticals, but none have yet been put to practical use as therapeutic agents. Therefore, an object of the present disclosure is to provide a novel anti-human CCR7 antibody that has superior properties as a pharmaceutical ingredient.

[0019] The present inventors humanized a monoclonal antibody that blocks the CCR7 signaling mechanism, as described in Patent Document 1, using a CDR grafting method. However, this method failed to produce an antibody with the desired low immunogenicity for pharmaceutical use. Therefore, the inventors modified the amino acid sequences of the variable regions, including the CDRs, of known monoclonal antibodies that block the CCR7 signaling mechanism in various ways and examined their function-inhibiting activity and immunogenicity. As a result, they succeeded in obtaining a novel anti-human CCR7 antibody that has a CDR amino acid sequence different from that of existing antibodies and has low immunogenicity. Furthermore, they found that this antibody has cytocidal activity through ADCC activity against cancer cells, particularly leukemia cells that highly express CCR7. Furthermore, they unexpectedly succeeded in enhancing the ADCC activity by defucosylation of the glycosylation of the antibody.

[0020] An antibody according to one embodiment of the present disclosure specifically binds to the extracellular domain of human CCR7, and has 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: 27, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 29, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 35, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 37, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 39.

[0021] Preferably, the antibody has a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:21 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:31.

[0022] Preferably, the antibody has an Fc region, the Fc region contains an N-glycoside-linked sugar chain, fucose is not bound to position 6 of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain, and the antibody has antibody-dependent cellular cytotoxicity.

[0023] An antibody according to one embodiment of the present disclosure specifically binds to the extracellular domain of human CCR7, and has a heavy-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 21 in which 1 to 10 amino acids have been substituted, added, or deleted, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 21, and a light-chain variable region comprising an amino acid sequence represented by SEQ ID NO: 31 in which 1 to 10 amino acids have been substituted, added, or deleted, or an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 31; and an Fc region comprising an N-glycoside-linked sugar chain in which fucose is not bound to position 6 of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain; and the antibody has antibody-dependent cellular cytotoxicity.

[0024] An antibody according to one embodiment of the present disclosure specifically binds to the extracellular domain of human CCR7, and has a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 45, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 49, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 55, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 57, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 59.

[0025] Preferably, the antibody has a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO:41 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO:51.

[0026] Preferably, the antibody has an Fc region, the Fc region contains an N-glycoside-linked sugar chain, fucose is not bound to position 6 of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain, and the antibody has antibody-dependent cellular cytotoxicity.

[0027] An antibody according to one embodiment of the present disclosure specifically binds to the extracellular domain of human CCR7, and has a heavy-chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence shown in SEQ ID NO: 41, or an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 41, and a light-chain variable region comprising an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence shown in SEQ ID NO: 51, or an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 51; the antibody has an Fc region, wherein the Fc region comprises an N-glycoside-linked sugar chain in which fucose is not bound to position 6 of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain; and has antibody-dependent cellular cytotoxicity.

[0028] A nucleic acid according to one embodiment of the present disclosure encodes the above-described antibody.

[0029] Preferably, the nucleic acid comprises a first nucleic acid encoding the amino acid sequence represented by SEQ ID NO:21 and a second nucleic acid encoding the amino acid sequence represented by SEQ ID NO:31.

[0030] Preferably, the nucleic acid comprises a first nucleic acid encoding the amino acid sequence represented by SEQ ID NO:41 and a second nucleic acid encoding the amino acid sequence represented by SEQ ID NO:51.

[0031] A cell according to one embodiment of the present disclosure comprises the nucleic acid described above.

[0032] A pharmaceutical according to one aspect of the present disclosure contains the above-described antibody as an active ingredient.

[0033] Preferably, the medicament is used for the treatment of cancer.

[0034] Preferably, the cancer is a blood cancer.

[0035] Preferably, the blood cancer is acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, or non-Hodgkin's lymphoma.

[0036] Preferably, the non-Hodgkin's lymphoma is B-cell lymphoblastic leukemia, T-cell lymphoblastic leukemia, chronic lymphocytic leukemia, follicular lymphoma, MALT lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, mature T / NK cell derived, peripheral T-cell lymphoma, adult T-cell leukemia, extranodal NK / T-cell lymphoma, or cutaneous lymphoma.

[0037] Preferably, the cancer is a solid cancer.

[0038] Preferably, the solid cancer is breast cancer, malignant breast tumor, gastric cancer, melanoma, non-small cell lung cancer, lung adenocarcinoma, cervical cancer, hepatocellular carcinoma, urothelial carcinoma, renal cell carcinoma, or squamous cell carcinoma.

[0039] Preferably, the squamous cell carcinoma is oral squamous cell carcinoma, esophageal squamous cell carcinoma, or pharyngeal squamous cell carcinoma.

[0040] According to the present disclosure, it is possible to provide a novel anti-human CCR7 antibody that has superior properties as a pharmaceutical raw material.

[0041] 1 is an explanatory diagram showing the alignment of the heavy chain variable regions of multiple humanized anti-CCR7 antibodies obtained in the Examples. FIG. 2 is an explanatory diagram showing the alignment of the light chain variable regions of multiple humanized anti-CCR7 antibodies obtained in the Examples. FIG. 3 is an explanatory diagram showing the alignment of the intracellular Ca of antibodies. ２＋ Graph showing the relationship between inhibitory activity against signals and antibody concentration. Graph showing the number of immunogenicity-positive donors for each antibody. Graph showing the relationship between antibody concentration and fluorescence intensity of flow cytometry histogram in hCCR7 gene-transfected cells. Graph showing the relationship between antibody concentration and fluorescence intensity of flow cytometry histogram in Granta-519 cells. Graph showing the relationship between antibody concentration and fluorescence intensity of flow cytometry histogram in MJ cells. Intracellular Ca in hCCR7 gene-transfected cells stimulated with CCL21. ２＋ 1 is a graph showing the results of evaluating the inhibitory activity of NB007-01 against signaling. ２＋ 1 is a graph showing the results of evaluating the inhibitory activity of NB007-01 against signaling. ２＋ 1 is a graph showing the results of evaluating the inhibitory activity of NB007-01 against signaling. ２＋8A is a graph showing the results of evaluating the inhibitory activity of CAP-100 against signaling. 8B is a graph showing the results of evaluating the inhibitory activity of NB007-01 against CCL19-induced cell migration in Granta-519 cells. 8C is a chromatogram of HPLC analyzing the sugar chain structure of defucosylated NB007-01. 8D is an explanatory diagram showing the abundance ratio of each sugar chain species calculated from the chromatogram of FIG. 8A. 8E is a graph showing the results of evaluating the cytotoxic function of each antibody by the ADCC Reporter Bioassay method. 8F is a graph showing the results of evaluating the cytotoxic function of each antibody by a cytotoxic activity measurement method using human PBMC. 8G is a graph showing the results of evaluating the antitumor effect of antibodies using a Granta-519 cell transplant model. 8H is a graph showing the results of evaluating the inhibitory effect of antibodies on cell infiltration into lymph nodes using a Granta-519 cell transplant model. 1 is a graph showing the results of evaluating the inhibitory effect of antibodies on cell infiltration into the liver using a Granta-519 cell transplant model.

[0042] In the present disclosure, complementarity determining region is abbreviated as CDR. In the present disclosure, the heavy chain variable region may be abbreviated as VH, the heavy chain constant region as CH, the light chain variable region as VL, and the light chain constant region as CL. In the present disclosure, the term "antibody" may be replaced with "immunoglobulin." In the present disclosure, the term "nucleic acid" may be replaced with "DNA" or "gene."

[0043] In the present disclosure, a humanized antibody refers to an antibody in which the CDRs are derived from a non-human animal and the other regions (framework regions, constant regions, etc.) are derived from humans. In the present disclosure, a chimeric antibody refers to an antibody in which the heavy chain variable region (VH) and light chain variable region (VL) are derived from a non-human animal and the other regions, such as the heavy chain constant region (CH) and light chain constant region (CL), are derived from humans.

[0044] <Human CCR7> CCR7 is a type of G protein-coupled receptor (GPCR) that penetrates the cell membrane seven times and is present with its N-terminus facing extracellularly and its C-terminus facing intracellularly. The gene (cDNA) encoding human CCR7 has already been isolated, and the amino acid sequence of human CCR7 is also known. This sequence information can be obtained, for example, from databases such as GenBank (e.g., GenBank: EAW60669.1). As an example, SEQ ID NO: 81 shows the nucleotide sequence of the human CCR7 gene. SEQ ID NO: 82 shows the amino acid sequence encoded by this nucleotide sequence.

[0045] Each domain of human CCR7 is considered to correspond to the following portion of the amino acid sequence shown in SEQ ID NO: 82. The left side indicates the amino acid number, and the right side indicates each domain. Note that the boundaries between each domain may vary slightly.

[0046] 1-24: Membrane transport signal peptide sequence (cleaved and removed after expression) 25-59: N-terminal domain 87-95: Intracellular loop 1 domain 117-130: Extracellular loop 1 domain 153-170: Intracellular loop 2 domain 192-219: Extracellular loop 2 domain 248-263: Intracellular loop 3 domain 290-313: Extracellular loop 3 domain 332-378: C-terminal domain

[0047] Various variants of human CCR7, such as amino acid substitutions, are known in addition to the variant shown in SEQ ID NO: 82. The "human CCR7" of the present disclosure includes such variants, as long as they have an extracellular domain and the function of CCR7.

[0048] <Anti-CCR7 Antibody> The antibody disclosed herein specifically binds to the extracellular domain of human CCR7. The antibody according to one embodiment has 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: 27, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 29, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 35, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 37, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 39. Preferably, the antibody is a humanized antibody having a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 21 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 31. An example of the antibody of this embodiment is NB007-01, which is described in the Examples below.

[0049] The heavy chain variable region (SEQ ID NO: 21) comprises the heavy chain CDRs 1 to 3 (SEQ ID NOs: 25, 27, and 29), with the regions other than the CDRs derived from a human antibody. Similarly, the light chain variable region (SEQ ID NO: 31) comprises the light chain CDRs 1 to 3 (SEQ ID NOs: 35, 37, and 39), with the regions other than the CDRs derived from a human antibody.

[0050] An antibody according to another embodiment has a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 45, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 49, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 55, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 57, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 59. Preferably, the antibody is a humanized antibody having a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 41, and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 51. An example of an antibody according to this embodiment is NB007-02, which is described in the Examples below.

[0051] The heavy chain variable region (SEQ ID NO: 41) comprises the heavy chain CDRs 1 to 3 (SEQ ID NOs: 45, 47, and 49), with the regions other than the CDRs derived from a human antibody. Similarly, the light chain variable region (SEQ ID NO: 51) comprises the light chain CDRs 1 to 3 (SEQ ID NOs: 55, 57, and 59), with the regions other than the CDRs derived from a human antibody.

[0052] The present disclosure further includes an antibody that specifically binds to the extracellular domain of human CCR7, having a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 5, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 7, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 9, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 15, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 17, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 19. Preferably, the antibody is a chimeric antibody having a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 1 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 11. Examples of such antibodies include the VH0 / VL0 described in the Examples below.

[0053] The present disclosure further includes an antibody that specifically binds to the extracellular domain of human CCR7, having a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 65, a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 67, a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 69, a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 75, a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 77, and a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 79. Preferably, the antibody is a humanized antibody having a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 61 and a light chain variable region comprising the amino acid sequence represented by SEQ ID NO: 71. An example of such an antibody is NB007-03, which is described in the Examples below.

[0054] The antibody may be a functional fragment of an antibody. Here, "functional fragment of an antibody" refers to a partial fragment of an antibody (i.e., immunoglobulin) that retains at least one action against an antigen. Examples of such partial fragments include F(ab')2, Fab, Fv, disulfide-linked Fv, single-chain antibodies (scFv, VH-VL), etc. Furthermore, the antibody of the present disclosure may be a multispecific antibody such as a diabody.

[0055] When the antibody is a functional fragment, it has the following effects, for example. That is, when applying the anti-human CCR7 antibody of the present disclosure to a pharmaceutical such as that described below, if a full-length antibody such as an IgG type is used, in addition to inhibiting the signal of the target receptor, damage to the target tissue may occur, which may lead to side effects. In such cases, employing a "functional fragment of an antibody" using only the variable region makes it easier to avoid the side effects described above.

[0056] The antibody preferably has the activity of blocking a CCR7-dependent intracellular signal transduction mechanism stimulated by a CCR7 ligand.

[0057] The antibody preferably has antibody-dependent cellular cytotoxicity (ADCC) activity, which makes the antibody particularly suitable as an active ingredient in a cancer therapeutic agent.

[0058] The present disclosure includes antibodies that are "functionally equivalent" to the above-described anti-human CCR7 antibodies. For example, the present disclosure discloses an antibody that specifically binds to the extracellular domain of human CCR7, and has a heavy chain variable region comprising an amino acid sequence represented by SEQ ID NO: 21 in which 1 to 10 amino acids have been substituted, added, or deleted, or an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 21, and a light chain variable region comprising an amino acid sequence represented by SEQ ID NO: 31 in which 1 to 10 amino acids have been substituted, added, or deleted, or an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 31, and that has antibody-dependent cellular cytotoxicity.

[0059] Similarly, an antibody that specifically binds to the extracellular domain of human CCR7 is disclosed, which has a heavy chain variable region comprising an amino acid sequence in which 1 to 10 amino acids have been substituted, added, or deleted in the amino acid sequence shown in SEQ ID NO: 41, or an amino acid sequence that has 90% or more identity to the amino acid sequence shown in SEQ ID NO: 41, and a light chain variable region comprising an amino acid sequence in which 1 to 10 amino acids have been substituted, added, or deleted in the amino acid sequence shown in SEQ ID NO: 51, or an amino acid sequence that has 90% or more identity to the amino acid sequence shown in SEQ ID NO: 51, and which has antibody-dependent cellular cytotoxicity.

[0060] The number of the above-mentioned substituted, added, or deleted amino acids is preferably 1 to 8, more preferably 1 to 5, and particularly preferably 1 to 3. The identity of the above-mentioned amino acid sequences is preferably 92% or more, more preferably 95% or more, and particularly preferably 97% or more.

[0061] <Defucosylation of Fc Region> The anti-human CCR7 antibody preferably has an Fc region, and the Fc region contains an N-glycoside-linked glycan, wherein fucose is not bound to position 6 of the N-acetylglucosamine at the reducing end of the N-glycoside-linked glycan. Simply put, it is preferable that the N-glycoside-linked glycan of the Fc region is defucosylated. In other words, it is preferable that core fucose is not bound to the N-glycoside-linked glycan of the Fc region. Defucosylation of the N-glycoside-linked glycan of the Fc region is expected to enhance the ADCC activity of the antibody and reduce the immunogenicity of the antibody.

[0062] An example of a method for producing an antibody in which the N-glycoside-linked sugar chains of the Fc region have been defucosylated (defucosylated antibody) is to use a host cell in which the activity of an enzyme involved in fucose synthesis is reduced or deleted. Examples of such enzymes include GDP-mannose 4,6-dehydratase (GMD), GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase (Fx), GDP-β-L-fucose pyrophosphorylase (GFPP), and alpha-1,6-fucosyltransferase (FUT8). An example of such a host cell is a CHO cell.

[0063] In actual production of a defucosylated antibody, it may be obtained as an antibody composition consisting of a mixture of a defucosylated antibody and a non-defucosylated antibody. The molar ratio of the defucosylated antibody to the non-defucosylated antibody in the antibody composition is not particularly limited, but preferably the defucosylated antibody accounts for 50% or more, more preferably 70% or more, and particularly preferably 80% or more. In other words, of the N-glycoside-linked sugar chains contained in the antibody composition, preferably 50% or more, more preferably 70% or more, and particularly preferably 80% or more are defucosylated.

[0064] <Nucleic Acid> The present disclosure includes nucleic acids (DNA) encoding the above-described antibodies. The nucleic acids include, for example, a first nucleic acid encoding a heavy chain variable region and / or a second nucleic acid encoding a light chain variable region. Specific examples include those containing a first nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 21 and a second nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 31. Other examples include those containing a first nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 41 and a second nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 51.

[0065] The nucleic acid may be incorporated into a vector. The vector is appropriately selected depending on the type of host cell to be introduced, etc. Vectors include vectors for gene therapy.

[0066] <Cells> The present disclosure encompasses cells containing the nucleic acid. For example, a cell containing a vector into which the nucleic acid has been incorporated is an example of such a cell. The type of cell is not particularly limited as long as it is capable of expressing the nucleic acid, for example, in which the vector functions. Examples include animal cells (COS cells, CHO cells, etc.), yeast, bacteria (Escherichia coli, etc.), plant cells, insect cells, etc.

[0067] <Method for Producing Antibodies> The antibodies can be produced using genetic recombination techniques. That is, recombinant cells that express the nucleic acids can be constructed, and the antibodies can be obtained from cultures of the cells.

[0068] An example of a method for constructing and producing a humanized antibody is given below. First, DNAs encoding the heavy chain CDR1 to 3 and the light chain CDR1 to 3 are prepared, each encoding the amino acid sequences shown in SEQ ID NOs: 25, 27, 29, 35, 37, and 39. Examples of such DNAs include the nucleotide sequences shown in SEQ ID NOs: 26, 28, 30, 36, 38, and 40, although other nucleotide sequences may also be used.

[0069] In another example, DNAs encoding heavy chain CDR1 to 3 and light chain CDR1 to 3 are prepared by encoding the amino acid sequences shown in SEQ ID NOs: 45, 47, 49, 55, 57, and 59. Examples of such DNAs include the nucleotide sequences shown in SEQ ID NOs: 46, 48, 50, 56, 58, and 60, but other nucleotide sequences may also be used.

[0070] Next, these DNAs are used to prepare DNAs encoding variable regions in which heavy chain CDRs 1 to 3 are grafted into the framework regions (FR) of VH of any human antibody. Similarly, DNAs encoding variable regions in which light chain CDRs 1 to 3 are grafted into the FR of VL of any human antibody are prepared. Each of the prepared DNAs is inserted into a vector containing a sequence encoding the CH or CL of a human antibody to construct a humanized antibody expression vector. The constructed expression vector is introduced into host cells to obtain recombinant cells that express the humanized antibody. The recombinant cells are then cultured, and the desired humanized antibody is obtained from the culture.

[0071] The method for purifying the antibody is not particularly limited, and any known method can be used. For example, the culture supernatant of the recombinant cell can be collected, and the antibody can be purified by a combination of known methods such as various types of chromatography, salting out, dialysis, and membrane separation.

[0072] <Drug> The present disclosure encompasses a pharmaceutical comprising the anti-human CCR7 antibody as an active ingredient. The pharmaceutical may be a pharmaceutical composition comprising the anti-human CCR7 antibody and a pharmaceutically acceptable carrier. Preferably, the pharmaceutical blocks a CCR7-dependent intracellular signal transduction mechanism induced by CCR7 ligand stimulation. Preferably, the pharmaceutical has antibody-dependent cellular cytotoxicity (ADCC) activity.

[0073] In a preferred embodiment, the medicament is used to treat cancer. For example, the medicament is used as an anticancer agent. The cancer may be either a blood cancer or a solid cancer.

[0074] Examples of blood cancers include acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, etc. Examples of non-Hodgkin's lymphomas include B-cell lymphoblastic leukemia, T-cell lymphoblastic leukemia, chronic lymphocytic leukemia, follicular lymphoma, MALT lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, mature T / NK cell-derived lymphoma, peripheral T-cell lymphoma, adult T-cell leukemia, extranodal NK / T-cell lymphoma, cutaneous lymphoma, etc.

[0075] Examples of solid cancers include breast cancer, malignant breast tumor, gastric cancer, melanoma, non-small cell lung cancer, lung adenocarcinoma, cervical cancer, hepatocellular carcinoma, urothelial carcinoma, renal cell carcinoma, squamous cell carcinoma, etc. Examples of squamous cell carcinomas include oral squamous cell carcinoma, esophageal squamous cell carcinoma, pharyngeal squamous cell carcinoma, etc.

[0076] Here, the term "treatment" means preventing or alleviating the progression and worsening of the pathological condition of a disease in a mammal that is at risk of contracting or is contracting the disease, and is used to mean a therapeutic procedure aimed at preventing or alleviating the progression and worsening of the symptoms of the disease.

[0077] <Administration Method> The above-mentioned pharmaceuticals can be administered orally or parenterally, systemically or locally. Examples of administration forms include injections, intranasal administrations, pulmonary administrations, and transdermal administrations. Injections can be administered systemically or locally, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous injection. The administration method can be selected appropriately depending on the age and symptoms of the patient. The dosage of the above-mentioned antibody can be selected, for example, from the range of 0.0001 mg to 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from the range of 0.001 to 100,000 mg of antibody per patient. However, the dosage of the above-mentioned antibody is not limited to these ranges.

[0078] <Formulation> The above-mentioned pharmaceuticals can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, USA). The above-mentioned pharmaceuticals can contain pharmaceutically acceptable carriers and additives. Examples of the carriers and additives include surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), colorants, flavorings, preservatives, stabilizers, buffers (phosphate, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonicity agents, binders, disintegrants, lubricants, flow enhancers, and flavoring agents, but are not limited to these. Other commonly used carriers and the like can also be used as appropriate. Specific examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts, etc. Furthermore, the composition may contain other low-molecular-weight polypeptides; proteins such as serum albumin, gelatin, immunoglobulins; and amino acids such as glycine, glutamine, asparagine, arginine, lysine, etc.

[0079] When preparing an aqueous solution for injection, examples include physiological saline, isotonic solutions containing glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride, and these may be used in combination with appropriate solubilizing agents, such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), and nonionic surfactants (polysorbate 80, HCO-50). If necessary, the antibody can also be encapsulated in microcapsules (such as microcapsules made of hydroxymethylcellulose, gelatin, poly(methyl methacrylate), etc.) or in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) (see, for example, "Remingto's Pharmaceutical Science 16th edition," Oslo Ed. 1980).

[0080] Furthermore, techniques for sustained drug release are known and may be applied to the above-mentioned pharmaceuticals (Langer et al., J. Biomed. Master. Res. 15:167-277 (1981); Langer, Chem. Tech. 12:9(8-105 (1982); U.S. Pat. No. 3,773,919; European Patent Application Publication No. 58,481; Sidman et al., Biopolymers 22: 547-556 (1983); European Patent Application Publication No. 133,988).

[0081] The above-mentioned drugs can be administered as antibody-drug conjugates (ADCs), which can be combined with antitumor drugs such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

[0082] <Application to Gene Therapy> The nucleic acid may be incorporated into a gene therapy vector to produce a gene therapy drug. Methods for administering the gene therapy drug (recombinant vector) include direct administration using a naked plasmid, administration by packaging the nucleic acid in a liposome or the like, administration by incorporating the nucleic acid into various viral vectors such as retroviral vectors, adenoviral vectors, vaccinia virus vectors, poxvirus vectors, adeno-associated virus vectors, and HVJ vectors (see Adolph, "Virus Genome Methods," CRC Press, Florida (1996)), and administration by coating the nucleic acid on a bead carrier such as colloidal gold particles (e.g., WO 93 / 17706).

[0083] The gene therapy drug may be administered by any method as long as the antibody is expressed in vivo and can exert its effect. Preferably, a sufficient amount is administered via an appropriate parenteral route, such as intravenous, intraperitoneal, subcutaneous, intradermal, intraadipose tissue, intramammary tissue, inhalation, or intramuscular route, including injection, infusion, gas-induced particle bombardment (using an electron gun, etc.), or mucosal administration such as nasal spray. Furthermore, the gene therapy drug may be administered ex vivo to cells using liposome transfection, particle bombardment (U.S. Patent No. 4,945,050), or viral infection, and then the cells may be reintroduced into an animal.

[0084] <Other Disclosures> The present disclosure includes a method for treating cancer, comprising administering to a cancer patient an effective amount of the antibody. The present disclosure includes the antibody for use in cancer treatment. The present disclosure includes use of the antibody for manufacturing a medicament for use in cancer treatment.

[0085] Example 1: Preparation of humanized anti-CCR7 antibodies To prepare a humanized version of the murine anti-CCR7 antibody R7-18 (described in International Publication No. 2012 / 043533 and Japanese Patent No. 5315495), a chimeric anti-CCR7 antibody "VH0 / VL0" was first prepared by fusing the heavy and light chain variant regions of R7-18 with the IgG1 Fc region of a human antibody. Furthermore, human frameworks were selected based on the homology between R7-18 and human germline VH and VK genes. Based on computer modeling, multiple variable region sequences were designed by adding mutations to the selected human germline VH and VK or grafted R7-18 CDR sequences so that they could support the predicted antibody conformation of R7-18. Of the designed humanized antibodies, "VH5-2 / VL1-3," "VH5-1 / VL1," and "VH5 / VL5," which maintained CCR7 neutralizing activity, were selected and named "NB007-01," "NB007-02," and "NB007-03," respectively. NB007-01, NB007-02, NB007-03, and CAP-100 (described in Patent Document 2) as a reference antibody, were produced using the QMCF method (described in International Publication No. 2006 / 084754 and Published Japanese Translation of PCT International Publication No. 2008-529510).

[0086] An alignment of the heavy chain variable region VH0 (SEQ ID NO: 1) of VH0 / VL0, the heavy chain variable region VH5-2 (SEQ ID NO: 21) of designed NB007-01, the heavy chain variable region VH5-1 (SEQ ID NO: 41) of designed NB007-02, and the heavy chain variable region VH5 (SEQ ID NO: 61) of designed NB007-03 is shown in Figure 1A. An alignment of the light chain variable region VL0 (SEQ ID NO: 11) of VH0 / VL0, the light chain variable region VL1-3 (SEQ ID NO: 31) of designed NB007-01, the light chain variable region VL1 (SEQ ID NO: 51) of designed NB007-02, and the light chain variable region VL5 (SEQ ID NO: 71) of designed NB007-03 is shown in Figure 1B. In Figures 1A and 1B, the region covering all of the CDRs defined by the KABAT and IMGT methods is enclosed in a dashed box.

[0087] The amino acid sequences (AA) of the heavy chain variable region, heavy chain constant region, heavy chain CDR1 to CDR3, light chain variable region, light chain constant region, and light chain CDR1 to CDR3 of each antibody obtained, as well as the nucleotide sequences of the DNA encoding them, are summarized in Tables 1-1 to 1-8.

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096] [Example 2] Efficacy of humanized anti-CCR7 antibodies Human CCR7 gene-transfected cells (described in WO 2012 / 043533 and Japanese Patent No. 5315495) were placed in a 96-well microplate at 2 × 10 per well. ４ The cells were seeded at an initial cell density of 100 μL and cultured for 2 days. After 2 days, the culture medium was replaced with a solution containing 3 μM Cal-520 (AAT, Bioquest), 0.05% Pluronic-F127, and 2.5 mM probenecid (Invitrogen). After 1 hour, 10 cells were added to each well. －６ ~10 －１０ The humanized anti-CCR7 antibodies NB007-01, NB007-02, or NB007-03 were added at a concentration range of 10 M. As a positive control, chimeric antibody VH0 / VL0 was added at a concentration of 10 M. －６ ~10 －１０ The concentration range of M was 1000.

[0097] After 15 minutes, 5 x 10 －８ Each cell was stimulated with CCL21 (R&D Systems) containing M. ２＋ The concentration was measured by Ca ２＋The measurement was performed using a signal measurement device (FDSS / μCELL; Hamamatsu Photonics), and the intracellular Ca of each additive (antibody) was measured. ２＋ The inhibitory activity of the antibody against intracellular Ca signaling was analyzed. The results are shown in Figure 2 and Table 2. ２＋ 1 is a graph showing the relationship between inhibitory activity against signals and antibody concentration. Inhibitory activity was calculated as a relative value by standardizing the inhibition rate of "no antibody but with ligand" to 0% and the inhibition rate of "no antibody and no ligand" to 100%. Table 2 shows the 50% inhibitory concentration (IC50) of each antibody calculated from the analysis results of FIG.

[0098] As shown in Figure 2, VH0 / VL0, NB007-01, NB007-02, and NB007-03 all increased intracellular Ca in a concentration-dependent manner. ２＋ This is because each additive inhibited the binding of CCL21 to human CCR7, resulting in an increase in intracellular Ca ２＋ This indicates that signal transduction was inhibited. The IC50 values ​​were 16.2 nM for VH0 / VL0, 18.2 nM for NB007-01, 16.4 nM for NB007-02, and 12.5 nM for NB007-03 (Table 2). These results demonstrate that the obtained humanized anti-CCR7 antibodies all have CCR7 inhibitory activity equivalent to that of the chimeric antibody VH0 / VL0.

[0099]

[0100] [Example 3] Immunogenicity of humanized anti-CCR7 antibodies The immunogenicity of NB007-01, NB007-02, and NB007-03 against human PBMCs was evaluated.

[0101] (1) Preparation of human PBMCs Human PBMCs were isolated from whole blood collected from healthy volunteers using a density gradient method, and then cryopreserved in human AB serum or fetal bovine serum supplemented with 10% dimethyl sulfoxide. The human PBMCs were stored at −180°C until use.

[0102] (2) DC-T cell assay. Monocytes were isolated from human PBMCs by magnetic separation and cultured for 5 days in DC medium containing interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The monocytes were differentiated into immature DCs (iDCs). The iDCs were collected, seeded onto cell culture plates, and the test substances NB007-01, NB007-02, or NB007-03 were added. The cells were then cultured overnight in DC medium containing interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α) to differentiate into mature DCs. NB007-01, NB007-02, and NB007-03 and the cytokine cocktail were removed by washing.

[0103] CD4+ T cells were isolated from human PBMCs using magnetic separation by negative selection (StemCells: EasySep ＴＭ CD4+ T cells were isolated using the Human CD4+ T Cell Enrichment Kit; 19052). CD4+ T cells and mature DCs were co-cultured in serum-supplemented medium for 6 days. 5-Ethynyl-2'-deoxyuridine (EdU) was added to the co-culture medium of mature DCs and T cells. After 16 hours, live and dead cells were separated by fluorescent labeling and further stained with T cell surface markers CD3 and CD4. After fixation and membrane permeabilization, EdU incorporation was stained with fluorescent azide and analyzed by flow cytometry (LSR Fortessa, BD). Proliferating Th cells were defined as CD3-positive, CD4-positive, and EdU-positive cells and analyzed using FlowLogic software.

[0104] (3) Statistical Analysis Positive responses to the test substance (NB007-01, NB007-02, or NB007-03) in donors were evaluated by calculating the stimulation index (SI), defined as the ratio of the mean response of the test substance to the mean response of the control or reference substance. KLH (Keyhole Limpet Hemocyanin) was used as the control or reference substance. The number of proliferating Th cells per well or the number of CD3-positive, CD4-positive, and EdU-positive cells was evaluated as a signal response. Donors with an SI > 2 were considered positive. Furthermore, to reduce the risk of false negatives, a statistical equivalence test was also performed. Here, the distribution-free resampling (DFR) method described by Moody et al. was used.

[0105] The results are shown in Table 3 and Figure 3. The number of donors who were statistically immunogenic positive for NB007-01 was 2 out of 20, indicating lower immunogenicity compared to the control substance. Similarly, the number of donors who were statistically immunogenic positive for NB007-02 was 7 out of 20, indicating lower immunogenicity compared to the control substance. Similarly, the number of donors who were statistically immunogenic positive for NB007-03 was 9 out of 20, indicating lower immunogenicity compared to the control substance. In particular, NB007-01, which had improved CDRs, had unexpectedly improved immunogenicity compared to NB007-03, which had no CDR improvements.

[0106]

[0107] [Example 4] Evaluation of cell binding by flow cytometry Binding to hCCR7 transfected cells and binding to MJ cells, a human lymphoma cell line derived from T cell lymphoma, was evaluated using NB007-01 labeled with the fluorescent dye Allophycocyanin (APC). Binding to Granta-519 cells, a human lymphoma cell line derived from B cell lymphoma, was evaluated using unlabeled NB007-01 and an Alexa Fluor 647-labeled anti-human IgG antibody (Thermo Fisher Scientific) as the secondary antibody for detection. For APC labeling, an APC Conjugation Kit - Lightning-Link (Abcam) was used. 100 μL of antibody solution diluted with PBS to 1 mg / mL was added to 10 μL of Modifier Reagent, and this was reacted with APC Conjugation Mix. After storing at room temperature in a dark place for 3 hours, 10 μL of Quencher Reagent was added and the mixture was used as a fluorescently labeled antibody.

[0108] The hCCR7 gene-transduced cells were detached from the culture dish using Cell Dissociation Buffer, enzyme-free, Hanks' Balanced Salt Solution (Thermo Fisher Scientific) and washed with PBS. Meanwhile, the required amount of suspended cells, Granta-519 cells and MJ cells, were each collected and washed with PBS. 1 × 10 cells were added to PBS. ７ An equal volume of goat serum (Thermo Fisher Scientific) was added to the cell suspension, which was suspended to a concentration of 100 μg / mL, and the mixture was left to stand at 4°C for 30 minutes for blocking. The mixture was centrifuged at 200 g for 5 minutes, the supernatant was removed, and 4 × 10 cells were collected. ６The cells were suspended in FACS buffer (PBS containing 1% FBS) to a concentration of 1 / mL, and 50 μL of the suspension was dispensed into a 96-well plate. Fluorescently labeled NB007-01 antibody or unlabeled NB007-01 diluted in FACS buffer was mixed and allowed to stand at 4°C for 1 hour. The cells were collected by centrifugation at 200g for 5 minutes and washed with 100 μL of FACS buffer. This washing procedure was repeated twice. The hCCR7 transfected cells and MJ cells that had been contacted with labeled NB007-01 were suspended in 100 μL of FACS buffer, and the binding of the labeled antibody to the cells was measured using a CytoFLEX flow cytometer (Beckman Coulter). Granta-519 cells contacted with unlabeled NB007-01 were suspended in 50 μL of Alexa Fluor 647-labeled anti-human IgG antibody diluted 800-fold with FACS buffer and allowed to stand for 1 hour at 4°C. After washing twice, the cells were suspended in 100 μL of FACS buffer and antibody binding was measured using a flow cytometer.

[0109] Graphs in which the horizontal axis represents antibody concentration and the vertical axis represents the geometric mean of fluorescence intensity of FACS histograms are plotted are shown in Figures 4A to 4C. These graphs confirmed the specific binding of NB007-01 to hCCR7 transfected cells (Figure 4A), Granta-519 cells (Figure 4B), and MJ cells (Figure 4C).

[0110] [Example 5] Intracellular Ca ２＋ Functional evaluation of NB007-01 by signal measurement hCCR7 gene-transfected cells were seeded in a 96-well microplate and cultured for 2 days in the same manner as in Example 2. The culture medium was replaced with a solution containing 3 μM Cal-520 (AAT Bioquest), 0.05% Pluronic-F127, and 2.5 mM probenecid (Invitrogen). After 1 hour, 10 －６ ~10 －１０ NB007-01 was added in a range of concentrations from 10 to 15 min. After 15 min, 5 × 10 －８ M's CCL21 (R&D Systems) or 1.5 x 10 －８ Each cell was stimulated with M CCL19 (R&D Systems). ２＋Using a signal measurement device, intracellular Ca ２＋ The inhibitory activity of NB007-01 on the signal was analyzed. The results are shown in Figures 5A and 5B. ２＋ 5A and 5B are graphs showing the relationship between inhibitory activity against signals and antibody concentration, with Fig. 5A representing the case where stimulation was performed with CCL21 and Fig. 5B representing the case where stimulation was performed with CCL19. Inhibitory activity was calculated as a relative value by standardizing the case where "no antibody, but with CCL21 or CCL19" was used as an inhibition rate of 0% and the case where "no antibody, but with CCL21 or CCL19" was used as an inhibition rate of 100%.

[0111] As shown in Figures 5A and 5B, NB007-01 inhibited CCL21- and CCL19-induced intracellular Ca ２＋ The IC50 values ​​were 17.8 nM for CCL21 stimulation and 21.4 nM for CCL19 stimulation. These results suggest that NB007-01 inhibits CCL21- and CCL19-induced intracellular Ca2+ signaling. ２＋ It was shown to inhibit signaling.

[0112] [Example 6] Intracellular Ca using human lymphoma cell lines ２＋ Evaluation of signal transduction inhibitory activity MJ cells were cultured in HBSS (CaCl ２ , MgCl ２ Wash with assay buffer containing 1.16 x 10 ６ The cells were mixed at a cell concentration of 1 × 10 cells / mL with a solution containing 1 μM Cal-520 (AAT Bioquest) and 0.05% Pluronic-F127, and incubated at 37°C. After 45 minutes, the cells were washed twice with assay buffer and diluted to 1 × 10 cells / mL. ６ The cell concentration was adjusted to 100 / mL, and 80 μL of the solution was seeded into each well of a 96-well black PDL-coated microplate (CELLCOAT, Grenier). NB007-01 or CAP-100 was added to each well at 8 concentrations, 3-fold serially diluted, within the concentration range of 250 nM to 0.11 nM, and the mixture was incubated for 15 minutes. ２＋The cells were placed in a signal analyzer (FDSS μCELL; Hamamatsu Photonics) and stimulated with 20 nM CCL21 (R&D Systems). ２＋ The inhibitory activity of each antibody against the signal was analyzed. The results for NB007-01 are shown in Figure 6A, and the results for CAP-100 are shown in Figure 6B. As shown in Figure 6A, NB007-01 inhibited CCL21-induced intracellular Ca ２＋ CAP-100 inhibited CCL21-induced intracellular Ca signaling in a concentration-dependent manner, with a 50% inhibitory concentration (IC50) of 14.8 nM. On the other hand, as shown in Figure 6B, CAP-100 inhibited CCL21-induced intracellular Ca ２＋ Although signal transduction was inhibited in a concentration-dependent manner, the maximum inhibition rate was about 50%.

[0113] [Example 7] Evaluation of cell migration inhibitory activity using a human lymphoma cell line Granta-519 cells were stained with CytoRed (Dojindo Laboratories) and then washed twice with RPMI + 0.5% BSA. ６ NB007-01 (0.3 μg to 100 μg / mL) or a negative control human IgG1 antibody (Icosagen) (1 μg / mL or 100 μg / mL) was added to a cell suspension at 100 μg / mL, and the mixture was left to stand at 37°C for 30 minutes.

[0114] 50 ng / mL CCL19 was added to the wells of a receiver plate equipped with an insert-integrated 96-well Transwell with a 3.0 μm polycarbonate membrane (Corning). After placing the Transwell insert on the receiver plate, 50 μL of the cell suspension described above was seeded into the insert and allowed to stand at 37°C. After 4 hours of standing, the insert was removed, and each well was photographed in three fields using a 4x fluorescent lens to count the number of migrated cells. The inhibitory activity was calculated as a relative value, normalized by setting the inhibition rate of "no antibody, with CCL19" as 0% and the inhibition rate of "no antibody, without CCL19" as 100%. Figure 7 shows the inhibitory effect of NB007-01 on CCL19-dependent cell migration. The IC50 of NB007-01 was 25.5 nM. The inhibition rates of the negative control human IgG1 antibody at 1 μg / mL and 100 μg / mL were 3.0% and 3.3%, respectively.

[0115] [Example 8] Preparation of Defucosylated NB007-01 The GlymaxX method (WO 2011 / 035884, Japanese Patent No. 5746183) was used as a method for defucosylation of the antibody. Defucosylated NB007-01 was prepared using the same procedure as in Example 1 for preparing NB007-01 by the QMCF method, except that a CHO cell line (CHOEBNALT85-RMD C4) modified by the GlymaxX method was used as the CHO cells for antibody gene expression. The antibody was purified.

[0116] AdvanceBio Gly-X-N-glycan prep with InstantPC Kit (Agilent), sialidase (AdvanceBio Sialidase A) (Agilent, GK80040), β1-3,4 galactosidase (β1-3,4 galactosidase; BTG) (NEB, P0746S), β-N-acetylglucosaminidase S (β-N-acetylglucosaminidase S; GUH) (NEB, P0744), and α1-2,4,6 fucosidase O (α1-2,4,6 fucosidase O) were used. N-glycans were cleaved from purified antibodies and labeled using PEG-1000 (FucO; FucO) (NEB, P0749) according to the manufacturer's instructions. The labeled N-glycans were analyzed by HPLC (Agilent 1260 Infinity II) using an AdvanceBio Glycan 2.7 μm column (Agilent) according to the manufacturer's instructions. The HPLC chromatogram is shown in Figure 8A, and the abundance of each glycan species calculated from the chromatogram is shown in Figure 8B. In Figure 8A, "Undig" indicates untreated, "Sialidase A" indicates sialidase treatment, "Sial+BTG" indicates sialidase + BTG treatment, "Sial+BTG+GUN" indicates sialidase + BTG + GUN treatment, and "Sial+BTG+GUN+FucO" indicates sialidase + BTG + GUN + FucO treatment. As shown in Figures 8A and 8B, the majority of the separated peaks were assigned to specific glycans based on their relative retention times (glucose units), accounting for 89% of the peak area for all samples. Furthermore, cleavage experiments with FucO showed little change in the peaks, indicating that more than 99% of the identified glycan species were defucosylated.

[0117] Example 9 Evaluation of the Cytotoxic Function of Defucosylated NB007-01 (1) ADCC Reporter Bioassay The ADCC function of the defucosylated antibody was evaluated using the ADCC Reporter Bioassay, V Variant (Promega). 1.4 mL of Low IgG Serum included in the kit was mixed with 33.6 mL of RPMI 1640 Medium to prepare an ADCC Assay Buffer. 5 × 10 Granta-519 cells were cultured in the buffer. ５ The cells were suspended to a cell density of 1000kJ / mL. Furthermore, a diluted solution of the antibody to be used as the test substance was prepared using ADCC Assay Buffer. NB007-01, defucosylated NB007-01, CAP-100, and a negative control human IgG (manufactured by Icosagen) were used as test substances. 630 μL of the ADCC Bioassay Effector Cells included in the kit were taken and added to 3.6 mL of ADCC Assay Buffer to prepare an effector cell suspension. 25 μL each of the antibody dilution solution, Granta-519 cell suspension, and effector cell suspension were dispensed into a Culture Plate-96 (PerkinElmer), and incubated at 37°C, 5% CO ２ The plate was then left to stand for 6 hours under a dark environment. The luciferase assay substrate included in the kit was dissolved in the luciferase assay buffer, and 75 μL of the solution was added to each well of the plate. After standing for 15 minutes at room temperature in the dark, the relative luminescence intensity (RLU) was measured using an ARVO microplate reader (PerkinElmer). Figure 9 is a graph plotting antibody concentration on the horizontal axis and RLU on the vertical axis. RLU is an index of effector cell activation, and this bioassay is a functional measurement method based on the ADCC mechanism of action. As shown in Figure 9, defucosylated NB007-01 (EC50 = 16.1 pM) had significantly improved ADCC function compared to NB007-01 (EC50 = 123 pM) and was superior to CAP-100 (EC50 = 57.1 pM).

[0118] (2) Cytotoxicity Measurement Method Using Human PBMCs (a) Preparation of Human PBMCs Frozen human PBMCs were thawed in a water bath at 37°C. After washing with complete medium, 2 × 10 ６ The cells were resuspended in complete medium to a concentration of 1 mL / mL and incubated at 37°C in 5% CO ２ The cells were cultured overnight under the conditions.

[0119] (b) Pretreatment of Granta-519 cells. Granta-519 cells were cultured in ADCC medium at a viable cell concentration of 3 × 10 ４ The resulting solution was prepared to a volume of 80 μL per well and seeded into each well of a 96-well round-bottom plate. 20 μL of a test substance diluted with ADCC medium was then added to each well and allowed to stand at room temperature for 30 minutes. The test substances used were defucosylated NB007-01 (1000-0.0001 ng / mL), CAP-100 (10,000-0.1 ng / mL), and Rituximab (10,000 ng / mL, Roche).

[0120] (c) Measurement of ADCC activity Human PBMCs were used as effector cells (E) and Granta-519 cells as target cells (T) at an E:T ratio of 50:1. Specifically, the human PBMCs in (a) were washed with ADCC medium, and the cell concentration was adjusted to 1.5 × 10 ６ 100 μL of cells were added to each well of (b). After incubation at 37°C for 5 hours, the cells were centrifuged at 250 × g for 4 minutes, and 50 μL of the supernatant was collected into each well of a clear flat-bottom 96-well plate. Additionally, 45 minutes before collecting the supernatant, 20 μL of lysis solution (CytoTox 96 Non-Radioactive Cytotoxicity Assay kit, Promega, G1780) was added to the wells to provide a control for maximum cytotoxicity.

[0121] 50 μL of substrate mixture (CytoTox 96 Non-Radioactive Cytotoxicity Assay kit, Promega, G1780) was added to each well, and the plate was mixed for 30 seconds. The plate was then left to stand at room temperature for 30 minutes. 50 μL of stop reagent (CytoTox 96 Non-Radioactive Cytotoxicity Assay kit, Promega, G1780) was added to each well, mixed for 30 minutes, and absorbance was measured at 490 nm. Cytotoxicity (%) was calculated as a relative value, normalized to 100% for maximum cytotoxicity and 0% for no test substance. A graph plotting the calculated values ​​is shown in Figure 10. In FIG. 10, "aNB007" means defucosylated NB007-01.

[0122] The results of this test also showed that defucosylated NB007-01 (EC50 = 1.9 ng / mL, 12.6 pM in IgG equivalent) exhibited superior cytotoxic activity to CAP-100 (EC50 = 26.3 ng / mL, 175 pM in IgG equivalent). Furthermore, Rituximab, which is clinically used for its antitumor activity based on ADCC activity, only exhibited 18.6% cytotoxic activity at a concentration of 10,000 ng / mL, suggesting the superiority of defucosylated NB007-01 in tumor therapeutic effects.

[0123] Example 10 Antitumor Activity of Humanized Anti-CCR7 Antibodies The antitumor activities of NB007-01, defucosylated NB007-01, and CAP-100 were evaluated using a tumor transplant model using Granta-519 cells. Female CB17 / SCID mice aged 6-8 weeks were used as model animals.

[0124] Granta-519 cells were cultured in RPMI-1640 medium containing 20% ​​FBS at 37°C and 5% CO ２ The cells were cultured under 1 × 10 ７ Granta-519 cells at a concentration of 0.1 mL per cell were transplanted intravenously into 6-8 week-old CB17 / SCID mice. The day of transplantation was designated as day 0. On day 1 after transplantation, the mice were divided into groups based on their body weight. The grouping was performed using the matched distribution method (Study Director). ＴＭThe test was performed using the following software:

[0125] Administration of the test substances began two days after cancer cell transplantation. The dose was 3 mg / kg for NB007-01, CAP-10, and isotype control human IgG1, and 0.03 mg / kg for defucosylated NB007-01. Each test substance was administered in a volume of 10 μL / g, twice a week.

[0126] After cancer cell transplantation, each animal was monitored daily for morbidity and mortality. Additionally, the animals were monitored for abnormalities in mobility, food and water consumption, weight gain or loss, and eye and coat condition due to tumor growth or treatment. Body weight was measured twice a week. Deaths and clinical signs were recorded in detail for each animal. Weight was recorded by the Study Director. ＴＭ The antitumor activity was evaluated by Kaplan-Meier survival curves based on the survival rate of each individual.

[0127] In addition, livers and lymph nodes were harvested from some animals in each group 24 days after cancer cell transplantation. The harvested organs were fixed in 10% formalin buffer, and paraffin blocks were prepared. The prepared blocks were cut into 4 μm sections and treated at 60°C for 30 minutes. The infiltration rate of Granta-519 cells was measured by immunostaining. Granta-519 cells were identified using an antibody against human CD45 (Cell Signaling Technology, Cat#13917), a hematopoietic cell surface marker. The sections were activated using EDTA, pH 9.0, at 100°C for 20 minutes. The antibody was diluted 1:800. All slides after immunostaining were scanned at 40x magnification using the NanoZoomer-HT 2.0 / Pannoramic SCAN Image system and saved. For immunostaining scoring, the number of CD45-positive cells was measured using the HALO™ platform and the images were analyzed by 1 mm ２ The number of cells was evaluated per 100 cells.

[0128] The time course of survival rates, as expressed by Kaplan-Meier survival curves, is shown in Figure 11 . The number of CD45-positive cells in lymph nodes is shown in Figure 12A, and the number of CD45-positive cells in the liver is shown in Figure 12B. As shown in Figure 11 , NB007-01 extended survival compared to the isotype control group, and even extended survival more than CAP-100. As shown in Figures 12A and 12B, NB007-01 potently inhibited cancer cell infiltration into various organs, and its effect in the liver was stronger than that of CAP-100. This suggests that NB007-01 exerts its antitumor effect by inhibiting lymphoma infiltration into various organs. Furthermore, defucosylated NB007-01 exhibited a survival-prolonging effect comparable to that of CAP-100 at a dose 1 / 100 of that of NB007-01. This indicates that defucosylated NB007-01 with enhanced cytotoxic activity can be expected to have an antitumor effect at low doses.

Claims

1. An antibody that specifically binds to the extracellular domain of human CCR7, 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: 27; a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 29; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 35; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 37; and having a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 39; A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 21, and An antibody having a light chain variable region comprising the amino acid sequence represented by sequence number 31.

2. having an Fc region, wherein the Fc region comprises an N-glycoside-linked sugar chain; fucose is not bound to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain; The antibody according to claim 1, which has antibody-dependent cellular cytotoxicity.

3. An antibody that specifically binds to the extracellular domain of human CCR7, a heavy chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 45; a heavy chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 47; a heavy chain CDR3 comprising the amino acid sequence represented by SEQ ID NO: 49; a light chain CDR1 comprising the amino acid sequence represented by SEQ ID NO: 55; a light chain CDR2 comprising the amino acid sequence represented by SEQ ID NO: 57; and having a light chain CDR3 comprising the amino acid sequence represented by SEQ ID NO:59; A heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 41, and An antibody having a light chain variable region comprising the amino acid sequence represented by sequence number 51.

4. having an Fc region, wherein the Fc region comprises an N-glycoside-linked sugar chain; fucose is not bound to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chain; The antibody according to claim 3, which has antibody-dependent cellular cytotoxicity.

5. A nucleic acid encoding the antibody of any one of claims 1 to 4.

6. The nucleic acid of claim 5, comprising a first nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 21 and a second nucleic acid encoding the amino acid sequence represented by SEQ ID NO:

31.

7. The nucleic acid of claim 5, comprising a first nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 41 and a second nucleic acid encoding the amino acid sequence represented by SEQ ID NO:

51.

8. A cell comprising the nucleic acid of claim 5.

9. A cell comprising the nucleic acid of claim 6.

10. A cell comprising the nucleic acid of claim 7.

11. A pharmaceutical comprising the antibody according to any one of claims 1 to 4 as an active ingredient.

12. The pharmaceutical composition according to claim 11, which is used for the treatment of cancer.

13. The pharmaceutical composition of claim 12, wherein the cancer is a blood cancer.

14. The pharmaceutical composition of claim 13, wherein the blood cancer is acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, or non-Hodgkin's lymphoma.

15. The pharmaceutical composition of claim 14, wherein the non-Hodgkin's lymphoma is B-cell lymphoblastic leukemia, T-cell lymphoblastic leukemia, chronic lymphocytic leukemia, follicular lymphoma, MALT lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, mature T / NK cell-derived lymphoma, peripheral T-cell lymphoma, adult T-cell leukemia, extranodal NK / T-cell lymphoma, or cutaneous lymphoma.

16. The pharmaceutical composition of claim 12, wherein the cancer is a solid cancer.

17. The pharmaceutical composition of claim 16, wherein the solid cancer is breast cancer, malignant breast tumor, gastric cancer, melanoma, non-small cell lung cancer, lung adenocarcinoma, cervical cancer, hepatocellular carcinoma, urothelial carcinoma, renal cell carcinoma, or squamous cell carcinoma.

18. The pharmaceutical composition of claim 17, wherein the squamous cell cancer is oral squamous cell carcinoma, esophageal squamous cell carcinoma, or pharyngeal squamous cell carcinoma.