Antibodies, nucleic acids, cells and drugs

Abstract

The invention provides an antibody specifically binding to an extracellular domain of human CCR7. The present invention relates to a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO.25, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO.27, a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO.29, a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO.35, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO.37, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO.39.

Description

Technical Field

[0001] The present invention 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 drug containing the antibody as an active ingredient. Background Art

[0002] Chemokines are proteins that regulate the migration of various cells and cell functions. Abnormal functions of chemokines and their receptors cause various diseases such as fibrosis, autoimmune diseases, acute and chronic inflammation, and cancer. So far, although agents that control the activities of chemokines and their receptors have been developed and clinically applied, it is difficult to say that the problems have been fully solved.

[0003] In order to activate activities such as the migration of specific chemokine-expressing cells and the regulation of cell functions, it is necessary to bind selectively to a cell membrane receptor for chemokines. Approximately 20 chemokine receptors have been discovered, and all chemokine receptors are seven-transmembrane proteins (GPCRs) that bind to trimeric G proteins. When a chemokine binds to a receptor, the Gα unit of the trimeric G protein is released. As a result, the intracellular Ca concentration increases, or phosphatidylinositol 3-kinase (PI3K, phosphatidylinositol 3 kinase), the small Rho GTPases pathway, and other pathways are activated to exert functions. Any chemokine receptor is activated by a relatively selective chemokine, but the primary structure of the protein or the intracellular activation mechanism is 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 in physiology and pathology is controlled by the expression of their respective proteins in specific cells and tissues at specific times (during inflammation) (Non-Patent Document 1).

[0004] Human CC motif receptor 7 (CC MOTIF, RECEPTOR 7; alias: EBI1, CMKBR7; hereinafter referred to as "CCR7") was initially discovered as a GPCR selectively expressed in lymphocytes by Epstein-Barr virus infection (Non-Patent Document 2). Then, it was found that CCR7 is a selective chemokine receptor for CCL19 (alias: ELC) and CCL21 (alias: 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 under physiological conditions. It is known that such cells are guided by CCR7 to lesions such as inflammatory sites, and the inflammatory response and immune response are enhanced. In addition, abnormal activation of CCR7 causes various diseases such as autoimmune diseases, fibrosis after acute and chronic inflammation, and cancer metastasis.

[0005] In cancer treatment, it is important to inhibit the proliferation of primary cancer and prevent recurrence accompanied by distant metastasis. In addition to conventional surgical and chemotherapy, although the efficacy has been improved by molecularly targeted drugs (e.g., kinase inhibitors), antibody-drug therapies, and cancer immunotherapies, there is a further expectation for the development of therapeutic drugs that prevent recurrence accompanied by distant metastasis. As mechanisms of distant metastasis, there are cases via blood vessels from the primary cancer and cases via lymphoid tissues. To date, although extracellular matrix protease inhibitors (MMP inhibitors) and the like have been developed as metastasis preventive drugs, there have been no cases of clinical application.

[0006] Various studies have shown that CCR7 is expressed in various tumor cells such as B-cell chronic lymphocytic leukemia, non-Hodgkin lymphoma, breast cancer cells, and malignant breast tumors. In addition, it has been clarified 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 ligands of CCR7, are highly expressed in lymph nodes, selective inhibition of CCR7 function can be expected to inhibit lymphatic metastasis of cancer cells.

[0007] As a selective CCR7 function inhibitor, small molecule compounds and monoclonal antibodies that selectively bind to the extracellular region of the receptor and block the signal transduction mechanism of CCR7 based on CCL19 / CCL21 stimulation can be considered as candidates.

[0008] Patent Document 1 discloses eight monoclonal antibodies that specifically bind to the extracellular domain of human CCR7. In addition, the amino acid sequences of the complementarity determining region (CDR), heavy chain variable region, and light chain variable region of each monoclonal antibody are disclosed. These anti-human CCR7 antibodies are particularly effective for the treatment of fibrosis.

[0009] Patent Document 2 discloses a humanized antibody of a monoclonal antibody that specifically binds to human CCR7. In addition, it is disclosed that the Fc mutant of the humanized antibody or complement-dependent cytotoxicity enhanced thereby kills various tumor cells of blood cancer via its effector function.

[0010] Patent Document 3 discloses an antibody-drug conjugate in which a monoclonal antibody that specifically binds to human CCR7 and a mutant that does not have an effector function are conjugated with an anticancer agent.

[0011] However, the antibodies described in Patent Documents 1 to 3 have not reached practical use as therapeutic drugs. In this technical field, another or improved anti-human CCR7 antibody with more excellent characteristics is required as a drug raw material.

[0012] The characteristics of monoclonal antibodies required as raw materials for antibody drugs that inhibit the function of CCR7 include high selectivity for the receptor, strong function inhibitory activity, high solubility, thermal stability, low aggregation, and low immunogenicity. All of these are characteristics required for the practical application of antibody drugs. In particular, low immunogenicity in humans is important in determining the safety and administration period of antibody drugs. The necessity of low immunogenicity is also shown to be important in the guidelines for the development of biopharmaceuticals by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA).

[0013] As a general method for achieving low immunogenicity of antibodies, antibody humanization can be cited. Specifically, for antibodies obtained using rodents, in order to maintain high selectivity for the receptor and strong function inhibitory activity and reduce immunogenicity, the antibody can be humanized using the CDR transplantation method while maintaining the amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 at 100%. However, regarding antibodies against GPCRs, only two have been marketed as drugs, and it is not easy to reduce immunogenicity while maintaining high target molecule specificity and strong function inhibitory activity.

[0014] In addition, it is known that the in vivo anti-tumor activity of antibody drugs is caused not only by directly blocking the intracellular signal transduction mechanism of the receptor but also by antibody-dependent cell-mediated cytotoxicity (ADCC) activity and complement-dependent cellular cytotoxicity (CDC) activity. 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 or natural killer (NK) cells with Fc receptors that recognize the Fc region of the antibody are attracted to the target cell and kill the cancer cell or target cell bound by the antibody. It is considered that in cancer treatment, antibodies with ADCC activity in addition to neutralizing activity exhibit anti-tumor activity with a smaller dosage, and thus are useful. The strength of ADCC activity is determined by the expression level of the antigen in the target cell, the strength of the binding between the antigen and the antibody, the selectivity of the antibody, and the strength of the affinity between the Fc region of the antibody and the Fc receptor. It is not clear whether all GPCR antibodies have ADCC activity, and the strength of ADCC activity varies for each antibody.

[0015] As a method for artificially enhancing the ADCC activity of antibodies, there are methods for changing the amino acid sequence of the Fc region of the antibody and methods for controlling the sugar chain structure of the antibody. As a method for controlling the sugar chain structure of the antibody, it is known to remove fucose at the reducing end of the N-type complex sugar chain of the antibody (afucosylation).

[0016] Prior art documents

[0017] Patent documents

[0018] Patent Document 1: WO 2012 / 043533

[0019] Patent Document 2: WO 2017 / 025569

[0020] Patent Document 3: WO 2021 / 220199

[0021] Non-patent documents

[0022] Non-patent Document 1: Viola A, Luster AD “Chemokines and their receptors: drug targets in immunity and inflammation”, Annu Rev Pharmacol Toxicol. 2008;48:171-197

[0023] Non-patent Document 2: Birkenbach, 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. Summary of the invention

[0024] Technical problems to be solved by the invention

[0025] As described above, antibodies that inhibit the function of CCR7 are expected to be useful as antibody drugs, but there are no antibodies that have been put into practical use as therapeutic drugs. Therefore, an object of the present invention is to provide a novel anti-human CCR7 antibody having more excellent properties as a pharmaceutical raw material.

[0026] Technical means for solving the problems

[0027] The present inventors humanized a monoclonal antibody that blocks the signal transduction mechanism of CCR7 described in Patent Document 1 using the CDR grafting method. However, in this method, an antibody with low immunogenicity, which is desired for a drug, could not be obtained. Therefore, the inventors made various modifications to the amino acid sequences of the variable regions containing CDRs that constitute a known monoclonal antibody that blocks the signal transduction mechanism of CCR7, and studied the function inhibitory activity and immunogenicity. As a result, a novel anti-human CCR7 antibody with an amino acid sequence of CDRs different from those of existing antibodies and low immunogenicity was successfully obtained. Furthermore, it was found that this antibody has a cytotoxic effect based on ADCC activity on cancer cells, particularly leukemia cells highly expressing CCR7. In addition, by defucosylating the sugar chains of this antibody, the ADCC activity was unexpectedly enhanced.

[0028] An antibody according to one embodiment of the present invention is an antibody that 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.

[0029] 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.

[0030] Preferably, the antibody has an Fc region that contains an N-glycoside bond sugar chain and does not have fucose bound to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside bond sugar chain, and the antibody has antibody-dependent cell inhibitory activity.

[0031] An antibody according to one embodiment of the present invention is an antibody that 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 represented by SEQ ID NO. 21, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 21; 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 represented by SEQ ID NO. 31, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 31. The antibody has an Fc region that contains an N-glycoside bond sugar chain, and fucose is not bound to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside bond sugar chain. The antibody has antibody-dependent cell inhibitory activity.

[0032] An antibody according to one embodiment of the present invention is an antibody that 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.

[0033] 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.

[0034] Preferably, the antibody has an Fc region that contains an N-glycoside bond sugar chain, and fucose is not bound to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside bond sugar chain. The antibody has antibody-dependent cell inhibitory activity.

[0035] Regarding the antibody of one aspect of the present invention, it is an antibody that specifically binds to the extracellular domain of human CCR7 and has: a heavy chain variable region that contains an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO. 41, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 41; and a light chain variable region that contains an amino acid sequence in which 1 to 10 amino acids are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO. 51, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 51. The antibody has an Fc region that contains an N-glycoside bond sugar chain, and fucose is not bound to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside bond sugar chain. The antibody has antibody-dependent cell inhibitory activity.

[0036] A nucleic acid of one aspect of the present invention encodes the above-mentioned antibody.

[0037] Preferably, the nucleic acid contains a first nucleic acid and a second nucleic acid. The first nucleic acid encodes the amino acid sequence represented by SEQ ID NO. 21, and the second nucleic acid encodes the amino acid sequence represented by SEQ ID NO. 31.

[0038] Preferably, the nucleic acid contains a first nucleic acid and a second nucleic acid. The first nucleic acid encodes the amino acid sequence represented by SEQ ID NO. 41, and the second nucleic acid encodes the amino acid sequence represented by SEQ ID NO. 51.

[0039] A cell of one aspect of the present invention contains the above-mentioned nucleic acid.

[0040] A drug of one aspect of the present invention contains the above-mentioned antibody as an active ingredient.

[0041] Preferably, the drug is used for the treatment of cancer.

[0042] Preferably, the cancer is hematological cancer.

[0043] Preferably, the hematological cancer is acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, or non-Hodgkin lymphoma.

[0044] Preferably, the non-Hodgkin 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 lymphoma, mature T / NK cell-derived, peripheral T-cell lymphoma, adult T-cell leukemia, extranodal NK / T-cell lymphoma, or cutaneous lymphoma.

[0045] Preferably, the cancer is a solid cancer.

[0046] 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.

[0047] Preferably, the squamous cell carcinoma is oral squamous cell carcinoma, esophageal squamous cell carcinoma or pharyngeal squamous cell carcinoma.

[0048] Advantages of the Invention

[0049] According to the present invention, a novel anti-human CCR7 antibody having more excellent properties as a pharmaceutical raw material can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1A is an explanatory diagram showing the alignment of the heavy chain variable regions of multiple humanized anti-CCR7 antibodies obtained in the examples.

[0051] Figure 1B is an explanatory diagram showing the alignment of the light chain variable regions of multiple humanized anti-CCR7 antibodies obtained in the examples.

[0052] Figure 2 is a graph showing the relationship between the inhibitory activity of the antibody against intracellular Ca 2+ signals and the antibody concentration.

[0053] Figure 3 is a graph showing the number of immunogenicity-positive donors for each antibody.

[0054] Figure 4A is a graph showing the relationship between the antibody concentration in hCCR7 gene-introduced cells and the fluorescence intensity of the histogram of flow cytometry.

[0055] Figure 4B is a graph showing the relationship between the antibody concentration in Granta-519 cells and the fluorescence intensity of the histogram of flow cytometry.

[0056] Figure 4C is a graph showing the relationship between the antibody concentration in MJ cells and the fluorescence intensity of the histogram of flow cytometry.

[0057] Figure 5A is a graph showing the results of evaluating the inhibitory activity of NB007-01 against intracellular Ca 2+ signals upon CCL21 stimulation in hCCR7 gene-introduced cells.

[0058] Figure 5B ​​​​​​​​​is a graph showing the results of evaluating the inhibitory activity of NB007-01 on intracellular Ca 2+ signal upon CCL19 stimulation in cells transfected with the hCCR7 gene.

[0059] Figure 6A is a graph showing the results of evaluating the inhibitory activity of NB007-01 on intracellular Ca 2+ signal upon CCL21 stimulation in MJ cells.

[0060] Figure 6B is a graph showing the results of evaluating the inhibitory activity of CAP-100 on intracellular Ca 2+ signal upon CCL21 stimulation in MJ cells.

[0061] Figure 7 is a graph showing the results of evaluating the inhibitory activity of NB007-01 on CCL19-induced cell migration in Granta-519 cells.

[0062] Figure 8A is an HPLC chromatogram analyzing the sugar chain structure of afucosylated NB007-01.

[0063] Figure 8B is a diagram showing the proportion of each sugar chain species calculated based on the Figure 8A chromatogram.

[0064] Figure 9 is a graph showing the results of evaluating the cytotoxic function of each antibody obtained by the ADCC Reporter Bioassay method.

[0065] Figure 10 is a graph showing the results of evaluating the cytotoxic function of each antibody obtained by the cell inhibition activity assay using human PBMC.

[0066] Figure 11 is a graph showing the results of evaluating the anti-tumor effect of an antibody using a xenograft model of Granta-519 cells.

[0067] Figure 12A is a graph showing the results of evaluating the inhibitory effect of an antibody on cell infiltration into lymph nodes using a xenograft model of Granta-519 cells.

[0068] Figure 12B is a graph showing the results of evaluating the inhibitory effect of an antibody on cell infiltration into the liver using a xenograft model of Granta-519 cells. Detailed implementation mode

[0069] ​​​​​​​​​​In the present invention, the complementarity determining region is abbreviated as CDR. In the present invention, the heavy chain variable region is sometimes 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 invention, the term "antibody" can be replaced by "immunoglobulin". In the present invention, the term "nucleic acid" can be replaced by "DNA" or "gene".

[0070] The humanized antibody of the present invention refers to an antibody in which the CDR is derived from an animal other than human, and other regions (such as the framework region, constant region, etc.) are derived from human. The chimeric antibody of the present invention refers to an antibody in which the heavy chain variable region (VH) and the light chain variable region (VL) are derived from an animal other than human, and other regions such as the heavy chain constant region (CH) and the light chain constant region (CL) are derived from human.

[0071] <human CCR7>

[0072] CCR7 is a kind of G protein-coupled receptor (GPCR), which traverses the cell membrane 7 times, with its N-terminus facing the outside of the cell and its C-terminus facing the inside of the cell. The gene (cDNA) encoding human CCR7 has been isolated, and the amino acid sequence of human CCR7 is also known. This sequence information can be obtained from databases such as GenBank (for example, GenBank: EAW60669.1). As an example, SEQ ID NO.81 represents the base sequence of the human CCR7 gene. SEQ ID NO.82 represents the amino acid sequence encoded by this base sequence.

[0073] It is considered that each domain of human CCR7 corresponds to the following parts in the amino acid sequence shown in SEQ ID NO.82. The amino acid numbers are on the left, and the domains are on the right. It should be noted that there may be some deviations in the boundaries between the domains.

[0074] 1 - 24: Membrane transfer signal peptide sequence (cleaved / removed after expression)

[0075] 25 - 59: N-terminal domain

[0076] 87 - 95: Intracellular loop 1 domain

[0077] 117 - 130: Extracellular loop 1 domain

[0078] 153 - 170: Intracellular loop 2 domain

[0079] 192 - 219: Extracellular loop 2 domain

[0080] 248 - 263: Intracellular loop 3 domain

[0081] 290 - 313: Extracellular loop 3 domain

[0082] 332 - 378: C-terminal domain

[0083] In human CCR7, various variants such as amino acid substitution mutants are known in addition to those shown in SEQ ID NO.82. In the "human CCR7" of the present invention, as long as it has an extracellular domain and has the function of CCR7, the said variants are included.

[0084] <Anti-CCR7 antibody>

[0085] The antibody disclosed in the present application is an antibody that specifically binds to the extracellular domain of human CCR7. The antibody of one mode 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. A humanized antibody is preferred, which 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. As an example of the antibody of this mode, NB007-01 described in the following examples can be cited.

[0086] The said heavy-chain variable region (SEQ ID NO.21) comprises the said heavy-chain CDR1 - 3 (SEQ ID NO.25, 27 and 29), and the region outside the CDR is derived from a human antibody. Similarly, the said light-chain variable region (SEQ ID NO.31) comprises the said light-chain CDR1 - 3 (SEQ ID NO.35, 37 and 39), and the region outside the CDR is derived from a human antibody.

[0087] Antibodies of other types have: 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, 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. As an example of the antibody of this type, NB007-02 described in the following examples can be cited.

[0088] The heavy-chain variable region (SEQ ID NO. 41) comprises the heavy-chain CDR1-3 (SEQ ID NO. 45, 47, and 49), and the regions outside the CDRs are from a human antibody source. Similarly, the light-chain variable region (SEQ ID NO. 51) comprises the light-chain CDR1-3 (SEQ ID NO. 55, 57, and 59), and the regions outside the CDRs are from a human antibody source.

[0089] In addition, the present invention encompasses an antibody that 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. 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, 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. As an example of this antibody, VH0 / VL0 described in the following examples can be cited.

[0090] In addition, the present invention includes an antibody that 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. 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. A humanized antibody is preferred, which has: 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. As an example of the antibody, NB007-03 described in the following examples can be cited.

[0091] The antibody may be a functional fragment of the antibody. Here, the "functional fragment of the antibody" refers to a partial fragment of an antibody (i.e., an immunoglobulin) that retains at least one function against an antigen. Examples of the partial fragment include F(ab’)2, Fab, Fv, disulfide-linked Fv, single-chain antibody (scFv, VH-VL), etc. In addition, the antibody of the present invention may be a multispecific antibody such as a bispecific antibody.

[0092] In the case where the antibody is a functional fragment, for example, it has the following effects. That is, when the anti-human CCR7 antibody of the present invention is applied to a drug as described below, when using a full-length antibody such as IgG type, in addition to inhibiting the signal of the target receptor, damage may sometimes be caused in the target tissue, resulting in side effects. In such a case, when using the "functional fragment of the antibody" that only uses the variable region, it is easy to avoid the side effects as described above.

[0093] The antibody preferably has the activity of blocking the CCR7-dependent intracellular signal transduction mechanism based on CCR7 ligand stimulation.

[0094] The antibody preferably has antibody-dependent cell cytotoxicity (ADCC) activity. Thus, the antibody is particularly suitable as an active ingredient of a cancer therapeutic agent.

[0095] The present invention encompasses antibodies that are "functionally equivalent" to the anti-human CCR7 antibody. For example, 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 are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO. 21, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 21; 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 represented by SEQ ID NO. 31, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 31, and the antibody has antibody-dependent cell cytotoxicity activity.

[0096] 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 are substituted, added, or deleted in the amino acid sequence represented by SEQ ID NO. 41, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by 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 represented by SEQ ID NO. 51, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 51, and the antibody has antibody-dependent cell cytotoxicity activity.

[0097] The number of amino acids substituted, added, or deleted is preferably 1 to 8, more preferably 1 to 5, and particularly preferably 1 to 3. The identity of the amino acid sequence is preferably 92% or more, more preferably 95% or more, and particularly preferably 97% or more.

[0098] <Fucose-free glycosylation of the Fc region>

[0099] Preferably, the anti-human CCR7 antibody has an Fc region that contains N-glycoside-linked sugar chains, and fucose is not bound to the 6-position of N-acetylglucosamine at the reducing end of the N-glycoside-linked sugar chains. Briefly, it is preferred that the N-glycoside-linked sugar chains of the Fc region are fucose-free glycosylated. Further in other words, it is preferred that core fucose is not bound to the N-glycoside-linked sugar chains of the Fc region. By fucose-free glycosylating the N-glycoside-linked sugar chains of the Fc region, an increase in the ADCC activity of the antibody and a decrease in the immunogenicity of the antibody can be expected.

[0100] A method for preparing an afucosylated antibody in which the N-glycan sugar chain in the Fc region is afucosylated, for example, can be exemplified by using a host cell in which the activity of an enzyme involved in the synthesis of fucose is reduced or absent. Examples of the enzyme include GDP-mannose 4,6-dehydratase (GMD), GDP-keto-6-deoxymannose 3,5-epimerase, 4-reductase (Fx), GDP-beta-L-fucose pyrophosphorylase (GFPP), alpha-1,6-fucosyltransferase (FUT8), etc. Examples of the host cell include CHO cells.

[0101] It should be noted that when actually producing an afucosylated antibody, it is sometimes obtained in the form of an antibody composition containing a mixture of an afucosylated antibody and an antibody that is not afucosylated. There is no particular limitation on the ratio (molar ratio) of the afucosylated antibody to the non-afucosylated antibody in the antibody composition. It is preferably 50% or more of the afucosylated antibody, more preferably 70% or more, and particularly preferably 80% or more. In other words, among the N-glycan sugar chains contained in the antibody composition, preferably 50% or more are afucosylated, more preferably 70% or more, and particularly preferably 80% or more.

[0102] <Nucleic acid>

[0103] The present invention includes a nucleic acid (DNA) encoding the antibody. For the nucleic acid, for example, it includes: a first nucleic acid encoding a heavy chain variable region, and / or a second nucleic acid encoding a light chain variable region. As a specific example, a nucleic acid containing the first nucleic acid and the second nucleic acid can be exemplified, where the first nucleic acid encodes the amino acid sequence represented by SEQ ID NO.21, and the second nucleic acid encodes the amino acid sequence represented by SEQ ID NO.31. In addition, a nucleic acid containing the first nucleic acid and the second nucleic acid can be exemplified, where the first nucleic acid encodes the amino acid sequence represented by SEQ ID NO.41, and the second nucleic acid encodes the amino acid sequence represented by SEQ ID NO.51.

[0104] The nucleic acid can be integrated into a vector. The vector is appropriately selected according to the type of host cell into which it is introduced, etc. The vector includes a vector for gene therapy.

[0105] <Cell>

[0106] The present invention includes cells containing the said nucleic acid. For example, a cell containing a vector integrated with the said nucleic acid is an example of such a cell. As the type of cell, as long as it can express the said nucleic acid, for example, a cell in which the said vector functions, there is no particular limitation. Examples include animal cells (such as COS cells, CHO cells, etc.), yeast, bacteria (such as Escherichia coli), plant cells, insect cells, etc.

[0107] <Method for manufacturing an antibody>

[0108] The said antibody can be manufactured by a genetic recombination method. That is, a recombinant cell expressing the said nucleic acid can be constructed, and the said antibody can be obtained from the culture of the said cell.

[0109] Taking the method for constructing and producing a humanized antibody as an example. First, as the DNA encoding heavy chain CDR1-3 and light chain CDR1-3, each DNA encoding the amino acid sequences represented by SEQ ID NO.25, 27, 29, 35, 37, 39 is prepared. As this DNA, the base sequences represented by SEQ ID NO.26, 28, 30, 36, 38, 40 can be cited, and other base sequences can also be used.

[0110] In another example, as the DNA encoding heavy chain CDR1-3 and light chain CDR1-3, each DNA encoding the amino acid sequences represented by SEQ IDNO.45, 47, 49, 55, 57, 59 is prepared. As this DNA, the base sequences represented by SEQ IDNO.46, 48, 50, 56, 58, 60 can be cited, and other base sequences can also be used.

[0111] Next, using these DNAs, a DNA encoding a variable region is prepared, in which heavy chain CDR1-3 is transplanted into the framework region (FR) of VH in any human antibody. Similarly, a DNA encoding a variable region is prepared, in which light chain CDR1-3 is transplanted into the FR of VL in any human antibody. Each prepared DNA is inserted into a vector having a sequence encoding CH or CL of a human antibody to construct a humanized antibody expression vector. By introducing the constructed expression vector into a host cell, a recombinant cell expressing the humanized antibody is obtained. And, this recombinant cell is cultured, and the required humanized antibody is obtained from this culture.

[0112] As the purification method of the said antibody, there is no particular limitation, and known methods can be adopted. For example, the culture supernatant of the said recombinant cell can be recovered, and various known methods such as chromatography, salting out, dialysis, membrane separation, etc. can be combined to purify the said antibody.

[0113] <Drug>

[0114] The present invention encompasses a drug containing the anti-human CCR7 antibody as an active ingredient. The drug may be a pharmaceutical composition comprising the anti-human CCR7 antibody and a pharmaceutically acceptable carrier. Preferably, the drug blocks the CCR7-dependent intracellular signaling mechanism based on CCR7 ligand stimulation. Preferably, the drug has antibody-dependent cell cytotoxicity (ADCC) activity.

[0115] In a preferred embodiment, the drug is used for the treatment of cancer. For example, the drug is used as an anticancer agent. The cancer may be any one of hematological cancers and solid cancers.

[0116] Examples of hematological cancers include acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, etc. Examples of non-Hodgkin lymphoma 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 lymphoma, mature T / NK cell-derived, peripheral T-cell lymphoma, adult T-cell leukemia, extranodal NK / T-cell lymphoma, cutaneous lymphoma, etc.

[0117] 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 carcinoma include oral squamous cell carcinoma, esophageal squamous cell carcinoma, pharyngeal squamous cell carcinoma, etc.

[0118] Here, "treatment" means, in a mammal that may have a disease or has a disease, to prevent or mitigate the development and deterioration of the morbid state of the disease, and thus is used in the sense of a therapeutic treatment aimed at preventing or mitigating the development and deterioration of each symptom, etc. of the disease.

[0119] <Administration Method>

[0120] The drug can be administered orally or parenterally, systemically or locally. As the administration methods, injection dosage forms, nasal administration dosage forms, pulmonary administration dosage forms, transdermal administration dosage forms, etc. can be cited. In the case of injection dosage forms, for example, it can be administered systemically or locally by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc. In addition, the administration method can be appropriately selected according to the age and symptoms of the patient. As the dosage of the antibody, for example, it can be selected in the range of 0.0001 mg to 1000 mg per 1 kg body weight each time. Or, for example, the dosage can be selected in the range of 0.001 to 100000 mg / body of the antibody for each patient. However, the dosage of the antibody is not limited to these ranges.

[0121] <Formulation>[Formulation is the process of preparing a drug into a suitable dosage form for administration. It involves various steps such as selecting appropriate excipients, determining the manufacturing process, and ensuring the stability and quality of the final product. The goal of formulation is to optimize the drug's delivery, absorption, and therapeutic effect while minimizing potential side effects. Different dosage forms, such as tablets, capsules, injections, and topical formulations, may be used depending on the drug's properties and the intended route of administration. Formulation also takes into account factors such as patient compliance, ease of manufacturing, and cost-effectiveness. In the pharmaceutical industry, formulation scientists play a crucial role in developing safe and effective drug products.]< / Formulation>

[0122] The drug can be formulated according to conventional methods (e.g., Remington's Pharmaceutical Science, latest edition, Mark Publishing Company, Easton, U.S.A). The drug may contain pharmaceutically acceptable carriers, additives. Examples of such carriers or additives include surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), coloring agents, flavoring agents, preservatives, stabilizers, buffers (phosphoric acid, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonic agents, binders, disintegrants, lubricants, flow promoters, taste modifiers, etc., but are not limited to these, and other commonly used carriers, etc. may be appropriately used. Specifically, examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, calcium carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglycerides, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, etc. In addition, it may contain other low molecular weight polypeptides; proteins such as serum albumin, gelatin, immunoglobulins; amino acids such as glycine, glutamine, asparagine, arginine, lysine.< / Formulation>

[0123] In the case of preparing an aqueous injection solution, examples include physiological saline, isotonic solutions containing glucose, other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and can be used in combination with appropriate solubilizers, such as alcohols (ethanol, etc.), polyols (propylene glycol, PEG, etc.), nonionic surfactants (polysorbate 80, HCO-50, etc.). In addition, if necessary, the antibody can be encapsulated in microcapsules (microcapsules of hydroxymethylcellulose, gelatin, poly[methyl methacrylate], etc.), or made into a colloidal delivery system (liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, etc.) (refer to "Remingto's Pharmaceutical Science 16th edition", Oslo Ed. 1980, etc.).< / Formulation>

[0124] In addition, techniques for sustained release of pharmaceuticals are known and can be applied to the aforementioned drug (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981); Langer, Chem. Tech. 12:98-105 (1982); U.S. Patent 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).

[0125] The aforementioned drug can be administered as an antibody-drug conjugate (ADC). Examples of the combined drugs include monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) as anti-tumor agents.

[0126] <Applications in gene therapy>

[0127] It is also contemplated to integrate the aforementioned nucleic acid into a vector for gene therapy as a gene therapy drug. As methods for administering the gene therapy drug (recombinant vector), in addition to direct administration based on naked plasmids, methods such as administration after packaging in liposomes, etc., and methods of integrating into various viral vectors such as retroviral vectors, adenoviral vectors, vaccinia virus vectors, poxvirus vectors, adeno-associated virus vectors, HVJ vectors, etc. (refer to Adolph, "Viral Genome Methods", CRC Press, Florida (1996)), and methods of administering after coating on bead vectors such as colloidal gold particles (International Publication Pamphlet No. 93 / 17706, etc.) can be cited.

[0128] As long as the gene therapy drug can express the aforementioned antibody in vivo and exert its effect, it can be administered by any method. It is preferably administered in a sufficient amount through appropriate parenteral routes, such as intravenous, intraperitoneal, subcutaneous, intradermal, within adipose tissue, within mammary tissue, inhalation, intramuscular, etc., through methods such as injection, infusion, gas-induced particle bombardment method (using an electron gun, etc.), mucosal routes such as nasal sprays, etc. In addition, the gene therapy drug can also be administered to cells by liposome transfection, particle bombardment method (U.S. Patent No. 4,945,050) or viral infection in ex vivo, and then the cells are reintroduced into an animal for administration.

[0129] <Other disclosures>

[0130] The present invention includes a method for treating cancer, which includes administering an effective amount of the antibody to a cancer patient. The present invention includes the antibody for treating cancer. The present invention includes the use of the antibody for manufacturing a drug used in cancer treatment.

[0131] Examples

[0132] [Example 1] Preparation of humanized anti-CCR7 antibody

[0133] To prepare a humanized antibody of mouse anti-CCR7 antibody R7-18 (described in International Publication No. 2012 / 043533 and Japanese Patent No. 5315495), first, a chimeric anti-CCR7 antibody "VH0 / VL0" was prepared by fusing the heavy chain and light chain variable regions of R7-18 with the Fc region of human antibody IgG1. In addition, a human framework (region) was selected based on the identity between R7-18 and human germline VH and VK genes. Based on computer modeling, a plurality of variable region sequences were designed by adding mutations to the sequences of the selected human germline VH and VK, or the CDRs of the transplanted R7-18, in a manner that could support the predicted three-dimensional structure of the antibody of R7-18. Among the designed humanized antibodies, "VH5-2 / VL1-3", "VH5-1 / VL1", and "VH5 / VL5" that maintained the 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 prepared using the QMCF method (described in International Publication No. 2006 / 084754 and Japanese Patent Application Laid-Open No. 2008-529510).

[0134] The 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 the designed NB007-01, the heavy chain variable region VH5-1 (SEQ ID NO.41) of the designed NB007-02, and the heavy chain variable region VH5 (SEQ ID NO.61) of the designed NB007-03 is as Figure 1A shown. The 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 the designed NB007-01, the light chain variable region VL1 (SEQ ID NO.51) of the designed NB007-02, and the light chain variable region VL5 (SEQ ID NO.71) of the designed NB007-03 is as Figure 1B shown. In Figure 1A and Figure 1BIn this case, the region that completely covers all CDRs defined by the Kabat method and the IMGT method is enclosed by a dashed box.

[0135] The amino acid sequences (AA) of the heavy chain variable region, heavy chain constant region, heavy chain CDR1-3, light chain variable region, light chain constant region, and light chain CDR1-3 of each obtained antibody, as well as the base sequences of the DNA encoding them, are summarized in Tables 1-1 to 1-8.

[0136] [Table 1-1]

[0137]

[0138] [Table 1-2]

[0139]

[0140] [Table 1-3]

[0141]

[0142] [Table 1-4]

[0143]

[0144] [Table 1-5]

[0145]

[0146] [Table 1-6]

[0147]

[0148] [Table 1-7]

[0149]

[0150] [Table 1-8]

[0151]

[0152] [Example 2] Potency of Humanized Anti-CCR7 Antibody

[0153] The human CCR7 gene was introduced into cells (described in International Publication No. 2012 / 043533 and Japanese Patent No. 5315495) and inoculated into a 96-well microplate at an initial cell concentration of 2×10 4 cells / 100 μL and cultured for 2 days. After 2 days, the 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 Humanized anti-CCR7 antibodies NB007-01, NB007-02, or NB007-03 within the concentration range of M. As a positive control, VH0 / VL0 as a chimeric antibody was added within the concentration range of 10 -6 ~10 -10 M.

[0154] After 15 minutes, each cell was stimulated with 5×10 -8 M of CCL21 (R&D Systems). The intracellular Ca 2+ concentration was measured at this time using a Ca 2+ signal measurement device (FDSS / μCELL; Hamamatsu Photonics), and the inhibitory activity of each additive (antibody) on the intracellular Ca 2+ signal was analyzed. The results are shown in Figure 2 and Table 2. Figure 2 is a graph showing the relationship between the inhibitory activity of the antibody on the intracellular Ca 2+ signal and the antibody concentration. Regarding the inhibitory activity, the case of "no antibody and with ligand" was set as an inhibition rate of 0% and the case of "no antibody and no ligand" was set as an inhibition rate of 100% for standardization, and calculated as a relative value. Table 2 shows the 50% inhibitory concentration (IC50) of each antibody calculated based on the Figure 2 analysis results.

[0155] As Figure 2 shown, VH0 / VL0, NB007-01, NB007-02, and NB007-03 all inhibited intracellular Ca 2+ signal transduction in a concentration-dependent manner. This indicates that each additive inhibited the binding of CCL21 to human CCR7, and as a result, inhibited intracellular Ca 2+ signal transduction. Regarding the IC50 value, it was 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). From the above, it is shown that the obtained humanized anti-CCR7 antibodies all have the same CCR7 inhibitory activity as VH0 / VL0 as a chimeric antibody.

[0156] [Table 2]

[0157]

[0158] [Example 3] Immunogenicity of Humanized Anti-CCR7 Antibody

[0159] Evaluate the immunogenicity of NB007-01, NB007-02, and NB007-03 on human PBMC.

[0160] (1) Preparation of human PBMC

[0161] Human PBMC was isolated from whole blood collected from healthy volunteers using the density gradient method, and cryopreserved by adding human AB serum or fetal bovine serum (FBS) and 10% dimethyl sulfoxide. Human PBMC was stored at -180 °C until use.

[0162] (2) DC-T cell assay

[0163] Monocytes were isolated from human PBMC by magnetic separation and cultured in DC medium containing interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for 5 days to differentiate monocytes into immature DC (iDC). The iDC was recovered, seeded on a cell culture plate, and NB007-01, NB007-02, or NB007-03 as the test substance was added, and further cultured in DC medium containing interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) overnight to differentiate into mature DC. NB007-01, NB007-02, NB007-03, and the cytokine cocktail were removed by washing.

[0164] CD4+ T cells were isolated from human PBM by magnetic separation based on negative selection (StemCells: EasySep TM Human CD4+T CellEnrichment Kit; 19052). CD4+ T cells and mature DC were co-cultured in a serum-supplemented medium for 6 days. 5-Ethynyl-2'-deoxyuridine (EdU) was added to the co-culture medium of mature DC and T cells. After 16 hours, live cells and dead cells were separated by fluorescence labeling, and further stained with CD3 and CD4, which are surface markers of T cells. Then, fixation and membrane permeabilization were performed, and the incorporation of EdU was stained with a fluorescent azide and analyzed by flow cytometry (LSR Fortessa, BD). Proliferative Th cells were defined as CD3-positive, CD4-positive, and EdU-positive cells, and analyzed using FlowLogic software.

[0165] (3) Statistical analysis

[0166] For the positive response of the test substances (NB007-01, NB007-02, or NB007-03) to the donors, evaluation was performed by calculating the stimulation index (SI) defined as the ratio of the average response of the test substance to the average response of the control or reference substance. As the object or reference substance, KLH (Keyhole Limpet Hemocyanin) was used. The number of proliferative Th cells in each well, or the number of CD3-positive, CD4-positive, and EdU-positive cells was evaluated by signal response. Based on the calculated SI, donors with SI > 2 were set as positive.

[0167] In addition, to reduce the risk of false negatives, an evaluation based on a statistical equivalence test was also performed. Here, the Distribution Free Resampling (DFR) method described by Moody et al. was used.

[0168] The results are shown in Table 3 and Figure 3 as follows. The number of donors for which NB007-01 was statistically immunogenic positive was 2 out of 20, showing lower immunogenicity compared to the control substance. Similarly, the number of donors for which NB007-02 was statistically immunogenic positive was 7 out of 20, showing lower immunogenicity compared to the control substance. Similarly, the number of donors for which NB007-03 was statistically immunogenic positive was 9 out of 20, showing lower immunogenicity compared to the control substance. In particular, NB007-01 with a modified CDR showed an unexpected improvement in immunogenicity compared to NB007-03 without a modified CDR.

[0169] [Table 3]

[0170]

[0171] [Example 4] Cell Binding Evaluation Based on Flow Cytometry

[0172] For the binding to cells transfected with the hCCR7 gene and the binding to the human lymphoma cell line derived from T cells, namely MJ cells, NB007-01 labeled with the fluorescent dye Allophycocyanin (APC) was used for evaluation. For the binding to the human lymphoma cell line derived from B cells, namely Granta-519 cells, unlabeled NB007-01 was used, and Alexa Fluor 647-labeled anti-human IgG antibody (Thermo Fisher Scientific) was used as the detection secondary antibody for evaluation. For APC labeling, an APC Conjugation Kit-Lightning-Link (Abcam) was used. To 100 μL of the antibody solution diluted to 1 mg / mL with PBS, 10 μL of the reaction initiation solution (Modifier reagent) was added, and it was reacted with the APC Conjugation Mix. After storing at room temperature in the dark for 3 hours, 10 μL of the reaction termination solution (Quencher Reagent) was added and used as the fluorescently labeled antibody.

[0173] The cells transfected with the hCCR7 gene were detached from the culture dish using Cell Dissociation Buffer, enzyme-free, Hanks’ Balanced Salt Solution (Thermo Fisher Scientific) and washed with PBS. On the other hand, appropriate amounts of Granta-519 cells and MJ cells as suspended cells were separately aliquoted and washed with PBS. An equal volume of goat serum (Thermo Fisher Scientific) was added to the cell suspension to make it 1×10 7 / mL and incubated at 4°C for 30 minutes for blocking. The supernatant was removed by centrifugation at 200 g for 5 minutes to make it 4×10 6Suspend the cells in FACS Buffer (PBS containing 1% FBS) at a density of / mL, and dispense 50 μL into each well of a 96-well plate. Mix the fluorescently labeled NB007-01 antibody or unlabeled NB007-01 diluted with FACS Buffer, and let it stand at 4 °C for 1 hour. Centrifuge at 200 g for 5 minutes to collect the cells, and wash the cells with 100 μL of FACS Buffer. Perform this washing operation twice. The hCCR7 gene-transfected cells and MJ cells that have come into contact with labeled NB007-01 are suspended in 100 μL of FACS Buffer, and the binding of the cells to the labeled antibody is measured using a flow cytometer CytoFLEX (BECKMAN·COULTER). The Granta-519 cells that have come into contact with unlabeled NB007-01 are suspended in 50 μL of a dilution of Alexa Fluor 647-labeled anti-human IgG antibody diluted 800-fold with FACS Buffer, and let it stand at 4 °C for 1 hour. After washing the cells twice, suspend them in 100 μL of FACS Buffer, and measure the binding to the antibody using a flow cytometer.

[0174] A graph with the antibody concentration on the horizontal axis and the geometric mean of the fluorescence intensity of the FACS histogram on the vertical axis is as Figures 4A - 4C shown. From this, it was confirmed that NB007-01 specifically binds to hCCR7 gene-transfected cells ( Figure 4A ), Granta-519 cells ( Figure 4B ), and MJ cells ( Figure 4C ).

[0175] [Example 5] Functional evaluation of NB007-01 based on intracellular Ca 2+ signal measurement

[0176] By the same procedure as in Example 2, seed hCCR7 gene-transfected cells in a 96-well microplate and culture for 2 days. Replace the medium with a solution containing 3 μM Cal-520 (AAT Bioquest), 0.05% Pluronic-F127, and 2.5 mM probenecid (Invitrogen). After 1 hour, add NB007-01 in the concentration range of 10 -6 ~10 -10 M to each well. After 15 minutes, stimulate each cell with 5 × 10 -8 M of CCL21 (R&D Systems) or 1.5 × 10 -8 M of CCL19 (R&D Systems). Use a Ca 2+ signal measurement device to analyze the inhibitory activity of NB007-01 on intracellular Ca 2+ signals. The results are asFigure 5A and Figure 5B as shown Figure 5A and Figure 5B are graphs showing the relationship between the inhibitory activity of an antibody against intracellular Ca 2+ signals and the antibody concentration. Figure 5A shows the case of stimulation with CCL21, Figure 5B shows the case of stimulation with CCL19. Regarding the inhibitory activity, the case of "no antibody, with CCL21 or CCL19" was set as the inhibition rate of 0%, and the case of "no antibody, no CCL21 or CCL19" was set as the inhibition rate of 100% and standardized, and calculated as a relative value.

[0177] As Figure 5A , Figure 5B shown, NB007-01 inhibited CCL21- and CCL19-induced intracellular Ca 2+ signaling in a concentration-dependent manner. The IC50 value was 17.8 nM for CCL21 stimulation and 21.4 nM for CCL19 stimulation. From the above, it is shown that NB007-01 inhibits CCL21- and CCL19-induced intracellular Ca 2+ signals.

[0178] [Example 6] Evaluation of inhibitory activity of intracellular Ca 2+ signaling using a human lymphoma cell line

[0179] The MJ cells were washed with a test buffer of HBSS (containing CaCl 2 , MgCl 2 ) + 0.1% BSA, and mixed with a solution containing 1 μM Cal-520 (AAT Bioquest) and 0.05% Pluronic-F127 at a cell concentration of 1.16×10 6 cells / mL, and incubated at 37°C. After 45 minutes, the cells were washed twice with the test buffer, adjusted to a cell concentration of 1×10 6 cells / mL, and 80 μL was inoculated into each well of a 96-well black PDL-coated microplate (CELLCOAT, Grenier). NB007-01 or CAP-100 at 8 concentrations diluted 3-fold in the concentration range of 250 nM to 0.11 nM was added to each well and incubated for 15 minutes. The test plate was set in a Ca 2+ signal analysis device (FDSSμCELL; Hamamatsu Photonics), and the inhibitory activity of each antibody against intracellular Ca 2+ signals was analyzed when the cells were stimulated with 20 nM of CCL21 (R&D Systems). The results of NB007-01 are as Figure 6AAs shown, the results of CAP-100 are as Figure 6B shown. As Figure 6A shown, NB007-01 inhibits CCL21-induced intracellular Ca 2+ signaling in a concentration-dependent manner, and its 50% inhibitory concentration (IC50) is 14.8 nM. On the other hand, as Figure 6B shown, CAP-100 inhibits CCL21-induced intracellular Ca 2+ signaling in a concentration-dependent manner, but the maximum inhibition rate is about 50%.

[0180] [Example 7] Evaluation of Cell Migration Inhibitory Activity Using Human Lymphoma Cell Lines

[0181] After staining Granta-519 cells with CytoRed (Dojindo Laboratories), they were washed twice with RPMI + 0.5% BSA. NB007-01 (0.3 μg to 100 μg / mL) or human IgG1 antibody (manufactured by Icosagen) as a negative control (1 μg / mL or 100 μg / mL) was added to a cell suspension of 4 × 10 6 cells / mL, and the mixture was allowed to stand at 37°C for 30 minutes.

[0182] 50 ng / mL of CCL19 was added to the wells of the receiving plate of an insert type 96-well Transwell with a 3.0 μm polycarbonate membrane (manufactured by Corning). After setting the insert of the Transwell on the receiving plate, 50 μL of the cell suspension was inoculated into the insert and allowed to stand at 37°C. After standing for 4 hours, the insert was removed, and three fields of view of each well were photographed with a 4-fold fluorescence lens to measure the number of migrated cells. Regarding the inhibitory activity, the case of "no antibody, with CCL19" was set as an inhibition rate of 0% and the case of "no antibody, no CCL19" was set as an inhibition rate of 100% for normalization, and the relative value was calculated. Figure 7 Shows the inhibitory effect of NB007-01 on CCL19-dependent cell migration. The IC50 of B007-01 is 25.5 nM. It should be noted that the inhibition rates of the human IgG1 antibody of the negative control at 1 μg / mL and 100 μg / mL are 3.0% and 3.3% respectively.

[0183] [Example 8] Preparation of Afucosylated NB007-01

[0184] As a method for afucosylating an antibody, the GlymaxX method (International Publication No. 2011 / 035884, Japanese Patent No. 5746183) was used. In the steps of preparing NB007-01 by the QMCF method in Example 1, as the CHO cells for antibody gene expression, a CHO cell line (CHOEBNALT85-RMD C4) modified by the GlymaxX method was used to prepare afucosylated NB007-01. The antibody was purified.

[0185] Using the AdvanceBio Gly-X-N-glycan prep with InstantPC Kit (AGILENT), sialidase (AdvanceBio Sialidase A) (AGILENT, GK80040), β1-3,4-galactosidase (β1-3,4Galactosidase; BTG) (NEB, P0746S), β-N-acetylglucosaminidase S (β-N-Acetylglucosaminidase S; GUH) (NEB, P0744), and α1-2,4,6-fucosidase O (α1-2,4,6Fucosidase O; FucO) (NEB, P0749), N-type sugar chains were excised and labeled from the purified antibody according to the manual. For the labeled N-type sugar chains, HPLC (Agilent 1260 Infinity II) analysis was performed using an AdvanceBio Glycan 2.7μm column (AGILENT) according to the manual. The results of the HPLC chromatogram are as Figure 8A shown, and the abundance ratios of each sugar chain type calculated from the chromatogram are as Figure 8B shown. Figure 8A In, "Undig" represents untreated, "Sialidase A" represents sialidase treatment, "Sial+BTG" represents sialidase + BTG treatment, "Sial+BTG+GUN" represents sialidase + BTG + GUN treatment, and "Sial+BTG+GUN+FucO" represents sialidase + BTG + GUN + FucO treatment. As Figure 8A , Figure 8B shown, most of the separated peaks were assigned to determined sugar chains based on the relative retention time (glucose units), accounting for 89% of the peak area of all samples. In addition, in the cleavage experiment based on FucO, there was almost no change in the peaks, and more than 99% of the determined sugar chain types were afucosylated.

[0186] [Example 9] Evaluation of the cytotoxic function of afucosylated NB007-01

[0187] (1) ADCC Reporter Bioassay

[0188] The ADCC function of afucosylated antibodies 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 the ADCC Assay Buffer, and Granta-519 cells were suspended at a cell density of 5×10 5 / mL. In addition, a dilution solution of the antibody to be the test substance was prepared using the ADCC Assay Buffer. As the test substances, NB007-01, afucosylated NB007-01, CAP-100, and human IgG as a negative control (manufactured by Icosagen) were used. 630 μL of the ADCC Bioassay Effector Cells included in the kit was added to 3.6 mL of the ADCC Assay Buffer to prepare the 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 left standing at 37 °C and 5% CO 2 environment for 6 hours. The Luciferase Assay Substrate included in the kit was dissolved with the Luciferase Assay Buffer, and 75 μL of it was added to each well of the plate. After leaving it standing in the dark at room temperature for 15 minutes, the relative luminescence intensity (Relative Luminescence Units; RLU) was measured using a microplate reader ARVO (PERKINELMER). Figure 9 It is a graph with the antibody concentration on the horizontal axis and the RLU on the vertical axis. RLU is an index of the activation of effector cells, and this bioassay is a functional assay based on the ADCC mechanism of action. As Figure 9 shown, the ADCC function of afucosylated NB007-01 (EC50 = 16.1 pM) was significantly improved compared to NB007-01 (EC50 = 123 pM), and it was superior to CAP-100 (EC50 = 57.1 pM).

[0189] (2) Cell Inhibition Activity Assay Using Human PBMC

[0190] (a) Preparation of Human PBMC

[0191] Frozen human PBMC was thawed in a 37 °C water bath. After washing with the complete medium, it was adjusted to 2×10 6Resuspend in complete medium at a concentration of / mL and culture overnight at 37°C and 5% CO 2 conditions.

[0192] (b) Pretreatment of Granta-519 cells

[0193] Prepare Granta-519 cells in ADCC medium to a viable cell concentration of 3×10 4 / 80 μL and inoculate into each well of a 96-well round-bottom plate. Further, add 20 μL of the test substance diluted with ADCC medium to each well and let stand at room temperature for 30 minutes. As the test substances, use afucosylated NB007-01 (1000 - 0.0001 ng / mL), CAP-100 (10000 - 0.1 ng / mL), and Rituximab (10000 ng / mL, Roche).

[0194] (c) Measurement of ADCC activity

[0195] Use human PBMC as effector cells (E) and Granta-519 cells as target cells (T) and prepare at E:T = 50:1. That is, after washing the human PBMC from (a) with ADCC medium, adjust the cell concentration with ADCC medium and add 1.5×10 6 cells / 100 μL to each well of (b). After standing at 37°C for 5 hours, centrifuge at 250×g for 4 minutes and collect 50 μL of the supernatant into each well of a transparent flat-bottom 96-well plate. In addition, 45 minutes before collecting the supernatant, prepare a control for the maximum cytotoxicity condition with 20 μL of Lysis buffer (CytoTox 96 Non-Radioactive Cytotoxicity Assay kit, Promega, G1780) added.

[0196] Add 50 μL of the substrate mixture (CytoTox 96 Non-Radioactive Cytotoxicity Assay kit, Promega, G1780) to each well and mix the plate for 30 seconds. Then, let stand at room temperature for 30 minutes. Add 50 μL of the stop reagent (CytoTox 96 Non-Radioactive Cytotoxicity Assay kit, Promega, G1780) to each well, mix for 30 minutes, and measure the absorbance at 490 nm. The cytotoxic activity (%) is normalized with the maximum cytotoxic condition set as 100% and the condition without the test substance set as 0% and calculated as a relative value. The graph of the calculated values is as Figure 10 shown. Figure 10In this context, "aNB007" refers to afucosylated NB007-01.

[0197] In the results of this experiment, afucosylated NB007-01 (EC50 = 1.9 ng / mL, equivalent to 12.6 pM in terms of IgG) also showed superior cell inhibitory activity compared to CAP-100 (EC50 = 26.3 ng / mL, equivalent to 175 pM in terms of IgG). Additionally, Rituximab, which is clinically applied under the anti-tumor effect based on ADCC activity, remained at 18.6% cytotoxic activity at a concentration of 10000 ng / mL, suggesting the advantage of afucosylated NB007-01 in tumor treatment efficacy.

[0198] [Example 10] Anti-tumor Activity of Humanized Anti-CCR7 Antibody

[0199] The anti-tumor activities of NB007-01, afucosylated NB007-01, and CAP-100 were evaluated using a tumor transplantation model of Granta-519 cells. Female CB17 / SCID mice at 6 - 8 weeks of age were used as model animals.

[0200] Granta-519 cells were cultured in RPMI-1640 medium containing 20% FBS at 37°C and 5% CO 2 2. Granta-519 cells at a density of 1×10 7 cells / 0.1 mL were transplanted into 6 - 8-week-old CB17 / SCID mice via intravenous administration. The transplantation day was set as day 0. On the first day after transplantation, the animals were grouped based on their body weights. The grouping was performed by the Matched distribution method (StudyDirector TM software, version 3.1.399.19).

[0201] The test substances were administered starting from the second day after cancer cell transplantation. The dosage was 3 mg / kg for NB007-01, CAP-10, and human IgG1 of the isotype control, and 0.03 mg / kg for afucosylated NB007-01. Each test substance was administered at a dosing volume of 10 μL / g with a dosing frequency of twice a week.

[0202] After cancer cell transplantation, the morbidity and mortality rates of each individual were confirmed daily. In addition, abnormalities such as tumor proliferation, motor ability, food and water consumption, weight gain or loss, eye and hair arrangement status caused by the treatment were confirmed. For weight, it was measured twice a week. In the case of death or clinical signs being confirmed, the details were recorded for each individual. Weight was measured using Study Director TM software (version 3.1.399.19). The effect of anti-tumor activity was evaluated by the Kaplan-Meier survival curve to determine the survival rate of individuals.

[0203] In addition, for a part of the individuals in each group, the liver and lymph nodes were collected on the 24th day after cancer cell transplantation. The collected organs were fixed with 10% buffered formalin to prepare paraffin blocks. The prepared blocks were sectioned at 4 μm and treated at 60 °C for 30 minutes. The infiltration ratio of Granta-519 cells was measured by immunohistochemistry. The identification of Granta-519 cells used an antibody against human CD45 (Cell Signaling Technology, Cat#13917) as a hematopoietic cell surface marker. The sections were activated under the conditions of EDTA, pH 9.0, 100 °C, and 20 minutes. The antibody used was diluted 1:800 for use. The immunostained slides were all scanned and saved at 40 times magnification using the NanoZoomer-HT 2.0 / Pannoramic SCAN Image system. The HALOTM platform was used to measure the number of CD45-positive cells in the immunohistochemical scoring and evaluate it as the number of cells per 1 mm 2 of cells.

[0204] The change in the survival rate represented by the Kaplan-Meier survival curve is as Figure 11 shown. In addition, the number of CD45-positive cells in the lymph nodes is as Figure 12A shown, and the number of CD45-positive cells in the liver is as Figure 12B shown. As Figure 11 shown, compared with the isotype control group, the survival period of NB007-01 was prolonged, and furthermore, it was found that the survival period was prolonged compared with CAP-100. As Figure 12A , Figure 12BAs shown, NB007-01 strongly inhibits the infiltration of cancer cells into various organs, and its effect in the liver is stronger than that of CAP-100. Thus, it is suggested that NB007-01 exerts an anti-tumor effect by inhibiting the infiltration of lymphoma into various organs. In addition, afucosylated NB007-01 showed a survival prolongation effect comparable to that of CAP-100 at a dosage of 1 / 100. This indicates that afucosylated NB007-01 with enhanced cell inhibitory activity can expect an anti-tumor effect at a low dose.

Claims

1. 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.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.

2. The antibody according to claim 1, which 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.

3. The antibody according to claim 1, which has an Fc region, wherein the Fc region comprises an N-glycosidic bond sugar chain, The 6-position of N-acetylglucosamine at the reducing end of the N-glycosidic bond sugar chain is not bound to fucose, The antibody has antibody-dependent cellular inhibition.

4. The antibody according to claim 2, which has an Fc region, wherein the Fc region comprises an N-glycosidic bond sugar chain, The 6-position of N-acetylglucosamine at the reducing end of the N-glycosidic bond sugar chain is not bound to fucose, The antibody has antibody-dependent cellular inhibition.

5. An antibody that specifically binds to the extracellular domain of human CCR7, having: 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 represented by SEQ ID NO. 21, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO. 21; 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 represented by SEQ ID NO.31, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO.31, The antibody has an Fc region, wherein the Fc region comprises an N-glycosidic bond sugar chain, The 6-position of N-acetylglucosamine at the reducing end of the N-glycosidic bond sugar chain is not bound to fucose, The antibody has antibody-dependent cellular inhibition.

6. 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.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.

7. The antibody according to claim 6, which 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.

8. The antibody according to claim 6, which has an Fc region, wherein the Fc region comprises an N-glycosidic bond sugar chain, The 6-position of N-acetylglucosamine at the reducing end of the N-glycosidic bond sugar chain is not bound to fucose, The antibody has antibody-dependent cellular inhibition.

9. The antibody according to claim 7, which has an Fc region, wherein the Fc region comprises an N-glycosidic bond sugar chain, The 6-position of N-acetylglucosamine at the reducing end of the N-glycosidic bond sugar chain is not bound to fucose, The antibody has antibody-dependent cellular inhibition.

10. An antibody that specifically binds to the extracellular domain of human CCR7, having: 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 represented by SEQ ID NO.41, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by 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 represented by SEQ ID NO.51, or an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO.51, The antibody has an Fc region, wherein the Fc region comprises an N-glycosidic bond sugar chain, The 6-position of N-acetylglucosamine at the reducing end of the N-glycosidic bond sugar chain is not bound to fucose, The antibody has antibody-dependent cellular inhibition. 11 . A nucleic acid encoding the antibody according to claim 1 .

12. The nucleic acid according to claim 11, comprising a first nucleic acid and a second nucleic acid, The first nucleic acid encodes the amino acid sequence represented by SEQ ID NO.21, The second nucleic acid encodes the amino acid sequence represented by SEQ ID NO.

31.

13. The nucleic acid according to claim 11, comprising a first nucleic acid and a second nucleic acid, The first nucleic acid encodes the amino acid sequence represented by SEQ ID NO.41, The second nucleic acid encodes the amino acid sequence represented by SEQ ID NO.

51.

14. A cell comprising the nucleic acid of claim 11.

15. A cell comprising the nucleic acid of claim 12.

16. A cell comprising the nucleic acid of claim 13.

17. A medicine comprising the antibody according to any one of claims 1 to 10 as an active ingredient.

18. The medicament according to claim 17, which is used for the treatment of cancer.

19. The drug according to claim 18, wherein The cancer is a blood cancer.

20. The drug according to claim 19, wherein The blood cancer is acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia or non-Hodgkin's lymphoma.

21. The drug according to claim 20, 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, peripheral T-cell lymphoma, adult T-cell leukemia, extranodal NK / T-cell lymphoma or skin lymphoma.

22. The drug according to claim 18, wherein The cancer is a solid cancer.

23. The drug according to claim 22, 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.

24. The drug according to claim 23, wherein The squamous cell carcinoma is oral squamous cell carcinoma, esophageal squamous cell carcinoma or pharyngeal squamous cell carcinoma.

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